sedimentation constants, molecular weights, and ...sedimentation constants, molecular weights, and...

SEDIMENTATION CONSTANTS, MOLECULAR WEIGHTS, AND ISOELECTRIC POINTS OF THE RESPIRATORY

PROTEINS

BY THE SVEDBERG

(From the Laboratory of Physical Chemistry, University of Upsala, Upsala Sweden)

(Received for publication, July 3, 1933)

The blood of a number of animal groups possesses t,he faculty of taking up oxygen, when exposed to a high partial pressure of this gas, and giving it up to the surroundings at lower oxygen concentrations. This important physiological role is played by certain pigments, the respiratory proteins, dissolved in the blood or transported by it packed in blood corpuscles (1). An ultracentrifugal study of the blood pigments throughout the animal kingdom has proved that these prot.eins are exceedingly homogene- ous and well defined with regard to molecular weight (2). Not only their mass but also their chemical composition as revealed by the electrophoretic behavior shows unusual distinctness (3).

The detailed study of the different kinds of respiratory proteins is therefore of great interest not only from a physiological but also from a physicochemical point of view. The present paper reports some recent work on respiratory proteins done in t.he writer’s laboratory. Three different properties have been st,udied: the sedimentation constant as derived from the measurement of the rate of settling of the molecules in strong centrifugal fields, the molecular weight as calculated from sedimentation equilibrium determinations in centrifugal fields of medium strength, and the isoelectric point as measured by means of t.he migration of the molecules in electric fields.

The technique for the determination of sedimentation constant and molecular weight has been described elsewhere (4). A small quantity of solution (0.1 to 0.8 cc.) enclosed in a sector-shaped cell with quartz windows is exposed to the influence of a strong field of force in a special centrifugal instrument (the ultracentri-

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312 Respiratory Proteins

fuge) and the concentration gradient determined by taking photo- graphs of the solution during centrifuging. The pictures are then registered by means of a microphotometer and the curves obtained are used for the calculations.

If the field of force is very strong, the rate of settling, dx/dt, can be measured and from this the sedimentation constant, SW, defined as the velocity of sedimentation in a field of unit strength reduced to water of 20” as solvent, can be calculated.

We have

~200 = dx/dt.l/w*x.q/qo.(L - Vpa)/(l - VP) where w = angular velocity

x = distance from the center of rotation 3 = viscosity of the solvent ?)* = “ “ water at 20”

V = partial specific volume of the solute p = density of solvent PO = “ “ water at 20”

The quantity ~~200 is a constant which is characteristic of the molecular species studied. It is a function of the mass and shape of the molecule.

If the centrifugal force is adjusted so as to produce only a very slow rate of settling of the molecules, a measurable state of equilibrium between sedimentation and diffusion is reached after pro- longed centrifuging at constant temperature. The molecular weight is then given by the formula

2RT In (dcJ a4 = (1 - Vp)w’ (4 - zf)

where R = gas constant, T = absolute temperature, cz and cl = concentrations at the distances 8 and x1 from the center of rotation.

An improved technique for the electrophoretic determination of the isoelectric points of the proteins has been worked out in this laboratory (5). The measurements of the movement of the boundary are made by photographing the solution and by expo- sure to a microphotometer, just as in t,he case of the sedimentation determinations in the ultracentrifuge.

Besides the light absorption at about 275 rnp, common to all proteins, the respiratory pigments possesss characteristic absorp-


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T. Svedberg 313

tion bands in the long wave ultra-violet or in the visible part of the spectrum. If a suitable wave-length is chosen for the determination of the concentrat,ion gradient, it is therefore always possible to study the sedimentation of the molecules of a respiratory prot,ein in centrifugal fields or their migration in electric fields undisturbed by the presence of other blood prot,eins. This is of vital importance because of the difficulty of obt,aining blood quantit,ies large enough for chemical separation of the various proteins in t.he case of a great many invertebrates. The fact that a sample of blood can be studied in t,he uhracentrifuge directly aft,er being drawn and diluted with a suitcable salt solution with- out any ot,her treatment, obviously eliminates serious chance of error, especially in the case of the more unstable pigments. If respiratory proteins of more than one molecular weight are present in the blood, the uhracentrifugal methods enable us to carry out an analysis of the different, molecular species.

