BIOCHEMICAL CHARACTERIZATION AND INTERSPECIES RELATIONSHIP OF PARVALBUMIN ISOFORMS IN

GENUS CHANNA

ABSTRACT THESIS SUBiHTTEO FOR THE DEGREE OF

Boctor of ||6tlo£opfip IN

ZOOLOGY

SYBD HASAN ARIF

SECTION OF GENETICS DEPARTMENT OF ZOOLOGY

ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)

2002

ABSTRACT

Name of the candidate Syed Hasan Arif

Title of the Ph.D. thesis Biochemical cliaracterization and inter­

species relationship of Parvalbumin

isoforms in genus Channa.

Ca " ion participates in several biochemical events; one of them

being the muscular contraction that is triggered by the release of micro

amounts of calcium in to the basic contraction unit, the sarcomere.

Relaxation is brought about by the withdrawal of calcium from the

system for which calcium exchange proteins exist both inside the

sarcomere and in the sarcoplasm outside it. Evidence is gathering that

demonstrates that in the sarcoplasm most likely calcium exchanger is a

water soluble protein, the parvalbumin (PA). This protein is specifically

abundant in the skeletal muscle of fish, where its isotypes or isoforms

(the protein bands of different elctrophoretic mobilities) discriminate

different types of muscle fibers. Parvalbumin binds calcium with high

affinity, to be precise two moles of calcium by one mole of protein. It is

also shown to serve as biomarker at various levels of taxa due to its

polymorphic nature.

The parvalbumin as a polymorphic protein has attracted specific

attention from geneticists to explore the use of polymorphs as

subpopulation markers. Being monomeric proteins of rather low

molecular weight and having only two extant lineages, they may be

suitable candidates for tracing phylogeny.

Though a lot of information is available on parvalbumins of

temperate fish species and more precise information involving cloning

and sequencing techniques is being published, the knowledge on the

parvalbumins of fish species of tropical waters is far fi^om adequate.

Several specialized groups of specific evolutionary lineages are

represented in the fish fauna of tropical countries, one such group

constituted by the species of genus Channa that has evolved obligate air

breathing as an adaptive mechanism to face water scarcities, has a

reasonable distribution in India. The present investigations were aimed at

partial biochemical characterization of the parvalbumin isotypes/

isoforms of four species of this genus, Channa that is : Channa punctatus,

C. marulius, C. gahua and C. striatus. On the basis of the present

investigations on parvalbumin isoforms of this evolutionarily distinct

group the following conclusions could be made:

1. PA isoforms in these species make up to 45% of the soluble

proteins of the muscle sarcoplasm.

2. Individually, C. punctatus. C. marulius, C. gachua and C striatus

have respectively 3, 3, 2 and 3 isoforms with one out of them in

each species is the major PA. Stoichiometry of the different

isoforms in each of the above species, in the same order is :

0.07:0.043:1,0.05:0.54:1 and 0.9:0.2:1, respectively.

u^

3. All the bands initially identified as PAs in PAGE patterns cross-

react with anti-PAII-rabbit antibodies in western blot developed by

a chemiluminiscent method.

4. The molecular mass of minor and major PAs may differ, but agrees

well with the reported values of 10 to 12 kD.

5. PA isoforms of Channa species are of the highest thermostability

reported so far. During heat treatment at 90°C interspecies and

intraspecies differences in their relative stabilities were observed. It

is proposed that the heat denaturation is an aggregation reaction

involving oxidation of thiols.

6. Species differences are exhibited by UV spectra taking into

account wavelengths of 259, 283 and 293 as indicative of the

presence of Phe, Tyr and Trp.

7. Coincident with the obvious increase in the electrophoretic

mobilities at 10 mM EDTA is the negative spectral perturbation

with in the band assigned to Tyr and Trp in UV spectra .

8. Fish PA treated at high concentration of EDTA (200) mM show

very low intensity and resembling perturbations in contrast with the

UV spectrum of chick PA that shows very high magnitude peak in

Phe and Trp range. This difference confirms that Tyr and Trp

contents offish PA are indeed very low.

9. In terms of affinity of the chelators for the sites of their interaction

EGTA exerts a predominant influence, as apparent by

predominantly EGTA type changes in PAGE patterns of PAs if

simultaneously treated with both the chelators.

10. Evolutionarily, PA isoforms of species of genus Channa are of P-

lineage and spectral and biochemical peculiarities of PA isoforms

of C. gachua suggest that they may be a good model for

phylogenetic studies. Phylogenetic relationship as derived by

similarity dendrogram and other biochemical characteristics

suggest that though C. gachua might have been the most

generalized form, the likely species of most remote origin is C.

punctatus.

^ ^ - > ^

BIOCHEMICAL CHARACTERIZATION AND INTERSPECIES RELATIONSHIP OF PARVALBUMIN ISOFORMS IN

GENUS CHANNA

/ • -

THESIS SUBMITTED FOR THE DEGREE OF

It Boctor of $t)tlO!e(op^?

Ky m ii

\ \ \ > ^ ^ ^ ZOOLOGY

^""*ij.

SYED HASAN ARIF

SECTION OF GENETICS DEPARTMENT OF ZOOLOGY

ALIGARH MUSLIM UNIVERSITY ALIGARH (INDIA)

2002

.^sis

JF d m Lcr/.puter

/ •V' ( -T-^G^2 , j

1 3 JUN 2005

T5883

(Dedicated to my

Barents

VncCe

-. I External: 700920/21-300/301 •^"""^M Internal: 300/301

D E P A R T M E N T OF Z O O L O G Y ALIGARH MUSLIM UNIVERSITY

ALIGARH—202002 INDIA

Stations: _ ^ _ 1. AGRICULTURAL NEMATOLOGY "' "°' I^^ 2. ENTOMOLOGY ^ . ^ ^n / o 1 ^i 3. fnSHERY SCIENCE &AQUACULTURE Datea.>^.V.:.'.vC...'!i.aD^ 4. GENETICS 5. PARASITOLOGY

This is to certify that Mr. Syed Hasan Arif has completed

t he t h e s i s ent i t led '^Biochemical character izat ion and

interspecies relationship of parvalbumin isoforms in genus

Channa** under my supervision. The work is original and has

been independently pursued by the candidate.

I permit the candidate to submit the thesis for the award of

Doctor of Philosophy in Zoology of the AHgarh MusUm University,

AUgarh.

Dr. Absar-ul Hasnain (Supervisor)

^^^W5

Acknowledgements

My vocabulary fails to express my heartfelt gratitude to my supervisor, Dr. Absar-ul Hasnain. without whose perception, technical expertise, advice, generous material help and moral support this work would never attain this form. Blessmgs of my mother, fether Mr. Syed M. Atyab, my uncle Mr. Syed M. Musaiyab and phupi were the spiritual force behind this achievement.

I am obliged to the former Chairperson Prof. Jamil A. Khan for unfailing support and sincere advice during his tenure and to Prof. A. K. Jafri, the present Chairman of the Department for the departmental facilities and material help that was always available to me. Kind permission by Prof. (Mrs.) Durdana S. Jairajpuri to use the tools and equipment of Parasitology Research Laboratory is also thankfully acknowledged.

Thanks are also due to my esteemed former teacher Dr. Tariq M. Haqqi who in spite of being a busy person as the Director of Research in Rheumatology at Case Western Reserve University, Cleveland (USA) always took keen interest and gave valuable advices during his short visits when the available time was so scarce. Without his material support several of the experiments would not have been satisfactorily completed.

I will fail in my duty if I do not fondly recall my favourite teacher, (Late) Prof. Wajih A- Nizami who always inspired me as a researcher and guided and helped me at the hour of need.

Sincere thanks are also due to my reputed teachers Prof. Asif Ali Khan and Dr. Iqbal Perwez who also made their laboratory facilities available and gave valuable suggestions. Constructive advices suggestions of Dr. Kamal A. Rizvi, Prof. Absar M. Khan, Dr. M. Afzal, Dr. Niamat AM, Dr. Asim Azfer, Dr. Gul Ahmad, and Dr. Khalid Saifiillah have helped me a lot.

I have to recall the help by Dr. Abbas A. Abidi with a sense of gratitude.

Gratitude is due to my laboratory colleagues Dr. (Mrs.) Nikhat Nabi, Mr. R B. Pandey, Ms. Mumtaz Jabeen and Mr. Riaz Ahmad. Their sincerity has been an asset throughout this work. The tracing of difference spectra was possible due to the kind permission by Dr. Javed Musarrat of the Faculty of Agriculture to allow the use of facilities there. Mr. Fazlur Rehman Khan and Dr. Qamar Ahmad helped in spectroscopy and in developing western blots. I shall remember their contribution with sincere thanks.

Lastly, I wish to express my thank Dr. Ammar Ibne Anwar whose help was available at very critical moments as well as to my senior colleagues Dr. (Miss) Anjum Nasreen and Dr. (Miss) Fauzia Sherwani. Ms. Kavita Singh and Mr. Abul Farah were always helpful in experimental work that I acknowledge with great appreciation.

Laboratory work always needed the help of Mr. Shakir Ali and Mr. Zheer Ahmad and lab. hygiene was taken care of by Mr. Ramesh, which I wish to mention with thanks.

r^yed Hasan Arif)

CONTENTS

Page No.

I. Introduction 01-09

II. Material and Methods 10-23

III . Results 24-41

IV. Discussion 42-52

V. Conclusion 53-55

VI. References 56-65

INTRODUCTION

Note : The three dimensional structure of model parvalbumin

INTRODUCTION

Ca"~- ion participates in the control of several physiological events in

the cell which also needs some intracellular exchangers to exert their

regulatory effects. At cellular level, there exist proteins involved in

interaction with calcium to exert this effect. The disturbance in calcium

homeostasis can lead to several disorders or clinical complications and,

abnormally high levels of Ca" -binding proteins may even lead to continued

activation of DNA synthesis (Heizmann, 1984). Increased Ca""

concentration is also known to make tumor cells invasive and enhance their

proliferationfHeizmann et al. 1977). Other molecular events where Ca"^ ion

related regulatory mechanisms operate include muscle contraction, homione

action, enzyme activation, neurotransmission, cell division and secretion

(Heizmann el al., 1987). The above mentioned important but highly diverse

roles in the biology of cell are perfomied by at least one of the specific

protein belonging to multigene family of calcium binding proteins more than

34 of which have already been listed.

The present work deals with an important member of calcium binding

family, the parvalbumin, which has been recognized as the calcium storage

protein of muscle sarcoplasm. It is because of the exceptional solubility in

water that Pechere (1968) had named it as "parvalbumin". Its occurrence

was first reported in fish and amphibian skeletal muscle by Deuticke (1934);

however, subsequent work showed it to be ubiquitous to skeletal muscle of

cold blooded vertebrates that also makes up the major fraction of their

soluble protein repertoire. Intraspecies variations in the occurrence of

parvalbumin in muscular tissue may, however, exist. As for instance, no

parvalbumin could be detected in the breast muscles of either embryonic or

adult chicken (Heizmarm and Strehler, 1973). Huriaux et al. (1990) showed

that heart muscle fibers of the European barbell, Barbus barbus are devoid

of any parvalbumin, a finding that may be extended to other red muscle

types including that of the lateral line as well. The cardiac muscle of

Antarctic fishes, Channichthys rhinoceratus and Chanpsocephalus gunnari

are exceptions where, as many as three parvalbumin isofonns have been

detected (Laforet et al., 1991). Among mammals, parvalbumin has been

detected in cardiac muscle of the shrew only. There are indications that

contrary to initial understanding skeletal muscle of higher vertebrates may

not be entirely devoid of parvalbumin.

With the help of X-ray diffraction and primary sequence data three-

dimensional structure of mainly fish parvalbumin molecule has been worked

out. It is comprised of six helices named A, B, C, D, E and F, arranged into

two identical protein motifs called EF-hands. These protein motifs were

named after the Ca" - binding region of calmodulin, formed by the helices E

and F. Thus, a conmion structural characteristic of Ca" -binding proteins is

the EF-hand which has a helix-loop-helix motif fonned by a sequence of

about 30 amino acids (Kretsinger and Nockolds, 1973). EF-hands naturally

occur in pairs, and this domain partnering gives proteins a more stable

structure and function. The EF-hand (motifs) binds one Ca"^-ion per protein

motif. Due to the identical nature of the EF and CD-hands, and the

symmetrical layout these structures are thought to have arisen after gene

duplication.

