differences in amino acid sequence between dolphin and sperm whale myoglobins

4
136 SHORT COMMUNICATIONS (IOO-I65). Three phyla, the Cnidaria (Virgularia, Metridium and Physalia), the Polyzoa and Echinodermata fall into the second group. This is interesting. In the Annelida there is extreme variation, with I4.6 hydroxyproline residues in Allolo- bophora at one extreme and 165 in Lumbricus at the other. The variation in the imino acid ratios (hydroxyproline to proline) is also interesting. In Annelida high ratios have been reported, from 4.7 :I to 12.5 :I in different species. In other groups the ratio is lower. In sponges and Ascaris the ratios are 1.38 : I and I : 14. 4. To decide whether these variations in the imino acid content of collagen can be interpreted in terms of phylogenetic considerations, it will be necessary to investi- gate sufficient number of representative species from the different invertebrate phyla. My thanks are due to Prof. R. V. Seshaiya for suggesting the problem and for his guidance and to Dr. G. N. Ramachandran for reading through the manuscript and for his advice. Centre of Advanced Study in Marine Biology, A nnamalai University, Portonovo (India) M. KALYANI 1 G. N. RAMACHANDRAN, Treatise on Collagen, Vol. i, Academic Press, London and New York, 1967, p. 46. 2 G. KRISHNAN AND C*. SUNDARA t~AJULU, Biol. Zentr., 84 (1965) 359- 30. H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951) 265. 4 J. E. EASTOE AND A. COURTS, Practical Analytical Methods for Connective Tissue Protein, E. and F.N. Spon, London, 1963, pp. i, 3, 7, io. 5 A. A. LEACH, Biochem. dr., 74 (196o) 7o. 6 W. TROLL AND J. LINDSLEY, J. Biol. Chem., 215 (1955) 655. 7 F. L. CAMPBELL, Ann. Entomol. Soc. Am., 22 (I929) 4oi. Received May I9th, 197o Biochim. Biophys. Aeta, 221 (197 o) 135-136 BBA 33237 Differences in amino acid sequence between dolphin and sperm whale myoglobins Several years ago the investigation of similarities in the microstructure of dolphin and sperm whale myoglobins was started l. The differences between them found by the fingerprint technique seemed to be quite significant, so we decided to obtain information about the covalent structure of the dolphin myoglobin molecule from its digests and cyanogen bromide cleavage. Experimental data obtained by investigation of the tryptic hydrolyzate2, a, chymotryptic hydrolyzatO, 5 and cyanogen bromide cleavage 6 were published in the course of several years. Together with the hydrolyzate products, attention was centered on some chemical modifications of dolphin myoglobinL In this paper we report some new results regarding the amino acid sequence of the protein and revise the structure of one tryptic peptide 2, T 3 (Table I), as a final stage of our investigation of the primary structure of this protein. Bioehim. Biophys. Aeta, 221 (i97 o) 136-130

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136 SHORT COMMUNICATIONS

(IOO-I65). Three phyla, the Cnidaria (Virgularia, Metridium and Physalia), the Polyzoa and Echinodermata fall into the second group. This is interesting. In the Annelida there is extreme variation, with I4.6 hydroxyproline residues in Allolo- bophora at one extreme and 165 in Lumbricus at the other. The variation in the imino acid ratios (hydroxyproline to proline) is also interesting. In Annelida high ratios have been reported, from 4.7 :I to 12.5 :I in different species. In other groups the ratio is lower. In sponges and Ascaris the ratios are 1.38 : I and I : 14. 4.

To decide whether these variations in the imino acid content of collagen can be interpreted in terms of phylogenetic considerations, it will be necessary to investi- gate sufficient number of representative species from the different invertebrate phyla.

My thanks are due to Prof. R. V. Seshaiya for suggesting the problem and for his guidance and to Dr. G. N. Ramachandran for reading through the manuscript and for his advice.