Four types of respiratory proteins from the blood of inverte- brat,es are known: a red pigment (for which the name erythrocruorin has been proposed (6), a green pigment (chlorocruorin), a blue pigment (hemocyanin), and a pigment of reddish brown color (hemerythrin). On the last type, only a few preliminary determinations have been made. From the blood of the higher vert,e- brat.es only one type of red pigment (hemoglobin) is known. In the blood of the lowest, class of vertebrates, the Cyclostomata, we have found a red pigment resembling that contained in the eryt,h- rocytes of t,he capitellid worms.

Sedimentation constants have been det,ermined on samples of blood from a great many different, species representing most of the animal groups which possess respiratory blood prot,eins.i Molec- ular weigh& and regions of pH stabi1it.y have so far been measured only in a few typical cases.a Det,erminations of isoelectric points have been carried out on blood proteins from polych&,e and oligochaete worms, crust,aceans, gastropods, cephalopods, and a few vertebrates”

1 The determinations of sedimentation constants have been carried out by Mrs. Astrid Hedenius.

a Recent work on pH stability curves and molecular weight determinations from equilibrium runs hrts been done by Miss Inga-Britta Eriksson.

3 The electrophoretic measurements have been carried out by Dr. K. 0. Pedersen.


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For the ultracentrifugal measurements the blood samples have as a rule been diluted from 4 to 30 times with 1 per cent NaCl solution in order to avoid retardation of the sedimentation by too high a protein concentration. In some cases buffer solutions have

FIG. 1, a FIG. 1, b

FIG. 1, a AND b. Sedimentation pictures (a) and photometer curves (6) of a mixture of Nereis and Subella blood; centrifugal force 75,000 times gravity (32,000 R.P.M.); time between exposures 5 minutes. The fact that only one boundary is visible shows that their respiratory proteins, erythrocruorin and chlorocruorin, are identical with regard to sedimentation constant.

FIG. 2, a FIG. 2, b

FIG. 2, a AND b. Sedimentation pictures (a) and photometer curves (b) of a mixture of Sepia and Octopus blood; centrifugal force 78,000 times gravity (33,000 R.P.M.); time between exposures 5 minutes. The fact that the boundary is double shows that their respiratory pigments, which are both of the hemocyanin type, have different sedimentation constants, viz. 51.1 and 57.1 X 10-‘3.

been used. The blood of most of the invertebrates contains respiratory proteins of high molecular weight and low diffusion. The sedimentation boundaries are therefore very sharp and even small differences between the sedimentation constants of two blood pigments can be detected if the proteins in question are centrifuged when mixed in about equal proportions. This


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T. Sved berg 315

“mixture test,” has been applied to all blood proteins of high molecular weight. Tf only one boundary shows up, the sedimentation Constants are taken as identical. The limit of error is about f2 per cent.

FIG. 3, a FIG. 3, b FIG. 3, a AND b. Sedimentation pictures (a) and photometer curves (b) of

a mixture of Planorbis and Helix blood; centrifugal force 53,000 times gravity (27,000 R.P.M.); time between exposures 5 minutes. In this case two boundaries are visible wide apart because of the great difference between the sedimentation constants, which are 34.1 and 99.8 X lo-** respectively.

FIG. 4, a FIG. 4, b FIG. 4, a AND b. Sedimentation pictures (a) and photometer curves (b)

of the red pigment from the blood corpuscles of Notomastus; centrifugal force 175,000 times gravity (49,000 R.P.M.); time between exposures 15 minutes. The boundary is blurred because of the rapid diffusion of these comparatively small molecules (sedimentation constant 2.0 X 10-l*).