In smaller amounts parvalbumin might occur in non-muscle tissues as

well. Among fishes, its differing amounts occur in various non-muscle

tissues of pike and in the brain of European carp (Gosselin-Rey, 1974). The

tissues where parvalbumin is reported to exist in higher vertebrates are brain,

spleen, kidney and ovary of rabbit (Baron el al., 1975) and the brain of chick

(Heizmann and Strehler, 1979). Following dot-blot immuno-assay against

parvalbumin antibodies, a faint immunocross- reactivity was detected in

adipose tissue, tendons and gonads and by RIA in endocrine glands such as

thyroid, mammary and lachrymal glands (Heizmann, 1984). Whereas

occurrence of parvalbumin in neurons has been confirmed by

immunochemical study carried out on rat, its association with axonal flow in

the central and peripheral nervous system that is a calcium dependent

phenomenon had already been demonstrated (Ochs and Worth, 1978). These

observations emphasize the role of parvalbumin in regulating the level of

free Ca"" in the neuroplasm.

In addition to the above, parvalbumin appears to play a crucial role in

the process of calcification also. An immuno-histochemical study on bone

has demonstrated the parvalbumin cross-reactivity in the calcification center

of bone (Celio et al., 1984) and in the neuroblasts of teeth. This review

therefore suggests that though parvalbumin is the most abundant in skeletal

muscle, it is a typical example of calcium exchanger that is involved in a

wide ranging regulatory function in a variety of other tissues, as well.

In most of the instances parvalbumin exists in multiple molecular

fornis or isoforms and it is on its skeletal muscle isoforms that the quantum

of available information is the largest. For instance, in muscle of adult

common snook Contropomus undecimalis as many as seven isofomtis are

reported to exist (Ross e( ai, 1997). In some cyprinids (Piront and Gosselin-

Rey, 1974) including Barbus barbus (Focant el al., 1992) and the brown

trout, Salmo trutta (Huriaux et ai, 1996) three to four and in pike (Declerq

et a/., 1990) and sea bass, Morone labrax (Huriaux ef ai, 1996) two to three

isoforms have been reported. Five parvalbumin isoforms have also been

detected in the muscle of carp (Pechere et al., 1971), lung-fish, Protopterus

spp. (Gerday el al., 1978) and the rainbow trout, Onchorhynchus mykiss

(Huriaux el al., 1996). In general, two to five parvalbumin isoforms are a

rule rather than exception. To facilitate comparisons, parvalbumin isoforms

have been designated Roman numericals in the decreasing order of

electrophoretic mobihties on non-denaturing polyacrylamide gels (Huriaux

et ai, 1997). In certain instances, parvalbumin polymorphism has been used

as genetic markers to study population substructure (Piront and Gosselin-

Rey, 1974; Pandey and Hasnain, 1994).

The isoform system is significant in demarcating functional variance

of different muscle fiber types (Hamoir et at., 1972). Iinmuno-histochemical

techniques reveal the occurrence of parvalbumin in fast twitch skeletal

muscle fibers of mammals suggesting that the protein should be concerned

with rapid muscle relaxation. Parvalbumin may facilitate the rapid muscle

relaxation by shuttling Ca"^ from troponin-C to the sarcoplasmic reticulum

that is rich in it. It, therefore, represents a system for Ca"^ homeostasis that

allows rapid cyclic relaxation in physically active muscle fibres (Celio and

Heizmann, 1982). Parvalbumin deficiency slows down the speed of twitch

relaxation in knock-out mice increasing fatigue resistance in fast twitch

muscle as compared to that of the wild type (Chen e/ ai, 2001). In

amphibian and mammahan muscle, relaxing fiinction of parvalbumin has

been demonstrated in vivo as well as in vitro.

Biochemically, various isoforms are invariably highly acidic

monomeric proteins, having pi values < 5.0 and low molecular weights in

the range of 10-13 kDa, beside showing a high affinity to calcium (Ross et

al. 1997; Permyakov et al. 1989). Isoforms of suitable pi values may serve

as choice markers in isoelectric focusing experiments in native and urea gels

(Rehbeine/o/.,2000).

One of the most distinguished characteristics is their pronounced

thermostabiUty as they can withstand high temperature without denaturing

(Filimonov et ai, 1978). They are also resistant to proteolytic degradation.

However, they have no enzymatic activity and have so far failed to interact

with other proteins (Berchtold et al, 1984). The remarkable thennostability

and specificity facihtates the species identification and quality monitoring of

heat-processed fish (Rehbein et ai, 2000). Some recent immunological

studies have brought to light yet another important property of parvalbumins

that it is the major allergen offish (Bugajaska-Schretter et ai, 1998).

There is some evidence suggesting that various isoforms are

expressed in accordance with a temporal schedule or stage specificity during

organogenesis or the changing functional demands in the course of fish

growth (Huriaux et al., 1997). A study of parvalbumin synthesis during

different developmental stages in barbels has shown that they follow a

transition in isoform distribution (Focant et al., 1992). Ontogenetic changes

in the expression of parvalbumin isofonns have been recorded in case of

catfish, Heteropneustes fossilis (Sherwani et al., 2001).

Parvalbumins of most of the fishes and amphibians contain a high

proportion of phenylalanine and low amount of tyrosine but no tryptophan

(Blum et al., 1977). Tyrosine and tryptophan being the chromophores

contributing to spectral peaks in the range of 280-290, the low tyrosine and

tryptophan distinctly affect the spectral feature of parvalbumin in ultraviolet

range. Whiting is an exception, where phenylalanine contents are high

though it consists of one tryptophan and three cystein residues (Joassin and

Gerday, 1977). An unusually high amount of alanine has been found in the

skeletal muscle parvalbumin of eel (Dubois and Gerday, 1988).

Genetically, parvalbumins are classified into two different categories,

a and P-lineages (Goodman and Pechere, 1977; Goodman et al. 1979). The

common sequence Lys-Ala is characteristic of a-parvalbumins in the

position corresponding to residues 13-14 in rabbit parvalbumins, whereas (3-

parvalbumins have the sequence ala-ala in that position. In addition to this,

parvalbumins of a-type contain few residues of acidic amino acids than the

P-type and hence the isoelectric points of a-parvalbumins are higher than

those of P- parvalbumins. More precisely, the p-lineage parvalbumins may

be characterized by pi values lower than 4.8 (Tanokura el al. 1987) and

those of a-lineages, higher than 4.8. p-type appear to bind Ca' -ions more

strongly than the a-type. Although most muscles contain only the

parvalbumin of either a-lineage or of p-lineage, frog skeletal muscles are

Gerday, 1977; GiUis ei al., 1979). In general, parvalbumins are not

evolutionarily conservative as they are rather divergent proteins in terms of

the primary structure and a number of isoforms can be present in single

muscle cell. In case of fishes, attempts have been made to exploit species

specificity of parvalbumin multiphcity to establish phylogenetic

relationships among different species (Focant et al., 1988, 1990, 1994;

Huriaux et al., 1992; Huriaux et al., 1996). Moreover, a comparison of

primary structures has revealed an interesting feature that rabbit parvalbumin

shares phylogeny with "alkali" light chains (LC-3) of skeletal muscle

myosin. This chain contains three similar regions that show homology to the

Ca"-binding region of carp muscle parvalbumin.

statement of the Problem

Among vertebrates, fishes constitute about 40% of the recorded fauna

which has highly diverse adaptive features and morphology. Out of two

lineages of bony fishes one lineage evolved into teleosts while the other led

to evolution of amphibians and other higher vertebrates. Among teleosts,

adaptation to airbreathing appears to have evolved independently in highly

diverse groups. The importance of supplementing the existing knowledge on

accessory air-breathing goes beyond teleosts to the evolution of aerial

respiration among vertebrates, in general. This is to further emphasize that

the information on evolutionary placement and correlation of species of

genus Channa within and with other genera is far fi-om adequate. It was,

therefore, considered worth investigating that a suitable protein from species

belonging to this genus, which constitutes a well-known but evolutionarily

distinct group of air-breathers, be characterized biochemically and an

attempt is made to estimate their phylogenetic relationship by biochemical

or immunological methods.

The underlying reasons to choose parvalbumin for the purpose of

aimed investigations are as follows. Muscle makes up bulk of the body mass

embracing not only contractile apparatus, but also the glycolytic enzymes of

sarcoplasm with additional input from electron transport system of

mitochondria. Such huge metabolic machinery has to be equally rich in

protein molecules informative of the events which decided survival of the

animal and, in turn, tiie course and direction of the evolution. Comparative

biochemical, immunological or molecular studies on such molecules are

likely to provide clues to phylogenetic events. Parvalbumin qualifies this on

several counts : 1) It is one of the important members of the multigene

family of calcium binding proteins involved in a myriad of metabolic

reactions; 2) it occurs as a major fraction specifically in fish muscle playing

the role of calcium exchanger in the key process of muscular contraction; 3)

it exists in muscle in several isoforms which exhibit species or tissue

specificity and genetic heterogeneicity and; 4) its three-dimensional

structure is well understood and duphcations in its specific sequences are on

record. Moreover, parvalbumin of a few primitive fishes are already known

to differ structurally from their homologues of higher teleosts. There thus

existed a fare possibility that in certain respect this protein of channids may

not be the same as those of other teleosts documented so far.

The investigations were carried out on parvalbumin isoforms of four

obligate air-breather species of genus Channa (Channidae : Gronov), which

are distributed around Aligarh, The death will follow if they are prevented

from gulping air. The species are: Channa punctatus (Bloch), C. marulius

(Bloch), C. gachua (Hamilton) and C. striatus (Bloch). The selected species

also constitute a food fish group of that is economically quite important. The

reports of parvalbumins being allergens add clinical aspect to food value of

the muscle contents.

MATERIALS AND

METHODS

MATERIALS AND METHODS

The materials and protocols used during the study were:

1. Source of Chemicals

2. Key to the Genus, Channa

3. Extraction of Protein

4. Estimation of Protein

5. Purification of Parvalbumin

(a) Acetone Fractionation

(b) DEAE-Cellulose Column

6. Polyacrylamide Gel Electrophoresis

(a) Non-denaturing

(b) Denaturing

7. Staining:

(a) Coomassie Brilliant Blue-R 250 (CBB)

(b) Silver stain

(c) Photography

8. Estimationof Molecular Weights

9. Isoelectric Focusing (lEF)

10. Immunological Characterization of Parvalbumin

(a) Production of Antibodies

(b) Collection and Decomplementation of Sera

(c) Double Immuno-diffusion (DID) Test

(d) Western Blotting

(i) Transfer Buffer

10

(ii) Stain (Ponceau S)

(iii) Phosphate-buffered saUne

(iv) Blocking Solution

(v) Primary Reagent

(vi) Secondary Reagent

(vii) Wash Buffer

(viii) Protocol

11. Thermal Incubation/Thermal Treatment/Heat Treatment

12. Treatment of Parvalbumin with Chelating Agents

13. Ultraviolet Spectroscopy of Parvalbumin Isofonns

1. Source of Chemicals

Chemicals

Acrylamide

Acetone

Bis-acrylamide

Bovine Serum Albumin (BSA)

Bromophenol Blue

Coomassie Brilliant Blue G250

Calcium chloride (CaCl2)

DEAE-Cellulose

Ethylene Diamine Tetra Acetic acid (EDTA)

Ethyleneglycol-bis(-amino-ethyl ether)

N, N'-tetra acetitic acid (EGTA)

Source

Sigma Chemicals (USA)

s.d. fine-chem Ltd.