Centre of Advanced Study in Marine Biology, A nnamalai University, Portonovo (India)

M. KALYANI

1 G. N. RAMACHANDRAN, Treatise on Collagen, Vol. i, Academic Press, London and New York, 1967, p. 46.

2 G. KRISHNAN AND C*. SUNDARA t~AJULU, Biol. Zentr., 84 (1965) 359- 3 0 . H. LowRY, N. J. ROSEBROUGH, A. L. FARR AND R. J. RANDALL, J. Biol. Chem., 193 (1951)

265. 4 J. E. EASTOE AND A. COURTS, Practical Analytical Methods for Connective Tissue Protein,

E. and F.N. Spon, London, 1963, pp. i, 3, 7, io. 5 A. A. LEACH, Biochem. dr., 74 (196o) 7 o. 6 W. TROLL AND J. LINDSLEY, J. Biol. Chem., 215 (1955) 655. 7 F. L. CAMPBELL, Ann. Entomol. Soc. Am., 22 (I929) 4oi.

Received May I9th, 197o

Biochim. Biophys. Aeta, 221 (197 o) 135-136

BBA 33237

Differences in amino acid sequence between dolphin and sperm whale myoglobins

Several years ago the investigation of similarities in the microstructure of dolphin and sperm whale myoglobins was started l. The differences between them found by the fingerprint technique seemed to be quite significant, so we decided to obtain information about the covalent structure of the dolphin myoglobin molecule from its digests and cyanogen bromide cleavage. Experimental data obtained by investigation of the tryptic hydrolyzate2, a, chymotryptic hydrolyzatO, 5 and cyanogen bromide cleavage 6 were published in the course of several years. Together with the hydrolyzate products, attention was centered on some chemical modifications of dolphin myoglobinL In this paper we report some new results regarding the amino acid sequence of the protein and revise the structure of one tryptic peptide 2, T 3 (Table I), as a final stage of our investigation of the pr imary structure of this protein.

Bioehim. Biophys. Aeta, 221 (i97 o) 136-130

SHORT COMMUNICATIONS 137

T A B L E 1

S T R U C T U R E O F P E P T I D E S F R O M D O L P H I N M Y O G L O B I N C O M P A R E I ) \ V I T H C O R R E S P O N D I N G S I T E S I N

S P E R M \ V H A L E M Y O G L O B I N

Peptides from dolphin myoglobin (this paper) Corresponding sites in sperm whale myoglobinl o

T 3 ChB01 ChO02 ChO0a ChP20 ChM05

Gly-(His , Pro, Olx, T h r ) - L e u - G l u - L y s * 35-42 G l y - A l a - A s p - A l a - G l n - G l y - ( A l a , Met) i 2 4 - I 3 I L y s - I l e - P r o - I l e (Lys, Tyr) 98-1o 3 S e r - A r g - H i s - P r o - A l a - G l n - P h e 117 123 L y s - L y s - H i s - G l y - A s p - T h r - V a l - L e u 62 69 L y s - A l a - S e r - G l u - ( A s x , Leu, Lys, Lys, His, Gly, Asx, Thr , VaI, Leu) 56 69

* The incorrect s t ruc tu re pub l i shed in ref. 2 was Thr - (H i s , Pro, Glx, G l y ) - L e u - G l u - L y s .

The chymotryptic peptide ChM05 was purified. Its quantitative amino acid analysis showed a composition: Ly%, His 1, Asp2, Thr D Serl, Glul, Gly> Ala> Vall, Leu2. This peptide and some previously mentioned peptides2, 5 were subjected to sequential analysis using a modification of the Edman procedure 8. The N-terminal

T A B L E II

A M I N O A C I D S E Q U E N C E S O F D O L P H I N A N D S P E R M W H A L E 1° M Y O G L O B I N S

The pept ides isolated f rom t ryps in (TD) and c h y m o t r y p s i n (CD) digests and those isolated f rom b r o m o e y a n a t e c leavage (BC) are shown by bars. The solid line is used for sect ions wi th known s t ruc tu re and the do t t ed line for u n d e t e r m i n e d sequences . In the spe rm whale myoglob in s t ruc- ture , only the different and poss ib ly different ami no acids are shown.