In Fig. 1,~ and b, are reproduced the sedimentation pictures and photometer curves from an ultracentrifugal run with a mixture of Nereis and Sabella blood. The boundary is single and therefore the sedimentation constants are identical (57.1 X 10-l*) in spite of the fact that the erythrocruorin of Nereis and the chlorocruorin of Sabella are chemically different proteins. Fig. 2,~ and b, shows the double boundary of a mixture of Sepia and Octopus


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blood. Here t,he sedimemation constants are distinctly different (51.1 and 57.1 X lo-‘*) alt,hough t,he respiratory protein is in both cases a hemocyanin. Fig. 3,a and b, finally, gives the pictures of t,he sedimentat,ion of a mixture of two respiratory proteins of widely different constants; viz., t.he erythrocruorin of Planorbis and the hemocyanin of Helix.

In the few cases in which the sediment,ation constant is low, the mixture test cannot, be applied because of the rapid diffusion of t.he sedimenting molecules during centrifuging. Fig. 4,a and b, shows the pict,ures from an ultracentrifugal run wit,h t,he red respiratory protein contained in t,he blood corpuscles of Note- mastus (SW = 2.0 X lo-13). The blurring of the boundary is quite pronounced, although the time between exposures is not more than 15 minutes.

The light absorption in the visible part of the spectrum is due to the active group and therefore is almost identical for respiratory proteins possessing the same active group. This has been conclusively proved by earlier invest,igat,ors. During the present work, however, we noticed that the hemocyanin from Paludina differed in color from the ordinary hemocyanin from Helix. The light absorpt,ion was therefore measured and a marked shift in t,he position of t,he maximum, viz. from X 550 mp for the Helix hemocyanin to X 600 mp for t,he Paludina hemocyanin, was found.

In Table I the main data from the deberminations of sedimentation constants are given. In cases where the mixture test showed identity of sedimentation c’onstants, the mean value from all the determinations of the type of sedimentation constant in quest,ion has been used for all t,he species possessing this constant.

The following conclusions may be drawn from Table I. Respir- atory prot*eins contained in blood corpuscles have low sedimenta- t,ion constants and therefore low molecu1a.r weights. Hemo- globin charact,erized by the sedimentsCon const.ant 4.4 X lo-13, t,he high isoelectric point (pH 6.9), and the hemin group only occurs in the higher classes of the vertebrates; viz., Mammalia, Aves, Reptilia, Amphibia, Pisces. The blood corpuscles of the lowest class of the vertebrates, CyclostSomat,a, as well as the blood corpuscles of the capitellid worms (Notomastus) have a respirat,ory protein of a much lower sediment,at,ion constant (2.0 to 2.3). The glyceride worms (GEgcera) have a corpuscle prot,ein sedimen-


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T. Svedberg

TABLE I

Sedimentation Constants of Respiratory Proteins

- ____._-- Chzetopoda, Polychsta

Nereis virens

Lumbrinereis fro&is Arenicola marina Eumenia crassa Pectinaria belgica Polymnia nebulosa Sabella penicillus

Serpula vermicularis G&era go&i Notomastus latericius

Chtetopoda, Hirudinea Hirudo medin’nalis

Hwnopis sanguisuga Chzetopoda, Oligochreta

Lumbricus terrestris Cruetacea, Phyllopoda

Daphnia pulex Crustaces, Malacostraca

Palzmon fabrici

Pandalus borealis Palinurus vulgaris Eupagurus bernhardus Pagurus striatus Nephrops norvegicu-s Homarus vulgaris Astacus $uviatilis Hyas araneus Maja squinado Cancer pagurus Carcinus mzsnos Squilla mantis Calocaris macandre