Sigma Chemicals (USA)

Sigma Chemicals (USA)

Sigma Chemicals (USA)

Sigma Chemicals (USA)

Qualigens Fine Chemicals

Sigma Chemicals (USA)

Qualigens Fine Chemicals

Sigma Chemicals (USA)

11

Freund's Complete Adjuvant

Glycerol

Glutaraldehyde

lEF Marker

Methanol

Chemicals

Phannalyte(pH3-10)

Sodium Dodecyl Sulphate (SDS)

Silver Nitrate (AgNOj)

Trizma base

Tricine

TEMED

P-mercaptoethanol

Bangalore Genei (India)

Qualigens Fine Chemicals

Loba Chemie

Pharmacia Biotech (Sweden)

Merck / Qualigens Fine

Pharmacia Biotech (Sweden)

Sigma Chemicals (USA)

s.d. fme-chem Pvt. Ltd.

Sigma Chemicals (USA)

Loba Chemie

Sigma Chemicals (USA)

CDH Chemicals

Other chemicals and acids were of AR Grade.

(2) Key to the genus, Channa:

The fishes were identified/classified on the basis of the following key

given by Shrivastava (1980):

A. Channa punctatus (81.):

Ventral 2/3" to 3/4' length of pectoral. Pectoral without transverse

bands and with uniform coloration. Presence of several bands or

patches from back pass down the abdomen.

B. Channa marulius (Ham,);

12

Presence of 15 to 16 scales between the snout and the origin of the

dorsal fin; 9 to 10 scales are present the orbit and the angle of the

preopercle. (No band like C. striatus ). A large black ocellus present at

the upper part of the base of the caudal fin.

C. Channa gachua (Ham.):

Ventral 2/3"* length of pectoral. Pectorals with alternating orange and

blue transverse bands. No band present on the remaining body.

D. Channa striatus (BL):

(a) 15 to 20 scales present between the snout and origin of dorsal fin; 9

to 10 scales present between the orbit and the angle of preopercle

3(iii)

(b) Presence of 12 to 13 scales between the snout and origin of the

dorsal fin; presence of 4 to 5 scales between the orbit and the

opercle.

4(iv)

(c) Presence of 18 to 20 scales between the snout and the origin of the

dorsal fin; 9 scale present between the orbit and the angle of pre­

opercle. Grey and black bands descend fi-om the lateral side to the

abdomen without an/any ocellus at the tail.

13

3. Extraction of Proteins:

White dorso-lateral skeletal muscle from the anteriormost part just

behind the head of the live fish that was stunned with a cerebral blow was

dissected out and homogenized in chilled 50mM Tris-HCl, pH 7.5

containing ImM P-mercaptoethanol and 10% glycerol using electrical

homogenizer (Model: 985-370;Type 2; 5000-30,000 rpm; Biospec Products,

Inc.). The homogenate thus obtained was centrifiiged at 12,000 rpm for 20

minutes at 4°C, and either analyzed immediately or stored at -20 C till

further analysis.

4. Estimation of Protein:

Protein was estimated following the standard protocol of Lowry et al.

(1951), an outline of which is given below :

Aliquots of lOfil of protein solution were pipetted out in and then were

raised to 4 ml with distilled water. This was followed by the addition of an

equal volume mixmre of 5.5 ml of 2% Na2C03 in O.IN NaOH and freshly

prepared 0.5% CUSO4 in 1% sodium-potassium tartarate to each test tube.

Finally, Folin-Ciocalteau's reagent was added that was diluted with equal

volume of distilled water just prior to use. Before reading at 650 mn, the test

tubes were incubated at RT for 30 minutes.

5. Purification of Parvalbumins:

A. Acetone Fractionation:

14

The muscle extracts were first partly purified by acetone at two cuts,

55% and 85% of acetone. The precipitate obtained at first cut was

centrifuged at 10,000 rpm for 20 minutes at 4°C, and the supernatant thus

obtained was stepped for 85% cut. The mixture was centrifuged again at

10,000 rpm for 20 minutes at 4°C to pellet the precipitate. The pellet so

obtained was allowed to dry partly at room temperature and then dissolved

in chilled phosphate (NaH2P04.2H20; 0.0 IM, pH 8.0) or Tris-HCl buffer

(50mM, pH 7.5)

B. DEAE-Cellulose Column Chromatography:

The partly purified parvalbumin isoforms of each species were separated

on a DEAE (MN-cellulosepulver 300 DEAE, average particle size approx.

10 [i: Macherey, Nagel and Co., 516 Diiren, Germany) column (2.0x30 cm)

equilibrated with phosphate buffer (0.0 IM, pH 8.0). The column was eluted

with a linear NaCl at a flow rate of 20 ml/hour. Fractions of 2 ml each were

collected and screened electrophoretically. Similar fi-actions were pooled and

concentrated by acetone (85%), and the pellet dissolved either in 50 mM

Tris-HCl, pH 7.5, containing ImM p-mercaptoethanol or in normal saline.

6. Polyacrylamide Gel Electrophoresis (PAGE):

A. Non-Denaturing PAGE:

System of Laemmli's (1970) was followed with the modification no

solution contained SDS and the gels contained 10% glycerol. Screening of

the samples was done on 10% polyacrylamide slab gels (100 x 150 x Imm)

which were pre-run for about 30 minutes at 4mA/gel, 50V and IW. After

loading, the gel were initially ran at 4mA/gel, 50V, and IW until the

tracking dye (Bromo-phenol blue = BPB) entered into the separating gel.

Afterwards the runs were made at a constant supply of 8mA/gel, 70V and

IW throughout the entire duration of electrophoresis.

Solutions:

(i) Stock of Acrylamide : Acrylamide 30%+ bis-acrylamide 0.8%.

(ii) UpperTris, 4x(pH, 6.8) : 0.125MTris base, pH adjusted to 6.8.

(iii) Lower Tris, 4x (pH, 8.6) : Tris base, 0.375M adjusted to pH 8.6.

(iv) Running Buffer, (pH, 8.3) : Tris base, 0.025mM; glycine, 0.25M

(v) Ammonium per sulphate : 10%

(vi) N,N,N',N' tetra ethyl methylene diamine(TEMED).

B. Denaturing Polvacrvlamide Gel Electrophoresis (SDS-PAGE):

Sodium dodecyl sulphate gel electrophoresis was performed in a

discontinuous buffer system containing Tricine in the upper buffer, as

described by Schagger and Jagow (1987) for the separation of proteins

having molecular weights less than 5 kD. This system worked satisfactorily

in this laboratory for the resolution of low molecular weight proteins (1-100

kD). The 10% T, 3%C gels served the purpose to separate parvalbumins

effectively. The stock solutions required for this system are given below:

Solutions :

(i) Acrylamide Stock : Acrylamide-bisacrylamide: 49.5% T, 3% C

(ii) Anode Buffer : Tris 0.2 M, pH adjusted to 8.9.

(Hi) Cathode Buffer : Tris 0.1 M,pH adjusted to 8.2.

Tricine O.I M

SDS 0.1 %

(iv) Gel Buffer : Tris 3.0 M, pH adjusted to 8.4.

SDS 0.3%

Composition of Stacking and Separating Gels:

Stacking Gel Separating Gel

Acrylamide Stock (49.5 %T, 3%C)

Gel Buffer Glycerol Distilled water to

Some denaturing gels were also run in the system of Laemmli (1970) to

screen the myogen samples. The screening was done on 15 % (100 x 150 X

1mm) slab gels. The running buffer as well as the gel contained 0.1 % SDS.

The gels were pre-run at 4mA/gel, 50V and IW for about 15 minutes.

However, after the entry of the sample into the separating gel they were run

throughout at a constant supply of 8mA/ gel, 120 V and 1 W. After the

completion of the run the gels were washed overnight in 5 % acetic acid to

remove SDS and stained on the next day.

17

4 %T. 3 % C

0.4 ml

1.24 ml -

5.0 ml

10%.3%C

2.03 ml

3.33 ml 1.33 g 10 ml

7. Staining:

A. Commassie Brilliant Blue:

Routine screening of the gels was done by using coomassie brilliant blue

(CBB) R-250 dissolved in methanol acetic acid as per standard protocols.

The gels were destained in 7 % acetic acid.

B. Silver Stain:

Silver staining was performed using fixers and developers essentially

according to the method of Oakley et al. (1980).

After completion of electrophoretic run the gel was followed by

incubations in the fixatives, each of 20 minutes and thereafter the gel was

washed thoroughly by large volumes of distilled water and kept for

overnight washing. On the next day, the gel was kept in freshly prepared

stain for about 20 minutes or till the browning of the gel margins. It was then

stepped for brief rinsing with distilled water. After this, the developer was

added and the gel was allowed to develop till the bands of desired intensity

appear. As the bands of the desired level appear the reaction could be

stopped by keeping the gel in 7 % acetic acid.

The solution A and B were mixed in the ratio of 3:1 and then diluted with

an equal volume of distilled water. In order to clear any background the gel

was kept in 100 ml of this solution. The moment the background cleared it

was rinsed with distilled water and then incubated in solution C for about 30

minutes which was again followed by thorough rinsing with distilled water.

18

C. Photography:

All photographs were taken either on Kodak Gold color films or on 125

ASA black and white negative fihns using visible range transilluminator.

8. Estimation of iVIoiecuiar Weights :

In order to determine molecular weights of the purified isoforms of

parvalbumins, method of Schagger and Jagow (1987) was followed. The

molecular weights were calculated with the help of Gel-Pro software using

mostly chicken actomyosin along with some low molecular weight markers

as shown in respective figures.

9. Isoelectric Focusing:

The method used for determination of pi values was based on the

procedure described by the supphers Pharmacia Biotech, Sweden. The pi

values of the proteins were determined by using the pi Cahbration Kit marker

proteins focused on 1 mm thick polyacrylamide gel containing 5 % T, 3 % C,

pharmalyte of the range, pH 3-10 and 10 % glycerol. The focusing was done

at 20 mA, lOOOV and 4 W for a period of 1 hour. Plot to determine the

narrow range of PA stacking was constructed manually.

10. Immunological Characterization:

A. Production of Antibodies:

19

Antibodies were raised against the purified antigens of each species. The

antigen was administered intra-muscularly in white rabbits. Each injection

comprised of 500 mg of protein emulsified in a reasonable equal volume of

Freund's complete adjuvant. After the first injection, subsequent three booster

doses were given via the same route at an interval of 10 days each. During

the immunization period, the antibody titre was checked routinely by

Ouchterlony's double immuno-diffusion test (DID) after four days of each

booster, and it was found to be maximum after second booster. The rabbits

were bled by marginal vein of the ear, and the blood was allowed to clot at

room temperature to facihtate the collection of sera.

B. Collection and Decomplementation of Antisera:

After two hours, free serum was collected by centrifiigation at 1000 rpm

at room temperature for 15 minutes. The resulted antisera was

decomplemented by incubation at 56 C for 30 minutes and then centrifuged

at 20,000 rpm for 15 minutes (Talwar, 1983 and Champion el ai, 1974). The

supernatant was saved and stored at -20° C.

C. Double Immuno-diffusion (DID) Test:

The antigen-antibody complexes were visualized in 1 % agarose gel in

the form of precipitin arcs by following the technique of Ouchterlony's

double diffusion (1962). The gel was prepared in 0.05 M phosphate buffered

saline of pH 7.1. Three wells were punched in at a distance of 5 mm in a

triangular fashion and 100 ^g of antigen was loaded against 20|xl of

antiserum.

20

D. Western Blotting:

The transfer of proteins from polyacrylamide gels to nitrocellulose

membrane was performed according to the method of Sambrook et al.

(1987) except a few modifications. The electrophoretic separation of

proteins was carried out in native conditions in order to detect the level of

iiTununo-cross-reactivity of different parvalbumin isofomis distinctively.

(i) Transfer Buffer (1x; pH 8.3):

Tris base 48 mM

Glycine 39 mM

SDS 0.039 %

Methanol 20 %

Final volume made up to 1 liter with distilled water.