BC TD CD

Dolphin Mb Sperm whale Mb

No.

BC TD

Dolph in Mb Sperm whale Mb

No.

BC TD TD CD

Dolphin Mb Sperm whale Mb

No.

BC T D TD CD

Dolphin Mb Spe rm whale Mb

No.

G l y - L e u - S e r - A s p - G l y - G l u - T r p - G l n - L e u - V a l - L e u - A s n - V a l - T r p - V a l - - G l u His I "2 3 4 5 6 7 8 9 IO II I2 13 14

----- > ~-

Ala L y s - V a l - ( G l x , Ala, Asx, Val, Ala, Gly, His, Gly, Glx, Asx, Leu, I l e -

15 16 17 18 19 20 21 22 23 24 25 26 27 28

9 < - - - - - > ~6 >

-6 . . . . . . . > < - - - - -

Leu, I l e ) - A r g - L e u - P h e - L y s - G l y - H i s - P r o - G l u - T h r - L e u - G l u - L y s - Ser

29 3 ° 31 32 33 34 35 36 37 38 39 4 ° 41 42

<---__-------->

P h e - A s p - L y s P h e - L y s - H i s - L e u - L y s - T h r - G l u - A l a - A s p - M e t - L y s - - - Arg Glu 43 44 45 46 47 48 49 5o 51 52 53 54 55 56

Biochim. Biophys. Acta, 221 (197 o) 136-139

138 SHORT COMMUNICATIONS

BC TD CD CD

Dolphin Mb Sperm whale Mb

No.

BC TD CD

Dolphin Mb Sperm whale Mb

No.

BC TD CD

Dolphin Mb Sperm whale Mb

No.

BC TD TD CD

Dolphin Mb Sperm whale Mb

No.

BC TD TD CD CD

Dolphin Mb Sperm whale Mb

.No.

BC TD CD CD

Dolphin Mb Sperm whale Mb

No.

BC TD TD CD

Dolphin Mb Sperm whale Mb

.No.

. . . . . . . . . . . . . >

<-- - > ~ _ _ _

A la -Ser -Glu-Asx-Leu-Lys-Lys-His -Gly Asp -Thr -Va l -Leu -Thr - Asp Val

57 58 59 6o 61 62 63 64 65 66 67 68 69 7 °

%

- - > + - >

Ala-Leu-Gly-Ala- I le -Leu-Lys-Lys-Lys-Gly-His -His -Asp- (Ala , Glu - -

71 72 73 74 75 76 77 78 79 80 81 82 83 84

Glx) -Leu-Lys-Pro-Leu-Ala-Gln-Ser His -Ala-Thr -Lys His -Lys- Glu 85 86 87 88 89 9o 91 92 93 94 95 96 97 98

-> <-

< - -

-> < - - - - - > - ~

l l e -P ro - I l e -Lys -Tyr -Leu-Glu-Phe- I l e Ser-Glu-Ala- I le - I le -

99 ioo lOllO2 lO 3 lO 4 lO 5 lO6 lO71O8 lO 9 i i o t l i i i 2

%

His -Va l -Leu-His -Se r -Arg-His -P ro -Ala -Gln -Phe -Gly -Ala -Asp- Gly-Asn

I I 3 I I 4 I I 5 1 1 6 I I 7 i i 8 i i 9 I 2 O I 2 I I 2 2 1 2 3 1 2 4 I 2 5 I 2 6

Ala-Gln-Gly-Ala-Met -Asx-Lys-Ala-Leu-Glu Leu Phe-Arg-Lys Asn-

i27 i28 129 13o I3i i32 i33 134 135 136 137 138 139 14o

<- - - - ~

Asp- I l e -Ala -Ata -Lys -Tyr -Lys -Glu- I , eu -Gly-Phe-His - ( ; ly Tyr-Gln --

I4I 142 I43 I44 I45 146 147 148 149 15o I5I 152 153

a m i n o ac ids o f t h e m e n t i o n e d p e p t i d e s we re d e t e r m i n e d b y t h e m e t h o d of GREY AND HARTLEY 9. I n T a b l e I t h e s e q u e n c e s t h u s o b t a i n e d a re g iven .