Arachnomorpha, Xiphosura Limulua polyphemd

Arachnomorpha, Scorpionidea Euscorpius carpathicus

Type of respiratory protein -.-_-..-

Erythrocruorin dissolved i plasma

“ I‘ “ “ “ “ ‘I I‘ “ “

Chlorocruorin dissolved i: plasma

“ “ Erythrocruorin in corpuscle

‘L “ ‘C

“ dissolved il plasma

“ I‘

‘I “

‘I “

Hemocyanin dissolved ir plwma

“ ‘I ‘I ‘I ‘I “ “ ‘I “ I‘ “ “ “ “ “ ‘I ‘I “ “ ‘I “ I‘ “ “ “ ‘I

“ ‘I

“ ‘L

317

8300 x 1011

57.1

57.1 57.1 57.1 57.1 57.1 57.1

57.1 3.5 2.1

57.1

57.1

66.8

16.9

16.9

16.9 16.9 16.9 16.9 23.4 23.4 23.4 23.4 23.4 23.4 23.4 23.4 34.1

35.7

34.1


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Type of reepiratory protein


TABLE I-continued

Eutrachesta, Diptera Chironomus plumosus 2.0

“ SP. Amphineurrt, Placophors

Tonicella marmorea

Erythrocruorin dissolved in plasma

I‘ ‘I 2.0

Hemocyanin dissolved in plasma

60.8

Conchifera, Gastropod& Planorbis corneus

“ umbilicatua Paludina vivipara

“ contecta fittoriru8 littorea Buccinum undatum Neptunea antiqua timnea stagnalis Achatina julva Helix pomatia

“ arbustorum “ nemoralis ” hortensis

Limax mc&mus Agriolimax agrestis Arion empiricorum


“

Hemocyanin plasma

“ “ “ “ I‘ ‘I I‘ ‘I ‘I “ “ “ ‘I

34.1

“ 34.1 dissolved in 99.8

99.8 99.8 99.8 99.8 99.8 99.8 99.8 99.8 99.8 99.8 99.8 99.8 99.8 60.8

Conchifera, Cephalopoda Decapoda

Ldigo vulgaris Sepiola oweniana Rossia macrosoma Sepia o$icinulis

Octopoda Octopus vulgaris Eledone &rosa

I‘ moaehata Vertebrata, Cyclostomatn.

Maxine glutinosa Vertebrats, Pisces

Raja clavata Esox l&us Lun’opewa Sandra

“ “ ‘I I‘

“ ‘I ‘I

“ “ “ “ “ “ ‘I “ “ “ ‘I “ “

‘L “ “ I‘

“ “ I‘

57.1 57.1 57.1 57.1

51.1 51.1 51.1

Erythrocruorin in corpuscles 2.3

Hb in corpuscles “ “ “ “ “ “

4.4 4.4 4.4


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T. Svedberg

TABtE I<onc&&d

Vertebrata, Amphibia Rana temporaria

Vertebrata, Reptilia Chrysemys picta

Vertebrata, Aves Columba livia

Vertebrata, Mammalia Equus caballus

-

.- Type of regpirstory pmtkn

Hb in corpuscles

‘I “ ‘1

“ “ “

“ “ “

319

c?lP x 10s

4.4

4.4

4.4

4.4

* Svedberg, T., and Heyroth, F. F., J. Am. Chem. Sot., 61,639 (1929).

tation constant of 3.5 X 10-l*. Respiratory proteins dissolved in the blood have, as a rule, high sedimentation constants and therefore high molecular weights. The only exception is the blood pigment of the Chironomus larvs, which has a sedimentation constant identical with that of the Cyclostomatn and the capitellid worms.

Within a well defined animal group all the species have, as a rule, the same sedimentation constant. All the polychzeete and hirudine worms with the respiratory protein dissolved in the blood have t,he constant 57.1 X lo-la. Some of these proteins are red (erythrocruorin), others are green (chlorocruorin). The oligo- chste worms have a constant of 60.8 X lo-13, very close to that of the other worms. Some of the crust,acean families show the sedimentation constant of 16.9 X lo-l3 (hemocyanin and eryt,hro- cruorin), others 23.4 X lo-13, and one of them (Culocaris) 34.1 X 10-13. The xiphosurans (Limulus) and the scorpions (Euscor- pius) have probably the same constant, viz. 35.7 and 34.1 X lo-l3 respectively. In this case the mixture test has not yet been made. All the gastropods except Planorbis have the same constant, 99.8 x 10-13.