(ii) Outline of the Protocol:

The protein was transferred on to the nitrocellulose membrane in the

blot assembly (Genei Blot, mini dual) for an hour at a constant supply of 50

V; where the corresponding current and wattage were 150 mA and 8 W,

respectively. The protein transfer was checked by incubating the membrane

for 5 to 10 minutes in Ponceau S stain, after which it was subjected to

following the processing steps:

a. the nitrocellulose membrane was washed with several changes of distilled water,

b. incubation in blocking solution for 4 hours with gentle rocking in the incubator shaker at room temperature.

21

C. washed with three changes of wash buffer of 10 minutes each,

d. after this, the membrane was kept in the primary reagent for 4 hours at room temperature with gentle rocking,

e. the membrane was washed again with three changes of wash buffer of 10 minutes each,

f. it was then proceeded with immunological probing by incubating in the secondary reagent, goat anti-IgG coupled with horseradish peroxidase for 4 hours with gentle rocking,

g. after incubating with peroxidase labeled antibody the membrane was further washed with three changes of wash buffer of 10 minutes each,

h. wash buffer was di^ained off from the nitrocellulose membrane by tapping it over a filter paper towel,

i. then the membrane was immersed for 1-2 minutes in the substrate made by mixing th^ Chemiluminescent Substrates, A and B in equal volumes, and to this 20 |LI1 of 30% hydrogen peroxide was also added,

j . after 2 minutes of incubation in the substrate, the membrane was tapped again over the filter paper towel to drain off the extra substrate,

k. the illumination of the immunologically reactive sites was recorded on an ultrasound film (the membrane was layed in between the two sheets of transparencies to avoid the wetting of the film which may otherwise damage the film).

11. Thermal Incubation :

The parvalbumin solutions were subjected to heat treatment at a constant

temperature of 90 C for varying time intervals. During thermal incubation,

aliquots of the protein were saved after a time period of 15, 30, 60, 90, 120,

22

150 and 180 minutes, respectively were chilled immediately in ice. The

saved aliquots of proteins were centrifuged at 8000 rpm for 10 minutes at 4"

C and then electrophoressed on 10 % polyacrylamide gel with 10% glycerol

to screen their heat stability.

12. Treatment of Parvalbumin Isoforms with the Chelating Agents:

The purified parvalbumin isoforms solutions of each fish species were

subjected to overnight treatment with EDTA and EGTA at varying

concentrations: ImM, lOmM, 20mM, 30 mM, 40 mM, 50 mM, 100 mM and

200mM. They were also treated with both of these chelators at the same time

keeping concentrations of each one of them fi"om 1 mM to 200 mM. The

protein concentration in the solutions was 1 mg/ ml. After overnight

treatment, the protein samples were centrifiiged at 10,000 rpm for 15

minutes and the supernatant was saved for electrophoretic screening. The

samples were run in 10% polyacrylamide gels in Laemmli's system

containing 10% glycerol.

13. Ultraviolet Difference Spectroscopy of Chelater Parvalbumin Isoforms:

UV- absorbance spectra were recorded in the range, 190-320 nm on

double beam UV-visible spectrophotometer (UV-5704 SS; EC-Electric

corporation of India). Solution of the protein were 1 mg/ml.

23

RESULTS

RESULTS

The fish samples for the present studies were collected during 1998-

2001. The total number of samples of each species analyzed during the

period are given in Table 1. The four species of genus Channa Gronov

(Teleostomi: Channiformes: Channidae) selected for this study are well

distributed in the areas around Aligarh. Namely, the species are: Channa

punctatus Bloch, Channa marulius Ham., Channa gachua Ham. and

Channa strialus Bloch. Multiplicity of PA isoforms was investigated in

these species by polyacrylamide gel electrophoresis (PAGE) using soluble

muscle proteins/muscle extracts as the source of parvalbumins. Only dorsal

white muscle of the anterior most portion of the trunk just behind the head

were dissected out and lateral line red muscle was carefully removed. Other

experimental details are given in Materials and Methods.

A. General outline of Native PAGE patterns of soluble muscle extracts within the species of genus Channa :

It is to point out that the source of chemicals, in particular the

acrylamide and its cross-linker significantly influence the PAGE patterns.

The results being present here were obtained after using the consumables

purchased from the sources hsted under Materials and Methods.

Several differences in the relative intensities and the absence of some

bands of corresponding mobihties could be noted visually. However, the

PAGE patterns shown in Figure 1, where 10 representative lanes of each

24

species are shown, were analyzed with the help of Gel-Pro software (Table 2

and 3). According to this analysis a maximum number of 14, 10, 13 and 9

bands were recorded in C. punctatus, C. marulius, C. gachua and C. striatus,

respectively (Fig. 1: photographs a, b, c and d). The data presented in tables

also reveal the absence of some minor bands in one or the other lane of each

species.

Figure 1 and its corresponding tables show the presence of one major fast

migrating band in the patterns of each species. The biochemical

characterization to be presented and discussed in this chapter later on would

reveal that this major band occurring in PAGE patterns of muscle extracts of

each species is in fact the major parvalbumin isoform. Following the

conventional system of parvalbumin nomenclature, the isoforms have been

assigned Roman numericals as suffix (I, II or III, etc.) in the decreasing

order of their electrophoretic mobilities. According to this, PA II is the

major isoform in C. punctatus and C gachua, whereas, PA I is the major

isoform in C. marulius and C. striatus.

B. General outline of SDS-PAGE patterns of soluble muscle extracts within the species of genus Channa :

Polypeptide composition of soluble muscle proteins of all four species of

genus Channa as determined by SDS-PAGE protocol of Laemmli (1970) is

shown in Fig. 2. Though a few bands are prominent and occur consistently,

several others show quantitative variations of differing magnitudes. On the

basis of software analysis, the total number of polypeptides of different

25

molecular weights that could be counted in Fig. 2 is as follows. A total of 14

and 10 bands could be counted in PAGE patterns of C. punctatus and C

maruHus (Table 4: a and b; Table 5: a and b). As per evaluating by the

software system, the number of bands in SDS-PAGE patterns of C. gachua

and C striatus is 13 and 9 respectively (Table 6: a and b; Table 7 a and b).

Some major and a few minor bands appear to have a diagnostic value at

species level, which have been reproduced in Fig. 3 after deleting variable

ones.

C. Biochemical characterization of parvalbumin (PA) isoforms :

(i). Purification:

For biochemical characterization, the parvalbumin isoforms were

purified by a combination of three procedures:

1. Thermal incubation at 70° C for 30 minutes that apparently removes all other proteins,

2. Acetone fractionation between 55-85% saturation, and

3. Column chromatography on DEAE Cellulose or Cibachrome G-250.

The purified PA isoforms of four species have been compared in Fig. 3

along with the electrophoretically or column chromatographically purified

individual isoforms loaded in the subsequent lanes of each set. Whole

muscle extracts of each species is loaded in the preceding (1 *) lanes of the

set.

26

The PAGE patterns of thermal incubated and acetone fractionated lanes

of neither species reveal the presence of any impurity upon staining with

CBB. Whether, the yield of individual isoforms is compatible with its

amount in native muscle extracts is compared in Table 8 A. There is no

remarkable difference in the relative amount of either isofonn between

native and purified samples of any of the species.

(ii). Western Blotting of PA isoforms of Channa spp. :

Anti-P All-rabbit antisera raised in our own facility using highly

purified major PA II of C punctafus as an antigen were employed to screen

the western blots of probable PA candidates of each species as well as that

of the chick. The extent of immuno-cross-reactivity was documented

employing chemiluminiscence produced during the reaction with horse

radish peroxidase (HRP) mediated lumiGIow (KPL laboratories, USA). The

immunoblot thus prepared and shown in Fig. 4 confirms the parvalbumin

nature of the major as well as minor bands marked as PAs in Fig. 1 and 3

beyond doubt.

(ill) Molecular weights of purified PA isoforms of four species of genus Channa:

In the system of Laemmli (1970) band of the lowest molecular weight

stacks as 10 kD polypeptide and all PA isoforms stack at this position.

Therefore, Schagger and Jagow's system (1987) was opted to rule out the

possibility of inter or intra-species molecular variation in the molecular

27

weights of PA isoforms. In fact, intraspecies differences rather than the

inter-species differences exist in the molecular weights of PA isoforms. For

instance, PA isoforms of C marulius stack in 3 bands in the range 10-12 kD,

with the major one stacking as that of 10 kD (Fig. 5a: lanes 6 and 7). The PA

isofomis of C. punctatus, on the contrary, stack as a single broad band in the

range of 10-11.5 kD. It may be possible that the overloading has caused the

mix up of two bands of slightly different molecular weights. The case of PA

isofomis of C gachua and C. sthalus show the stacking as two bands of 10

and 11.5 kD (Fig. 5b: lanes 3 and 6k, respectively). The purified major band

of either species stacks as a band of 10 kD (Fig. 5b: lanes 4 and 7

respectively).

(iv) The effect of thermal incubation at 90° C on PA isoforms of genus Channa:

Thermostability is one of the most remarkable biochemical properties

of parvalbumin. In the experiments on themial treatment explained here, the

incubation was performed at a very high temperature of 90°C for duration of

3 hours (Fig. 7, 8, and 9). The heat treatment experiments were carried out in

the absence as well as the presence of thiol protector P-mercaptoethanol.

Clear species differences are observed in the behavior of PA isoforms of the

four species. The recorded changes were monitored by the native patterns

and further analyzed by Gel Pro software. Quantitative changes in the main

band have been presented in Fig. 12 and the changes in the appearance of

novel bands in Fig. 11. The PAGE pattern changes obtained by heat

28

treatment on the control PA isoform of the Chick are shown in Fig. 9. Both

of these aspects have been described in more detail below.

The two most remarkable changes characterizing the PAGE patterns of

the heat treated PAs are;

1. The appearance of several novel bands, which according to SDS-PAGE

analysis are aggregates of one or the other isofonns (Fig. 10) and,

2. Differences in the relative stabilities those are most conspicuous in case

of C. maruUus where the electrophoretic integrity is virtually lost by

120 minute of incubation (Fig. 7c).

The minimum changes are observed in the proteins stacking either as the

major PA isoforms of C. punctatus or costacking with it (Fig. 7a) and the

maximum in case of C. marulius (Fig. 7c). The extent of changes in heat

treated PA isoforms of C. siriatus (Fig. 8c) appear to be of intemiediate

rank.

(a) On PA isoforms oi Channa punctatus :

As shown by PAGE patterns (Fig. 7a), one of the most prominent effect

on PA isoforms of C. punctatus at 90°C is the appearance of several novel

bands of mild intensities (shown by arrows). One of them, appear at 30"

minute of incubation and persists during the entire incubation period of 3

hours as the fastest migrating band. With the appearance of this band

coincides a decrease in the intensity of PA III and as it gradually disappears,

a novel band of shghtly lesser electrophoretic mobility than the major Pa II

29

starts to appear at 30 "" minute of incubation. As PA III totally disappears,

this minor band virtually merges with PA II imparting the look of a broad

protein band with rather irregular boundary. Due to the conversion of

thermosensitive isoforms or their resultant aggregates the relative intensities

of other novel bands of higher electrophoretic mobilities continue to alter.

Addition of 5mM P-mercaptoethanol affects both the number and the

relative intensities of the PAGE patterns of heat treated PAs of C. puncfafus

(Fig. 7b). PA III remains as a sharp band up to 1 hour of incubation at 90°C,

indicating that it could resist the heat induced changes for additional 30

minutes as compared to Fig. 7a. Even by the 3" hour of incubation traces are

still visible. Due to the presence of P-mercaptoethanol two prominent

changes occur: 1) a delayed appearance of novel bands and, 2) the

sharpness of bands is retained as compared to the smearing effect noted in

figure 7a, in the absence of P -mercaptoethanol.

(b) On PA isoforms of Channa marulius :

The changes as a resuh of incubation at 90°C are more prominent in PA

isoforms of C. marulius, both in terms of the velocity of heat treatment

induced changes and their extent (Fig. 7c). Right from the 15* minute the

relative intensity of major PA I decreases substantially with a corresponding

increase in the intensity of a novel band that stacks in between PA I and II.