As a r e s u l t o f al l p r e s e n t s e q u e n c e d a t a , i t is pos s ib l e to c l a i m k n o w l e d g e of a l m o s t t h e c o m p l e t e p r i m a r y s t r u c t u r e o f d o l p h i n m y o g l o b i n , a s s u m i n g o n l y t h a t t h e

d o l p h i n c h y m o t r y p t i c p e p t i d e G l y - A l a - I l e - L e u occup ie s t h e s a m e p o s i t i o n (73 76)

Biochim. Biophys. Acta, 221 (197o) 136-139

SHORT COMMUNICATIONS 139

as the analogous peptide in sperm whale myogh~bin. All isolated peptides and the structure of dolphin nlyoglobin are shown in Table II. In Table I I the structure of sperm whale myoglobin and the different amino acid residues are also listed. Thirteen differences have been found. Only the position of one has not been exactly determined. Ile-28 or Ile-3o in sperm whale myoglobin is probably replaced by leucine in one of these positions in dolphin myoglobin. In each case the differences could have arisen from the change of one nucleotide base in a codon of mRNA, i.e. by a point mutation. A study of both mentioned myoglobins led to the conclusion that their spatial struc- tures are analogous n. This conclusion permits us to suggest that the last 15 amino acid residues in dolphin myoglobin, which have no exactly determined positions (18-3 o and 84, 85), occupy sites analogous to the same amino acids in sperm whale myoglobin.

Twelve replacements of amino acids take place at the surface or in surface crevices and one, in the interior of the globin molecule (Ser-35 replaced for Gly-35 in dolphin myoglobin). The last one increases the number of nonpolar residues in the interior of dolphin myoglobin molecule to 33. A nonpolar residue 12 (Val-66) is substi- tuted for a polar one (Asp-66 in dolphin myoglobin) at one site on the surface of dolphin myoglobin molecule. The other replacements do not essentially change the nature of amino acid residues. They also do not interact with the basic functional group heme.

Institute of Organic Chemistry, Bulgarian Academy of Science, Sofia z 3 (Bulgaria)

M. KARADJ OVA P. N•DKOV A. BAKARDJIEVA N. GeNOV

1 M. KARADJOVA, P. T. NEDKOV, B. ATANASOV AND B. KEIL, Coll. Czech. Chem. Commun., 2 9 (1964) 551 •

2 A. I~AKADJIEVA, B. MELOUN AND F. ~ORM, Collection Czech. Chem. Commun., 32 (1967) 196o. 3 A. P, AKADJIEVA AND g . MELOUN, Compt. Rend. Acad. Bulg. Sei., 18 (1965) 837. 4 P. NEDKOV, N. GENOV, M. KARADJOVA AND B. KEIL, Collection Czech. Chem. Commun., 33

(1968) 1974 . 5 N. GENOV, M. SHOPOVA AND M. I~ARADJOVA, F E B S Letters, I (1968) toS. 6 A. BAKARDJIEVA AND B. MELOUN, Collection Czech. Chem, Commun., 34 (1969) 215o. 7 P. NEDKOV AND B. MELOUN, Collection Czech. Chem. Commun., 34 (I969} 2o21. 8 P. NEDKOV AND N. GENOV, Bioehim. Biophys. Acta, 127 (1966) 544. 9 W. R. GREY AND B. S. HARTLEY, Biochem. J., 89 (1963) 59-

IO A. ]3. EDMUNDSON, Nature, 205 (1965) 883. i i P. NEDKOV, B. MELOUN AND B. I(EIL, Collection Czech. Chem. Commun., 34 (1969) 3597. 12 J. C. KENDREW, Brookhaven Syrup. Biol., 15 (I962) 21(,.

Received June 27th, 197o

Biochim. Biophys. Acta, 221 (197 o) 136-139