It is obvious from these regularities that biological kinship is usually accompanied by identity in the sedimentation constant. On the other hand, t,he number of different sedimentation constants observed is so small that the same constant must with necessity occur in more than one animal class and in respiratory proteins containing different active groups. It seems t,hat only a few molecular masses are stable and that it would depend upon


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the composition of the molecules with regard 60 various amino acids whether one or the other po&bility is realized. The constancy of the molecular weight within a certain animal group would then be a measure of the similarity of certain chemical processes leading to the formation of the respiratory protein.

TABLE II

Sedimentation Constants and Molecular Weights of Respiratory Proteins

Ery- thro-

cruorin Hb

60.8 57.1

34.1 222 16.9 11.5* 6.8* 4.4 3.5 2.1

:hloro ruorin

57.1

99.8 60.8 57.1 51.1 34.1 23.4 16.9

6.8’

No. of Fe atoms per mole-

cule in red pigment5

144

72 36 18 12 8 4 2 1

No. of units of

34,500

144

72

36 18 9 6 4 2

:

8,

L _-

5

2

2 1

-

Molecular weight

De?EltiY %dimentation equilibrium neaaurement

,l~,ooo

,750,OOO

,~,ooo ,310,000 640,000t 3sonJw ~8,C’W

8 68,000 34,500ll 25,000~

-

I I u

-

cs1culsted from No. of mitt3 of 34,50 and No. of

Fe atoms

4,970,ooo

1,240,OOO 620,000 310,000 207,000 138,000 69,000 34,500 17,250

-

0

-

4,800,090

2,250,OOO

1,820,OOO 1,020,OOO

581,000 357,000 220,000

94,000 a,OC@ 34,500 16,100

* Only as dissociation product; sedimentation constant not accurately known.

t Preliminary values only. $ From determination on other proteins with the same sedimentation

constant (e.g. phycoerythrin). $ Not determined. I/ From determination on ovalbumin which has the same sedimentation

constant. 7 Values from 17,300 to 33,000, indicating a mixture of units of 4 X

34,500 and 34,500.

Table II gives the molecular weights corresponding to the observed sedimentation constants as determined by means of equilibrium measurements, together with t,he values calculat,ed from the sedimentat,ion constants on the assumption Ohat. t,he molecules are spherical. The regulariCes in the molecular weights


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T. Svedberg 321

suggest certain simple relationships and Table II also contains the values computed on the basis of such a rule.

The number of iron atoms per molecule has been calculated on the assumption that the iron content is equal for all the proteins containing hemin and that the molecular weights are simple multiples of 34,500. The remarkable agreement between the observed and calculated values makes this hypothesis highly probable.’ Some observations concerning the dissociation of the respiratory proteins at the borders of the pH stability regions further strengthen the assumption about simple multiples. It has been found that the hemocyanin with the constant 99.8 at the alkaline border dissociates in two components of constants about 60 and 17; that is, in fragments 4 and & of the weight of the original molecule (8). The erythrocruorin with the constant 57.1 gives at the alkaline border fragments of const,ant 12, or & of the weight of the original molecule. The erythrocruorin of constant 34.1 dissociates at the acid end of the stability range into fragments with the constants around 23, 17, 12, and 7, corresponding to molecular weights +, 2, 6, and I’T of the stable molecule (9). The hemocyanin with the constant 23.4 is split up into 3 molecules of constant 16.9 and 2 molecules of about constant 7. The molecules 60.8 and 57.1 have probably the same weight but different shapes. The molecule 2.1 is at least in the case of Myxine a mixture of 34,500 and 4 X 34,500. For myoglobin, Theorell (7) found a slightly higher sedimentation constant and the molecular weight 34,500. Miss Laura Krejci (10) obtained a similar result for gliadin. The data of Table II further show that with increasing molecular weight the deviations from the spherical shape become more and more scarce.