The intensity of PA II also decreases visibly but a band comigrating with it

IS present up to 30 minute of incubation. A major increase in the intensity

of the intermediate continues up to 90* minute of thermal incubation with an

30

additional band appearing at 30" minute persisting for another 30 minutes.

Thereafter quantitative decrease in the novel bands follows that lasts up to

150* minute with the effect of heat denaturation displayed in the loss of

sharpness and decrease in the relative mobilities of the novel bands.

Similar to the behavior of C punctatus (Fig. 7b), the protective effect of

5mM P-mercaptoethanol on rather thermosensitive PA isofomis of C

marulius (Fig. 7d) is also apparent. The integrity of the bands, as defined by

the sharpness, relative electrophoretic mobility and the thickness of the

major PA I lasts by 180* minute. Though some decrease in PA II also

occurs, traces of the band are present even at 120 minute, whereas in its

absence it disappears 60 minutes earher. Further, heat treatment induced

appearance of novel bands shifts to more negatively charged species rather

than the heat treatment induced retardation in electrophoretic resolution that

is indicative of denaturation. In the presence of p-mercaptoethanol the

charges in the PAGE patterns resemble those of C. punctatus PA isoforms.

(c) On PA isoforms of Channa gachua :

The effect of thermal incubation on C. gachua resembles the general

pattern noted for C. punctatus PAs in the sense that novel bands of higher

electrophoretic mobility appear and intensity of some of them increases

coincident with an apparent progressive decrease in the major PA II during

the extended course of incubation (Fig. 8a).

31

In the presence of 5mM P-mercaptoethanol, only some changes in the

relative intensity of the fastest migrating novel bands are visible (Fig. 8b).

(d) On PA isoforms of Channa striatus :

During incubation, PA isoforms of C. striatus behave like those of C.

marulius in certain respects. For instance, the major changes during the first

90 minutes occur in the form of a novel band of intermediate mobility

appearing between PA I and III (Fig 8c). Traces of novel bands migrating

faster than the PA I apparently correspond to the changes in this band and

PA III which is probably converted at 15' minute of incubation into a

slightly less negatively charged species (Fig. 8c: lane-2). By 180' minute the

effect of thermal incubation as a loss in the sharpness of resolutions becomes

evident and only one band comigrating with PA 1 is clearly identifiable.

P-mercaptoethanol has a conspicuous protective on the relative mobility

of PA II of C. striatus, since in comparison with control of Fig. 7c, it has

lesser electrophoretic mobihty and stacks as a band of intermediate mobility

(Fig. Id). The sharpening effect on the major bands and a delayed change in

the minor ones, that was observed in heat treated PA isoforms of other three

species, was obvious in this case also.

(e) On the control, Chick PA isoforms :

Only one PA takes up CBB stain (Fig. 9), though with silver stain two

isofonns are detectable (Fig. 22 b). For heat treatment experiments, CBB

staining was employed that revealed the absence of novel bands that appear

32

due to heat treatment in fish Pas and only some loss in the relative intensity

was noted between 2-3 hours. The effect of P-mercaptoethanol was similar

to that observed so far.

(V) The graphical assessment of heat induced changes :

The observations made on the changes in PAGE patterns of heat treated

PA isoforms from different species of genus Channa have been assessed and

presented as graphs in Fig. 11 and 12. They demonstrate two aspects of the

observed changes : (i) an increase in the number of novel bands appearing as

a resuh of thermal incubation and, (ii) the quantitative change in the

intensity of the major band is maximum.

The increase in the number of novel bands is plotted in Fig, 11 that

demonstrates that in C. gachua very first increase observed at 15' minute

remains unchanged throughout the duration of incubation (also shown

photographically in Fig. 8 a). C. punctatus demonstrates a similar trend after

30 minute of thermal incubation as there is no further increase in the

number of novel bands (Fig. 11 and 7b). Heat treated PA isoforms of C.

striatus, however, show a sudden increase in the number of novel bands at

90' minute with no subsequent increase in their number. On the contrary,

those of C. marulius show a two step increase in the number of novel bands,

the first increase occurring at 30" minute and the second at 120" minute that

goes on increasing up to the last minute of incubation. According to this

assessment (Figs. 11 & 12), the maximum number of novel bands that

appear as a result of thermal incubation are of the following order and

33

magnitude : C. gachua (4) < C. punclatus (5) < C. striatus (6) < C. mamlius

(8).

When the graph based on the increase in the number of novel bands in

the presence of p-mercaptoethanol is constructed, rather smooth curves are

obtained w^hich clearly demonstrates the decelerating effect of this

compound on the increase in the number of the novel bands and its rate (Fig.

7, 8 and 9). According to it, the observed changes are of the order: C.

gachua < C. maruliuslC. striatus < C. punctatus. The two trends of

interspecies order apparently reflect inter and intraspecies differences in the

relative stabilities of PA isoforms and interconvertibility of the novel bands

as elaborated under "Discussion".

The plots of the changes in the intensity of major isoforms or the novel

major bands in are shown in Fig. 12. They show that without P-

mercaptoethanol in C. gachua instead of a decrease, an increase is observed

at the location of major PA II and no remarkable decrease is apparent even

after 3 hours of incubation. Similarly, in C. punctatus a shght decrease up to

120 minute is followed by a rather rapid decrease in the intensity. In C.

striatus, after a slight increase at 15* minute that persists up to 120' minute th

a sudden increase occurs at 150 minute followed by a return to the previous

level at 180 minute. Again a rapid decline is observed in case of C

marulius that only shows shght improvement between 90" and 120* minute

of incubation.

34

The presence of P-mercaptoethanol brings about a quantitative as well

as qualitative change in the patterns observed in the top figure of each

species. For example, almost all species show a sudden or progressive

decline in the intensity of the main PA bands (Fig. 7b and d; 8b and d; and

9b). These changes are most prominent in C. marulius followed by C.

gachua and C puncfafus in a decreasing order. In case of C. sirialus the

reduction of intensity is of the lowest magnitude.

(iv) The effect of chelaters on parvalbumin isoforms :

The result presented below compare the effect of general metal ion

chelator EDTA and Ca"* specific chelator EGTA on parvalbumin isoforms

of dorsal skeletal muscle of anterior most part of the trunk of all four

species of genus Channa with the chick PA included as a control.

(i) On PA isoforms of Channa punctatus :

It has already been shown in Fig. 4 that by PAGE, PA of muscle of C.

punctatus resolve into three isoforms wherein isoforai II is the major PA

(top of Fig. 14a, lane-0). Upon treatment with EDTA and EGTA

conspicuous changes occur in the electrophoretic behavior of multiple

isofomis showing the appearance of novel bands and the concentration

dependent changes in their relative intensities. These changes are more

remarkable in case of EGTA treated PAs.

35

In the PAGE patterns, no change in the electrophoretic mobihty of any

of PA isoforms is observed in the presence of ImM of EDTA (top of Fig.

14; lane-1). However, significant differences become apparent from EDTA

concentration of lOmM and beyond (Fig. 14; lane-2). At least two novel

bands (marked by arrows) appear with 10, 20 and 30mM of EDTA; out of

them one minor novel band is the fastest migrating band. Further, a gradual

increase follows in the intensity of the band comigrating with PA I, a change

that is concordant with the diminution of PA III.

The above figure also shows that with the differing concentrations of

EDTA rather minor changes occur in the UV-difference spectra. A major

negative shift can be noted in the spectrum of C. punclatus PA containing

lOmM EDTA wherein beside some minor peaks a major trough (O.D.= -0.9

units) appears in the range of 284-300 nm.

As shown in Fig. 18 , the changes in the PAGE patterns (inset of this

figure) are comparatively more pronounced in EGTA treated PA isoforms of

C. punctafus where a sudden and prominent shift in the relative mobility of

PA II occurs at 1 OmM EGTA that persists at its further high concentrations

(lanes 2-9). Below PA II, some minor but diffused bands also appear which

migrate faster than PA II. The observation remains visual because these

bands could not be reproduced in photographs. However, a prominent but

progressive decrease in the intensity of PA III takes place that is obvious

36

from lane 2-9 with the concomitant appearance and an increase in a single

minor but sharp band just above PA II.

(ii) On PA isoforms of Channa marulius:

In C marulius, PA III occurs as the major band (Fig. 15a, top, control

and as previously established and shown in Fig. 3). Unlike PAGE patterns of

C. punctatus, where the shift in the electrophoretic mobilities of protein

bands occurs form lOmM EDTA, PA isoforms of C. marulius exhibit a

prominent increase in the relative electrophoretic mobilities right from the

lowest concentration of ImM EDTA used in this study.

Of several novel bands of varying intensities that appear as a result of

EDTA treatment, the photographically reproduced major bands are three.

One of them shown by arrow migrates faster than even the PA I, whereas PA

II and III appear to gradually change into this band and/or the band that

comigrates with PA III. So far as the spectral changes are concerned, they

are siinilar to the case of C punctatus PAs with the most prominent shift

occurring at lOmM EDTA.

PAGE patterns of EGTA treated samples of C. marulius parvalbumins

resemble those of C. punctatus in demonstrating a shift in the relative

electrophoretic mobihties at lOmM EGTA (Fig. 18, inset of bottom

diagram). PAGE patterns of EGTA treated PA samples of this species reveal

37

that the band comigrating with PA II is of the highest intensity. It is also

evident that the band that comigrates with PA II is also most intense at the

very concentration of this chelator (lOmM EDTA, lane-2). Subsequently, it

shows a gradual decrease in other lanes (lanes 2-9). Concomitant with this

change is a gradual increase in the intensity of the fastest migrating band in

EGTA treated samples.

This is to remind that the prominent shift in the relative

electrophoretic mobilities of PA isoforms of C marulius is initiated at 1 mM

EDTA instead of 10 mM, (the concentration of a similar change in PAs of C

punctalus), but similar to other species a spectral shift is recorded at 10 mM

EDTA instead of 1 mM.

(iii) On PA isoforms of Channa gachua :

Similar to previous two examples a major shift in the elctrophoretic

mobility of PA isoforms of C. gachua are noticed with both the chelators,

though it appears to be of a high order and magnitude in EDTA treated

samples. In this case, the increase in the fastest migrating band is gradual

with a concomitant and corresponding decrease in the intensity of II (Fig.

16a ). A band of intermediate mobility (shown by arrow) also appears which

shows a similar gradual increase in the intensity.

38

The above mentioned changes in this fish species appear to be more

rapid upon treatment with EGTA (Fig. 19, inset of diagram). Similar to the

behaviour the PA of other two species discussed so far the most prominent

shift in UV spectrum occurs at a concentration of 10 mM.

(iv) On PA isoforms of Channa striatus :

C. striatus control consists of three PA isoforms, which differ in a

decreasing order from PA I-III quantitatively, the last being the major PA.

Only minor variations are evident in the PAGE patterns of EDTA treated PA

isoforms of this species. As for the shift in the electrophoretic mobilities,

only one major band shows a sUght increase within the entire range of

EDTA concentration used here (Fig. 17). With the disappearance of minor

isoforms PA-II and I, a novel minor band (shown by arrow) also appears that

becomes gradually more intense corresponding to the increasing

concentration of EDTA. The changes were, however, more prominent with

EGTA (Fig. 19, inset of bottom diagram), both an increase in the

electrophoretic mobility of the fastest migrating major band as well as the

relative intensities of the band that apparently corresponds to the

disappearance of PA II and III along with the minor band that appears upon

treatment with EDTA. As for the spectral changes, there is general similarity

with AP isoforms of other species including the negative shift at 10 mM

EDTA.

39

(v) On PA isoforms of the Control (Chick):

Two PA isoforms could be purified from chicken thigh muscle (Fig.

22 b, lane 0 of insets). The minor PA I is visualized by silver staining only.

Similar results were obtained by treating the chicken PA isofomis with

EDTA and EGTA. PAGE patterns show a minor decrease in the

electrophoretic mobilities of both isoforms that appear to be initiated at

ImM EDTA or EGTA and no further change occurs after lOmM in either

case. There is a gradual decrease in the relative intensity of both isoforms as

the concentration of the chelators increases. Similar to fish PAs, a prominent

negative shift of the spectrum between 284-305 nm at 10 mM EDTA was

again visible in this case. Where chick isoform differ from fish PAs is the

appearance of the peak measuring 2.5 O.D. units at 285 nm.