The scheme of simple multiples for the molecular weights of the respiratory proteins outlined above may seem rather fantastic and the writer has hesitated somewhat to advance this view. It is, however, supported by previous ultracentrifugal determinations on other native proteins. One gets the impression that the protein molecules are built up by successive aggregations of definite uIlits. It seems that the higher the molecular weight, the fewer are the possibilities of stable aggregation. The steps between the

* According to determinations by Theorell (7) the iron content of myoglobin, the red respiratory protein of the mammalian muscle, is identical with that of hemoglobin.


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molecular weights, therefore, become larger and larger as the weight increases. Especially the gap from 2,500,OOO to 5,000,OOO is very striking. No protein molecule has so’far been found in this large interval.

TABLE III

Isoelectric Points of Respiratory Proteins and Slopes of Mobility-pH Curves at Isoelectric Point

Chmtopoda, Polychmta Nereis v&-ens

Arenicola marina Chmtopoda, Hirudines

Hznwpis sanguisuga Chretopoda, Oligochreta

Lumbricus terrestris Crustacea, Malacostraca

Homarus vulgaris

AstacusJluviatilis Cancer pagurua

Conchifera, Gastropoda Planorbis corneus

Paludina viwipara

‘I contecta Ldtorina littorea Buccinum undatum Achatina fulva Helix pomatia

“ nemoralis “ hortensis “ arbustorum

Conchifera, Cephalopoda Eledone &rosa

Vertebrata, Aves Columba livia

Vertebrata, Mammalia Equus caballus

“ “ “ “

Type of protein


“ “

5.10

4.56

“ ‘I 5.01

“ ‘I 5.28


“ ‘I “ ‘I

4.95

4.93 12.1 4.65 16



“ I‘ “ “ “ “ “ “ “ “ “ “ ‘I “ I‘ “

4.77

4.71

4.63 11.4 4.34 12.8 4.61 13.7 5.03 7.8 5.05 8.1 4.63 11.4 4.57 12.1 5.50 7.6

“ “ 4.6

HbCO in corpuscles 7.23

HbOz “ “ HbCO “ “ Methemoglobin in cor-

puscles

6.92 7.2 6.92 7.2 7.04 6.7

3lope of mbility- H curve

9.5

16,

20

12.6

18

10.6

10.8


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T. Svedberg 323

The det.erminations of the isoelectric points of respiratory proteins carried out in this laboratory (2) together with the slope of the mobility-pH curve (du/dpH X 105) at the isoelectric point are summarized in Table III.

The measurements given in Table III show that the isoelectric point,s of the blood pigments of t,he invertebrates are all very low compared with the isoelectric point of hemoglobin, the respiratory protein of the vertebrates. Further relationships wit,h regard to various animal groups cannot be traced from t,he data so far avail- able. It is of great interest, however, to no&e that the isoelectric point varies from species to species. Even such nearly related forms a~ Paludina vivi~ara and Paludinu contecta have different isoelectric points. For a genus such as Helix, cont,aining several subgenera, the isoelectric point,s lie closer together within a subgenus. Thus Helix nemoralis and Helix hortenzis, belonging to the subgenus Z’aeheu, possess the isoelectric points 4.63 and 4.57 respectively, while Helix pornatia of the subgenus Heliwgena has its isoelectric point at pH 5.05 and Helix arbustorum of the subgenus Arionta at pH 5.50. The situation of the isoelectric point is therefore to a certain degree a measure of the kinship.

The above cataphoretic determinations, although not very numerous, show conclusively that the chemical composition of the blood pigments aa revealed by the position of the isoelectric point is constant for each species but varies from one species to the other. The sedimentation constant and the molecular weight may be used as group characteristics, the isoelectric points as a species characteristic. It is hoped that further studies along these lines will bring to light regularities which may help us t,o understand more thoroughly the peculiarities of the respiratory proteins.

The expenses connected with this investigation have been defrayed by grants from the Rockefeller Foundation, the Nobel Foundation, and the Foundation Therese och Johan Anderssons Minne.

Various persons and institutions have rendered great help in supplying material necessary for the work. The writer wants especially to express his thanks to Professor S. Ekman and Dr. I. Arwidsson, Upsala, to Professor R. Dohrn and Dr. L. Cslifano of the Zoological Station [at Naples, and to ,Professor [E. Lijnnberg and Dr. G. Gustafsson of the Zoological Station at Kristineberg, Sweden.


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SUMMARY

1. A systematic study of the sedimentation constants and molecular weight,s of the respiratory proteins throughout the animal kingdom has been carried out by means of the ultracentrifugal method. The isoelect,ric points of a number of these proteins have been determined by means of an improved electrophoretic method.

2. Bespirat,ory proteins contained in blood corpuscles have low sedimentation constants and comparatively low molecular weights. Hemoglobin only occurs in the higher classes of the vertebrates. The corpuscle pigment of the lowest vertebrate, Myxine, has a lower sedimentation constant and lower molecular weight than the hemoglobin of the higher vertebrates.

3. Respiratory prot,eins dissolved in the blood plasma have, as a rule, high sedimentation constants and high molecular weights. The only exception is the blood pigment of the Chironomus larvae.

4. Within a well defined animal group all species have, as a rule, the same sedimentation const,ant. Biological kinship, therefore, is usually accompanied by identity in the sedimentation constant.

5. Determinations of molecular weights by means of sedimenta- Con equilibrium measurements indicat,e that a system of simple multiples seems to obtain among the molecules of t,he blood pigments.

6. Determinations of the isoelectric points of the respiratory proteins show that the blood pigments of the invertebrates so far studied are all much less alkaline than the hemoglobin of the vertebrat.es. The isoelectric point varies from species to species. Even closely related forms have different isoelectric points. For a genus including several subgenera, the isoelectric points lie closer t,ogether wit,hin a subgenus. The sit,uation of the isoelectric point is therefore t,o a certain degree a measure of kinship.

Addendum-Recent determinations on the erythrocruorin from Area pexata (82~o = 3.5 X 10-r”) and Thyone briarewr (SXP = 2.6 X IO-“) have confirmed the rule that respiratory proteins contained in blood corpuscles have low sedimentation constants and low molecular weights.

A study of fresh timulus blood has shown that it contains three high molecular hemocyanins with constants 57.1, 34.1, and 16.9 X 10-r*. The second one is identical with the hemocyanin of EUSCOT~~W~.


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T. Svedberg 325

Another representative of the Cyclostomata, viz. Petromyzon jluviatilis, has now been studied. The respiratory pigment has a sedimentation constant of 2.0 X lo-la, or slightly lower than Ilfyxine.

The blood samples of Arca, Thyone, and Limulus were kindly put at our disposal by Dr. A. Redfield of the Oceanographic Institution at Woods Hole, Massachusetts.

BIBLIOGRAPHY

1. Redfield, A. C., Q26art. Rev. Biol., 8, 31 (1933). Jordan, H. E., Quart. Rev. Biol., 8, 58 (1933).

2. Svedberg, T., and Eriksson, I.-B., Nature, 130, 434 (1932). Svedberg, T., and Hedenius, A., Nature, 131, 325 (1933).

3. Pedersen, K. O., Kolloid-Z., 63, 268 (1933). 4. Svedberg, T., and coworkers, J. Am. Chem. Sot., 48-66 (192633). 5. Tiselius, A., Nova acta regis sot. scient. Upsaliensis, series 4;7, No. 4

(1930). 6. Svedberg, T., and Eriksson, I.-B., J. Am. Chem. Sot., 66,2834 (1933). 7. Theorell, H., Biochem. Z., 363,l (1932). 8. Pedersen, K. O., and Eriksson, I.-B., unpublished data. 9. Eriksson, I.-B., unpublished data.

10. Krejci, L., unpublished data.


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The SvedbergRESPIRATORY PROTEINS

ISOELECTRIC POINTS OF THEMOLECULAR WEIGHTS, AND

SEDIMENTATION CONSTANTS,

1933, 103:311-325.J. Biol. Chem.

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