To fiirther clarify the probable changes in the range of principles

chromophores (tyrosine, tryptophan and phenylalanine) in UV-range, spectra

of 200mM EDTA treated PA isoforms of all four fish species and the chick

were magnified between 251-31 Inm (Fig. 14 to 17 and Fig. 20). As the

respective figures show treatment with 200nm EDTA reveals some species

differences in this range as well. Chick PA spectra are self explanatory

without any magnification.

A low intensity shoulder may be present at 285nm in spectra of C.

punctatus, C. marulius and C striatus treated PAs, whereas, for that of C.

40

gachua the patterns or more or less a steep slope within the selected range,

200 mM EDTA does not induce any remarkable change around 285 mn or

beyond in any of the species (Fig. 20). On the contrary calcium-binding

appears to bring about very prominent changes in C punctatus being 0.07

O.D. units at 285 mn. In C. marulius a change 0.09 O.D. units is recorded at

this wavelength. The similar changes are apparent in the spectra of PAs of

the other two species also.

The cumulative effect of EDTA and EGTA on PAGE patterns of PA isoforms:

The apparent changes in native PAGE patterns of fish PAs treated

with 1 to 200 mM EDTA as well as EGTA (Fig. 21 and 22) exhibit the

prominence of the effect of EGTA. The control Chick EDTA+EGTA treated

PA is included with isoforms of C striatus in Fig. 22.

41

iininii M

^ r-»• WTTT'fTST7W

- " • ^ ^ r t ^ ' T ^ r t r * - - ? ^ - -nnrj^ l f n"i^

-PA-n^^

Fig. 1. Polyaciylatnide gel electrophoretic patterns of proteins of muscle sarcoplasm soluble in low ionic strength buffer (50 mM Itts-HCl, pH 7.5). The extract hereafter will be refa"red to as "soluble muscle proteins or muscle extract".

(a) Channa pimctatus

(b) Channa maruhus

(c) Channa gachua

(d) Channa stnatus

Labels PA-I to PA-III indicate protein bands biochemically characterized as parvalbumin isoforms.

Gels were 10% in acrylamide and run in modified protocol of Laemmli (1970) wherein no SDS was added to any solution or buffer. Other details as under "Materials & Methods".

53.5 46.5 43.0

. /

a

17 -11.5 "10 11.5

18.5

1 10 17,

Figure 2: SDS-PAGE patterns of whole myogen extracts run in 15 % gels according to the protocol of Laemmli (1970). (a) Channa punctatus (b) Channa marulius (c) Channa gachua (d) Channa striatus

Molecular weights of a few major bands are shown in diagram on the right (b and d). The calculation is based on Gel-Pro software analysis using the appropriate markers as shown in Tables 4 to 7. Based on subsequent biochemical characterization PA bands have been identified (marked).

PA I _

PA I I -

V-*/ '«

M C

- ^ « ~ >

i i ,#

PA I - I -PAIl PA III -J-

Figure 3: Native P A G E patterns showing the purified parvalbumin isoforms of Channa punctatus (a), Channa marulius (b), Channa gachua (c) and Channa striatus (d). Control soluble muscle extracts has been loaded in the preceding lane of each purified PA. Mixed purified PA isoforms of: C. punctatus, C. marulius, C. gachua and C. striatus in Lanes, 1 to 4 ( from left to right) (e).

Other details same as in Fig. 1 or under Materials and Methods.

1 5 6 7 8 10

FigurG 4: Immuno-blot (Western blot) showing the cross-reactivity of parvalbumin isoforms of chick (lanes, 1 and 2), Channa punctatus (lanes, 3 and 4), Channa marulius (lanes,5 and 6 ), Channa gachua (lanes, 7 and 8), and Channa striatus (lanes, 9 and 10). Anti-PA II rabbit antisera raised against highly purified PA II of Channa punctatus. Most dark bands corresf>ond to major PAs of each species which in C. punctatus, C. marulius, C. gachua and C striatus is PA II, PA I, PA II and PA I respectively . Depending on the location of the major band, the minor bands below and above (indicated by arrows) are PA I and PA III.

150 -

68 -

25 a 17

5

12.4 10 . 8 6

a

8

1 2 3 4 5 6 7 8 Figure 5: SDS-PAGE patterns in modified system of Schagger and Jagow (1987)

which facilitates the resolution of bands of less than 10 kDa. Molecular weights in kDa of Marker has been shown on the left of each photograph. a. Lanes are: 1: Marker, 2: Whole muscle extract of C punctatus, 3: All isoforms of C. punctatus, 4: Native PAGE purified (eluted) major isoform PA II ; 5: Whole muscle extract of C. marulius, 6: All isoforms of C. marulius, 7: Native PAGE purified (eluted) major isoform PA I and 8: Chick PA.

b. Lanes are: 1: Marker, 2: Whole muscle extract of C. gachua, 3: All isoforms of C. gachua, 4: Native PAGE purified (eluted) major isoform PA II; 5: Whole muscle extract of C. striatus, 6: All isoforms of C. striatus, 7: Native PAGE purified (eluted) major isoform PA I and 8: Whole muscle extract of C. punctatus.

9.3 -8.65 8.2-' 7.15 p 6.55 1

5 .85-

5.2

4.55 3.75

^ I

y

t- a

1 • - i » —

— - - >

M 1

1 a.

Standard graph of lEF pi values

10 T

8

6 -

4

2

0

^ ^ i j t 6 55 ^>»l585

^ S j 3.75

2 S5 2 62 3 73 5 19 7 37 7 92 8 46 e 56 8 64 8 65

Relative Mobilities b

Figure 6 : (a) isoelectric focusing on 5% polyacrylamide gel containing phamialyte of 3 to 10 pH range.

Lanes are: (1) pi marker (2) purified PA isoform (3) whole myogen extract of C. punctatus (4) whole myogen extract of C. marulius (5) whole myogen extract of C. gachua (6) whole myogen extract of C. striatus

Standard graph showing pi values of markers and parvalbumin isoform. (pl=4.55, indicated in red).

PA III

PA II —

PA I % i 4 |

V ^ V 'K

15 30 60 90 Time (minutes)

120 150 180

PA I

0 15 30 60 90 120 150 180 Time (minutes)

0 30 60 120 180 Time (minutes)

0 30 60 120 180 Time (minutes)

Figure 7 : P A G E patterns showing the effect of thermal incubation on parvalbumin isoforms of Channa punctatus at 90°C (a) in the absence of P-mercaptoethanol (b) in the presence of P-mercaptoethanol

The effect of thermal incubation on parvalbumin isoforms of Channa manilius at 90''C (c) in the absence of P-mercaptoethanol (d) in the presence of p -mercaptoethanol

PA II

PA I

15 30 60 90 120 150 180 Time (minutes)

0 30 60 120 Time (minutes)

180

PA III PA II PA I

PA l u ­ll -I - II.^**

0 15 30 60 90 120 150 180 0 30 60 120 180

Figure 8 : The effect of thermal incubation on parvalbumin isoforms of Channa gachua at 90°C (a) in the absence of P-mercaptoethanol (b) in the presence of p-mercaptoethanoi

The effect of thermal incubation on parvalbumin isoforms of Channa striatus at 90°C (c) in the absence of P-mercaptoethanol (d) in the presence of P-mercaptoethanol

PA I

0 15 30 60 90 120 150 180 Time (minutes)

PA I

0 30 60 120 180 Time (minutes)

Figure 9 : The effect of thermal incubation on parvalbumin isoforms of Chick at 90°C (a) in the absence of P-mercaptoethanol (b) in the presence of P-mercaptoethanol

150

68

25

2.4

8

" " *

4flHMli' ^"

/

a

/

M 0 1

150 —

68

25

12.4

8

-•J #

— - ^ •

b

M 0 1

Figure 10 : SDS-PAGE patterns ( in 15 % gels) showing the molecular weight of PA isoforms of C. marulius during heat treatment at 90°C in the absence (a) and in the presence of P-mercaptoethanol (b).

In either case a single band of 10 kDa appears which is degraded to almost invisibility after incubation of 3 hours, whereas it remains almost unaffected in the presence of p -mercaptoethanol.

Otamapuaaus

ChmamanJius

Otmigachia

Otwwaanaus

0 15 X 60 90 120 150 180

Twne (minutes) a

8 i

7-

- 6

| 5

1' £3 3

1

- • - ChtMwta mcnilaa

Chanagaehtio

-»-Chanta3rOoai$

^^ I / ^ ^

V 0 30 60 120 180

Time (minutes)

b

Figure 11 : The changes in the number of bands appearing in native PAGE patterns as a result of thermal incubation of parvalbumin isoforms of four species of genus, Channa (a) in the absence of P-mercaptoethanol (b) in the presence of p-mercaptoethanol

Further details as under "Results" and "Discussion'

Quantitative changes in the major band

V •C.punctatus

- C. marulius

- C.gachua

- C. striatus

90n

80

1 60 a •S 50

1 40 o

1 30

S 20 a S. 10

n

b

* ^ ^ ^ ^ ^

/V-

0 30 60 120

Time (minutes)

—•— C. punctatus

—•— C. marulius

—0- C. gachua

y. C. striatus

180

Figure 12 I The quantitative changes that appear in native PAGE patterns of thermal incubation of parvalbumin isoforms of four species of genus Channa : (a) in the absence of P-mercaptoethanol (b) in the presence of P-mercaptoethanol

Further details under "Results and Discussion".

Parvalbumin (controls)

Fig. 13. UV difference spectra of untreated (controls) of parvalbumin isoforms of four species of Genus Channa as identified by colour of the format legend.

PA III II I

0.4

0.2

(0 c o E-0.2 O

I -0.4

-0.6

-0.8

T - CD •<r • * CJ (N

-

-

. _ U1 CN

Channa punctatus

CO T- CD •<- CD \ - < ^ CD un CD CD h- h - 00 CO CN CN CN CM CN ^ CN

Wavelength \

— Control \

EDTMOmM I

-EDTA200mM \

1 1

en CN ^ CN

' ' / '

o o5 -ct CO r5 c>

Fig. 14. a : Changes in PAGE patterns oi Channa punctatus parvalbumin (PA) isoforms treated with 1-200 mM EDTA (lanes : 1-9). Lane marked '0 ' is control.

Below, UV difference spectra under similar conditions showing major perturbation between 275-305 nm with 10 mM EDTA (red line).

Other details under Materials & Methods.

"A HI.

I I -1 -

n t > 1

a

2 3 4 5 6 7 8

t mtLkkh^

9

im

Fig. 15.a Changes in PAGE patterns as Charma marulius parvalbumin (PA) isoforms treated with 1-200 mM EDTA (lanes : 1-9). T.ane marked '0* is control

Below, UV difference spectra under similar conditicMis showing major perturtmtion between 275-305 nm with 10 mM EDTA (red line).

Other details under Materials & Methods.

PA II • 1

a

0 1 2

Z

3 4 5 6 7 8 9

Z - - .

0.4

0.2

s o °-0.2

CJ

s o

-0.4

-0.6

-0.8

Channa gachua

to r- <D T- (D r- <D ^ m 35 to <o h- t eg CM CM CM CSI CN CM

Wavelength

- Control

EDTAIOmM

• EDTA200mM

Fig.l6.a Changes in PAGE patterns as Channa gachua parvalbumin (PA) isoforms treated with 1-200 mM EDTA (lanes : 1-9). Lane marked '0' is control

Below, UV difference spectra under similar conditions showing major perturbation between 275-305 nm with 10 mM EDTA (red line).

Other details under Nfaterials & Methods.

3 4 5 6 8

PA

lu­ll I

>» C M C N J f M C M C M M C M C g R C N I C M C M M

g -0.2 t

I -o

-0.6

-0.8

-1

Wavelength

-Control

EDTAIOmM

-EDTA200mM

Fig.l7.a Changes in PAGE patterns as Channa striatus parvalbumin (PA) isoforms treated with 1-200 mM EDTA (lanes : 1-9). Lane marked *0' is control

Below, UV difference spectra under similar conditions showing major perturbation between 275-305 nm with 10 mM EDTA (red line).

CHher d^ails under Materials & Methods.

0.3

Channa punctatus

0 1 2 3 4 5 6 7 8 9

0.4 Channa mamlius

0 1 2 3 4 5 6 7 8 9

0.05

Control EGTA 10 mM EGTA 200 mM

T f T f i n i o ( D < D r ^ h ~ o o o o o ) 0 ) o O ' ^ C M C M C M C N C M C M C N C N C S I C N C N C M C O C O C O

Wave length (nm)

Fig. 18. The effect of 1 mM-200 mM EGTA on PAGE patterns of PA isoforms (insets) and on difference spectra of the species as indicated.

I I I I I I I I I I I I I I I I I I R I I I [ I I

T - C D - « - C D T - C D T - C D T - C D ' » - C D T - C D T -

T i - T t i o t o ( D C D h - h - o o o o a ) a > o o ^ CMCMCNCNJCMCNCMCMCNCNJCMCNCOCOCO

Wavelength (nm)

Other details as described under materials and methods.

0.35

0.3

0.25 4

I 0.2

O 0.15 +

0.1 +

0.05

0 1 2 3 4 5 6 7 8 9 0.3

- Control

-EGTAIOmM

-EGTA200mM

T - C D - ^ C O ' ^ C O T - C D t - C O T - c D ' ^ C D T -

• ^ • < ; r i / ) i n c D c o r ^ r - - o o c o o 5 c n o o ^ C N r N j C N J C M C M C S J C M C g C N C N C M C V J C O C O C O

Wavelength (nm)

Fig. 19. The effect of 1 mM-200 mM EGTA on PAGE patterns of PA isoforms (insets) and on difference spectra of the species as indicated.

0 1 2 3 4 5 6 7 8 9

PA mi n I I

Channa stiiatus

- Cotrol

-EGTA 10 mM

-EGTA 200 mM

r I I I T I I I I I I I 1 I I I I I r I I I I [ -n—T

• t ' ^ J - l O i n C D f f i ^ - h - O O O O O J O S o O - i -CMCNICMCNrv lCNCNCNCNICNCNCMCOCOCO

Wavelength (nm)

Other details described under materials and methods.

Chick

2.5 ••

2

1,5 -•

1

0.5

0

-0.5 •

- 1 -

« 1*1

ffll

-Control

-EDTMOmM

EDTA200mM

B g a R s 8 S? Is K R \ 8 g- 8

W a ^ length (nm)

C h i c k

i 1

>

W j ; ; s „

• b)

. C o i trol

. CGTA 10 m M

. EGTA 2 0 0 in U

—I—r—I—1—I—

W a v e l e n g t h ( n m )

Fig.20: The effects of lmM-200 mM EDTA on difference spectra of chick parvalbumin isoforms (a). In the same range the effects of EGTA is shown in (b), corresponding PAGE patterns (insets).

0 1 2 3 4 5 6 7 8 9

PA lu­l l -1-

b

- ^ P W ^V 4^9 * * **^ 4i^ *

0 1 2 3 4 5 6 7 8 9

PA II-I-

0 1 2 3 4 5 6 7 8 9

Fig. 21. The combined effect of both EDTA+EGTA on native PAGE patterns of PA isoforms of:

(a) Channa punctatus

(b) C. marulius

(c) C. gachua

Other details of molar concentration of the chelators (1 to 200 mM) as in Fig. 14-19.

PA III -

a - rfcsrr

0 1 2 3 4 5 6 7 8 9

PA II I

^ ^

0 1 2 3 4 5 6 7 8 9

Fig. 22. The combined effect of both EDTA+EGTA on native PAGE patterns of PA isoforms of:

(a) Channa striatus

(b) The chick leg muscle

Other details of molar concentration of the chelators (1 to 200 mM)asin Fig. 14-19.

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

l _ _ l \ \ \ \ \ \ \ \ I

0.33

0.66

C. punctatus

C. marulius

C. gachua

C. striatus

Figure 23 I similarity dendrogram showing interspecies relationship between parvalbumins of four species within genus Channa based on OUT clustering.

Table 1 : The total number of samples of four species of genus Channa analyzed either for PAGE patterns or for isolating parvalbumin.

SPECIES

C punctatus

C. marulius

C. gachua

C. striatus

SITES OF COLLECTION

ALIGARH (No. of Samples)

510

251

370

240

MORADABAD (No. of Samples)

-

-

95

-

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DISCUSSION

DISCUSSION

Fish muscle consists of several types of fibers which determine its

biochemical and functional characteristics primarily depending on the

type of contractile apparatus they possess. Both of the characteristics are

determined mainly by the type of myofibrillar proteins and to some extent

with the participation of a few sarcoplasmic proteins. It is calcium that

triggers muscle contraction by release into sarcomeres through T-

channels and the relaxation depends on calcium removal fi^om the

contractile apparatus. This is carried out by Ca^-regulators or internal

and external exchangers of myofibrils. Parvalbumin that is located in the

muscle sarcoplasm is the most likely external exchanger or the regulator

of calcium ion levels (Gerday, 1982).

Parvalbumin (PA), a high affinity calcium binding protein of a

multigene family (Heizmann et al., 1987) is quite abundant in muscle of

cold blooded vertebrates specifically the fishes (Hamoir et al., 1972). It

some cases it is the major firaction of soluble protein repertoire of the

sarcoplasm. As supported by the published evidence and shown for

species of genus Channa in this study (Fig.l), PAGE of sarcoplasmic

proteins have largely served as species markers. A similar inference can

be made by SDS-PAGE pattern (Fig.2), if the number of bands showing

consistent presence are taken into account. SDS-PAGE has so far rarely

been used to study biomarkers.

Contrary to the above generalization, the PAGE pattern of PA

isotypes or isoforms have the application beyond species diagnostics

(Focant and Joyeaux, 1988), that is, the identification of fiber types as

42

initially shown by Hamoir (1972). Huriaux et al. (1990) showed that in

the tnink white fibers of European barbell PA II to PA IV occur while in

red fibers low amounts of only PA I and PA IV. Fiber discriminatory

pattern have been reported in other instances also (Focant et al., 1990;

Huriaux era/., 1992).

Occurrence of two to five PA isoform (or isotype) is common to

fish muscle (Boback and Slechta, 1988; Huriaux et al., 1992; 1997), but

in snook as many as seven isoforms have been discovered (Ross, et al.

,1997). The maximum number of PA isoforms in the species of genus

Channa is tliree. Each of the four species has one PA as the major and 1

to 2 or the minor ones (Fig.3). The ratio of minor to major isoforms is

species specific. In C. punctatus, C. marulius and C. striatus which have

three isoform each the ratio is of PA I : II : III is 0.07 : 0.043:1,

0.05:0.54:1 and 0.9:0.2:1, respectively. The value for C. gachua which

has two isoforms is 0.35:1. Since the ratios given above represent an

average of 40-50 samples of each species, they may be taken as a reliable

estimate for the muscle of the selected defined region. Pubhshed evidence

otherwise suggest differences in, the ratio of the musculature in different

regions of the trunk (Huriaux et al., 1991; Huriaux et al., 1993). In

addition, ontogenetic (Sherwani et al., 2001) and developmental changes

(Focant et al, 1992; Huriaux et al, 1996 and Huriaux et al., 1997) also

influence the intraspecies isoforms ratio.

Cumulatively the amount of PAs as a fi-action of soluble muscle

proteins, is characteristically high in all four fish species of Channa. On

an average the value for C. punctatus, C. marulius, C. gachua and C.

striatus are: 33%, 46%, 36% and 38%, respectively (Table 8 A and B).

43

A degree of species specially is exhibited by the number as well as the

type of the major isoforms that are typical to each species. Major isoform

in C. gachua and C. punctatus is PA II but the total number is different

because the former species lacks PA III (Fig.3). C. marulius and C.

striatus have PA I as the major isofomi but there is substantial difference

in the relative amount of PA II. PA II in C. marulius is 55% of the total

amount of all isoform as against 9% of C. striatus (Fig. 3).

During literature search, the report dealing with immunoblotting of

fish PAs could not be listed. In the present attempt Western blotting was

carried out with twin objective of : 1) identifying PA isofonns and, 2)

exploring the possibility of a quantitative assessment that would facilitate

an interspecies comparison of cross reactivity. By the results shown in

Fig. 4 based on screening with anti-CpPAII-rabbit antisera only the first

objective (the positive identification of entire set of isofonns in each

species) could be achieved. Since the cross-reactivity of anti-CpPAII-

rabbit antiserum with chick PA is ahnost of the same order and

magnitude as that of fish PAs, for tracing phylogeny further work of

quantitative nature is required to perfect the use of western blots

developed with chemiluminiscent techniques.

In general, there are scarce reports on immunological

characterization of fish parvalbumins. Gosselin-Rey et al. (1973)

characterized PA III of the pike while muscle and compared the results

with arginine residue modified conformational alternatives. In another

study following electrophoresis in agar gels, Gadidae PAs were cross-

reacted with anti-whitting-PAIII antibodies which recognized two groups

of PA isoforms within this family (Piront and Gosselin-Rey, 1974).

Identification of PA bands in gradient SDS-PAGE of snook muscle by

44

immuno cross-reactivity has been carried out in a later instance (Ross et

al, 1997). In this study possibility of using ELISA assay to type PAs

employing monoclonal antibodies was also explored.

Instead of the widely adopted method of Laemmli (1970) that

stacks all of the PAs as the band of 10 kD, the system of Schagger &

Jagow (1987) was preferred here, because in our laboratory it

satisfactorily resolves the polypeptides as low as 6 kD in 10% gels

(Fig.5). Gel-Pro analysis of the lanes show that the molecular weight of

PA isoforms of Channa species has a range of 10 to 12 kD (Fig. 5). The

purified PA isoforms of C. punctatus resolve as one broad band and at

this loading it could not be assured, if it is in fact is one band. However,

highly purified major isoform (eluted fi-om the band in native PAGE)

loaded in lane-4 stacks as the band of 10.5 kD (Fig. 5a). This is also true

for the highly purified major isoform of C. marulius, though the entire set

of its pure isoforms gives a broad band around 10 kD+ but with two

minor bands of up to 12.5 kD are also visible. No fiirther analysis was

performed to determine the molecular weight of minor bands by eluting

them individually. In the same figure the highly purified PA of chick

loaded in lane 8 gives a higher value of 13.5 kD which is in agreement

with its reported value (Heizmann and Strehler, 1979). Similar to C.

marulius purified PA isoform of C. gachua stacks as two band of 10 and

12 kD respectively (Fig. 5b, lane 3) while the highly purified major

isoform PA II stack as one band of 10 kD (Fig.5 b, lane 4). The results

obtained from purified and eluted major isoform PA I of C. marulius

((lanes 6 &7, respectively) are the same as those of C. gachua. It is thus,

Ukely that in this high resolution system 12 kD band represents the minor

isoforms of each species. These resuhs are to a certain extent in

45

confomiity with those reported in hterature. For instance, anti-PA

antibody precipitable band of snook PAs in SDS-PAGE gave a value of

around 11 kD (Ross et al., 1997). A relative molecular mass of about 11.2

to 11.5 kD has been reported for barbell PA isoform also (Huriaux et al.,

1997).

Following the initial observations by the group of Hamoir,

thermostability of parvalbumin has been principally used to help

purification of its isoforms (Hamoir et al., 1972). Fish PAs have not

otherwise been investigated for their behavior during heat treatment at

high temperatures. One recent report by Rehbein et al. (2000) compared

themiostabilities of several fish PAs by lEF but at the same temperature

range (70°C) that is commonly used to remove most of other

sarcoplasmic proteins during purification. Since it is the temperature

around which fish is baked in developed countries, they suggested the use

of this teclinique to identify cooked fish meat using PAs as the markers.

The extent of thermostability of PA isofomi at a temperature and

duration of incubation such as used in the present study has not so far

been reported. Results shown in Fig. 7 to 12 and as already explained

under "Results" highlight several new aspects of thermal stability of PAs

in general and, in particular those of Channa species. This is important

that the fish species of this genus inhabit rather high temperature tropical

waters and a correlation between the ambient temperature and

thermostability is a generally accepted view. The characteristic of heat

treated PAs under the conditions applied here demonstrate that:

(1) During heat treatment depending upon thermostability of each isoform, it

is converted into soluble aggregates (since they do not precipitate out),

migrating faster than the native ones resulting in the appearance of novel

46

bands, an observation supported by the fact that the appearance of

novel bands correspond to the gradual disappearance of the PA III and

PA II;

(2) The appearance of novel band during heat treatment depends on two

factors: (a) the lack or extent of multiplicity in the native PA (e.g. chick

where CBB stains only one isoform and no novel band appears vs fish

where multiple PAs exist and novel bands appear) and, (b) the relative

interspecies and intraspecies stability of PAs with the same number of

isoform as apparent by the order: C. punctatus > C. striatus > C.

marulius (Figs 7a, 8c and 7c, respectively);

(3) The novel bands are apparently soluble aggregates of different

conformational states of PA isoforms and/or their heteromers, because

their number and intensity is significantly affected by p-mercaptoethanoi;

(Fig. 7b and 8d & 7d respectively);

(4) The heat treatment leading to the appearance of novel aggregates and

retardation or smearing effect apparently caused by heat denaturation is

considerably reversed by p-mercaptoethanol.

It is, therefore, suggested that the heat induced changes are a SH-SS

mediated aggregation. More so, because none of the novel bands is a

degradation product due to total absence of any new band in SDS-PAGE

and only one band of 10 kD appears in all the heat treated PA samples.

The intensity of single band that stacks as 10 kD band in SDS-PAGE of

samples incubated for prolonged duration greatly depends on the absence

or presence of p-mercaptoethanol (Fig. 10a and b). The above

observations are also supported by plots of these changes based on

densitometric quantitation (Fig. 11 and 12). According to this data both

inter and intraspecies variations in themiostabilities of PA isoforms

between four fish species become evident.

47

This study is also the first of its kind where the effect of chelators on

fish PAs has directly being demonstrated by the changes in 10% PAGE

(Fig. 14 to 20). Some limited observations on of this nature on PA of

bullfrog on 4% polyacrylamide gels have been reported by Tanokura et

al., (1987). The results presented here demonstrate remarkable changes in

the electrophoretic mobilities of PA isoforms of all four fish species over

a wide range of EDTA (1 mM to 200 mM) or/and EGTA. With the

description available under "Results" the main inferences can be

summarized as:

(1) PA isoforms of all four species elicit a response to chelating affect

which differs from species to species;

(2) Whereas in fish species mobility shift is towards the anode, chick PAs

elicit an opposite response;

(3) Excepting EDTA treated PA isoforms of C. marulius where a mobility

shift is initiated at 1 mM of EDTA, in all other fish species and chick it

happens at 10mM EDTA;

(4) The observed changes are not due to salt effect {i.e. high chelator

concentrations up to 200mM) since no major differences exist between

the pattern throughout this range; and,

(5) The effect of EGTA is stronger than that of EDTA and if both of the

chelators are simultaneously added, the effect of EGTA remains

predominant.

The above changes to a limited extent are supported by difference

spectra (Figs 13 to 20). Fish as well as chick PA isoforms show a

prominent trough at 290 nm of ahnost of the same value of O.D. at only

10 mM EDTA.

48

Konosu et al, (1965) demonstrated that UV difference spectra of

carp white muscle PA is a classical example with a high Phe to other

aromatic amino acids while Tyr and Trp were absent. The absorbance at

259-269 nm indicates Phe. The difference spectra showing peaks at 259

and 270 nm have generally been reported for cyprinids and (Piront and

Gerday, 1973); cyprinids and the eel (Dubois and Gerday, 1988). Laforet

e? a/., (1991) showed that specific peaks of Tyr and Trp are not present in

cardiac muscle PA isoform of Antarctic fish.

Similar to typical difference spectra of reported cases, PA isoforms

of C punctatus, C. marulius and C. striatus also showed low intensity

peaks at 259 nm indicating the presence of Phe residues. In C. gachua

these residue are in negligible amount because only after stretching the

spectrum a low shoulder appears at this wavelength (Fig. 19). There are

no signs of Tyr or Trp in this species whereas in very low amounts Tyr

may be present in C punctatus and C. marulius and still less in C.

striatus. The later species may have Trp in very low amount as per height

of the shoulder at 293 nm. It is thus evident that the species differences

exist between amino acid compositions of PA isoforms of these four fish

species, in particular in the residues discussed with respect to difference

spectra.

Keeping in view that evolutionarily C. gachua has been the most

generalized form according to classical data (Chandy, 1955), an abnormal

profile of difference spectrum makes it an attractive model for ftirther

investigations to trace the phylogeny of fish parvalbumin isoforms. Its

specialized evolutionary placement is revealed by another unique feature

of UV spectrum of PA isoforms of this species and, where it differs fi-om

49

others, is the appearance of a sharp peak at 259 with 10 mM EDTA

indicating the presence of buried Phe.

It is also interesting that all fish PAs (Fig. 14 to 17) including that of

chick (Fig. 20) responded to 10 mM EDTA in a similar manner showing

a negative and prominent shift between 287 to 296 nm. The only

difference is that the intensity of negative shift in the spectrum of chick

PA is roughly half of that noted for PA isoforms of the fish species all of

which show a change of almost similar magnitude (Fig. 14 to 17). A

general similarity in the spectral behavior of the all the investigated

proteins confirms their parvalbumin nature as well as a degree of

structural homology; specifically, within the regions of the molecules

that interact with EDTA (where tyr and trp may be buried).

In the spectral profiles of PAs of all fish species, positive low

magnitudes shifts of a resembling pattern are characteristic of the change

at 200 mM EDTA. Chick PA was an exception where at 200 mM of

EDTA a sudden shift in O.D. of a magnitude of 2.5 units was noted after

233 nm that showed two maxima, one at 259-260 nm and the other at

270-280 nm showing the perturbation of the residues typical of these

peaks.

Electrophoretic changes (shown at the top of Figs. 14-17) are in

conformity with the spectral behavior of fish PAs, since the major shift in

electrophorefic mobility occurs at the same concentration of EDTA (10

mM) as that in UV spectra. The only exception is C. mamlius PA where

the shift in PAGE patterns is apparent at 1 mM EDTA, but the difference

spectrum at this concentration is not available at the moment. It is,

however, noteworthy, that during heat treatment C. mamlius PA shows

50

an early and more extensive response (Fig. 7c) and our unpublished data

shows that during storage even with 2 mM P-mercaptoethanol additional

bands (multimers) appear further supporting that it is most labile PA out

of all four species.

The low intensity change or shift, though species specific, is

observed when Ca^^ specific chelater EGTA was used. EGTA induced

changes were in accordance with the increasing concentration of the

chelater. As shown in Fig. 18 and 19 the order of the change was, C.

marulius > C. striatus > C. punctatus or C. gachua. Another important

feature is the persistence of the peak of about that appeared in these

vertically expanded spectra of all species at about 259 nm. The

corresponding changes in the PAGE patterns were reflected by more crisp

resolutions as shown in the inset of each diagram. If the two chelators

were combined the changes in electrophoretic patterns become

predominantly of the type when EGTA alone was present (Fig. 20, 21).

This shows stronger affinity of EGTA with regions of PA isoforms it

interacts as well as a similarity in the mechanism of the interaction of the

two chelaters.

As for the evolutionary lineage, the PA isoforms of all Channa

species appear to be of p-lineage because their pi values are below 4.8

(Pechere et al., 1973; Goddman et al., 1977). Due to the lack of

comprehensive information about lineages some controversy exists

regarding the numerical typing (nomenclature) of PA isoforms. Some

authors have numbered the isoforms with Roman numericals on the basis

of over all numerical strength of PA isoforms present within a family or

51

genus (Boback and Slechta, 1988; Huriaux et al. 1997). Five PA isoforms

bands of differing mobilities can be counted within genus Channa. That

would mean that at least 2 to 3 isofomis do not exist in one or the other

species since the maximum number in any case is tliree (marked in this

study as PA I, PA II and PA III). If the presence or absence of

comigration is taken into account and the species-wise Roman numericals

assigned in this study are altered taking overall number of bands within

the genus as the final figure, an erroneous dendrogram is constructed.

It is contended here that the dendrogram constructed according to the

numbering system of PA isoforms used in the present study appears to be

reasonable (Fig. 23). According to it, C. punctatus is of the most remote

origin, and C. striatus and C. marulius being more similar as per OTU

clustering share a close phylogenetic relationship. PA isoforais of C.

gachua that according to classical data was the most generalized form

(Chandy, 1955), have a similarity with PA isofomis of C. marulius'C.

striatus and PAs of all the three species might have evolved fi^om those of

C. punctatus.

These results show that the evolution of PA isoforms took a different

course than that of transferrins {Tfs) which showed more identity between

C. gachua and C. marulius proteins. Immunologically, Tfs of C. gachua

and C. striatus have been shown to be more similar followed by C.

marulius and all these Tfs exhibit least similarity with Tf of C. punctatus

(Nabi,1999) •

52

CONCLUSIONS

CONCLUSIONS

On the basis of the data collected on Parvalbumin (PA) isoforms of

four species of the genus Channa, the following conclusions may be made;

1. PA isoforms in these species make up to 45% of the soluble muscle

protein of the sarcoplasm.

2. Individually, C. punctatus. C. marulius, C. gachua and C. stnatus

have respectively 3, 3, 2 and 3 isofonns with one out of them \w each

species is the major PA. Stoichiometiy of the different isoforms in

each of the above species, in the same order is : 0.07:0.043:1,

0.05:0.54:1 and 0.9:0.2:1 respectively.

3. Each of the band initially identified as PA by PAGE cross-react with

anti-PAII-rabbit antibodies in western blot developed by a

chemiluminiscent method.

4 It is likely that the molecular mass of the minor and major PAs may

not be the same but the range of 10 to 12 kD is in agreement with the

values reported in the literature.

5. PA isoform of Channa species are of the highest themiostability

tolerating upto 90°C for 3 hrs. That the highest temperature and

duration reported for fish PAs so far. During heat treatment inter and

intra species differences in the relative stabilities were observed. PAs

of C. marulius were most unstable. It is proposed that the heat

53

denaturation is basically an aggregation reaction involving oxidation

of thiols.

6. Inter species differences are also exhibited by UV difference spectra

taking into account specifically at wave lengths of 259, 283 and 293

indicative of the presence of Phe, Tyr and Trp, though in low

amounts.

7. Coincident with a definite increase in the electrophoretic mobilities of

PA isoforms at 10 mM EDTA is a prominent negative spectral

perturbation with in the UV spectral bands of Tyr and Trp.

8. Fish PA treated at high concentration of EDTA 200 mM show very

low intensity perturbations of similar order and magnitudes in contrast

with the UV spectra of chick PA which shows a very high magnitude

in the range of Phe and Trp peaks. This confirms that Tyr and Trp

contents of fish PA are indeed very low and that the interspecies

difference in the amino acids compositions exist.

9. In terms of the affinity of the chelators for the sites of their interaction

EGTA exerts a stronger influence, on the overall confirmation of the

molecule as apparent by predominantly EGTA type changes in PAGE

patterns of PAs simultaneously treated with both the chelators.

10. Evolutionarily, PA, of species of genus Channa are of p-lineage as

determined by the pi values and some spectral and biochemical

peculiarities of PA isoforms of C. gachua a suggest that it may be a

54

good model for phylogenetic studies on PAs. It appears that though on

the basis of classical studies the most generalized form might have

been C. gachua, C. punctatus might be the species of most remote

origin.

J -r-se(33^ /J'^

33

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