Biochimica et Biophysica Acta, 491 (1977) 23-28 © Elsevier/North-Holland Biomedical Press

BBA 37601 T H E M Y O G L O B I N OF T H E K I L L E R W H A L E ( O R C I N U S ORCAJ*

O. CAST1LLO, H. LEHMANN and L. T. JONES University Department of Clinical Biochemistry, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QR (U.K.) (Received September 1st, 1976)

SUMMARY Whereas the myoglobin of the dolphin surprisingly resembles that of the sperm whale more than that of the porpoise, the myoglobin of a killer whale does not show this feature. It differs from the myoglobins of the dolphin in 6, of the porpoise in 8, and of the sperm whale in 14 positions. The residues in which the cetaceans differ are localised in one particular area of the molecule.

INTRODUCTION In order to provide further information concerning the order Cetacea we have established the primary structure of the myoglobin of Orcinus orca (killer whale). The other cetaceans whose myoglobins we considered were: sperm whale, dolphin, Amazon River dolphin and porpoise. Of these the killer whale and dolphin [1] have the least number of amino acid differences, namely 6. We have noted that the amino acid residues in which the 5 cetaceans differ from one another are restricted to one particular area of the molecule. MATERIALS AND METHODS Skeletal muscle was obtained from a killer whale which had recently died and the myoglobin was extracted and purified as described previously [2]. 90 mg of pure myoglobin were obtained and then digested by trypsin, pepsin and chymotrypsin [3] and the resulting peptides were separated by two dimensional high voltage paper electrophoresis at p H 6.5 and chromatography. Some overlapping peptides were separated by re-electrophoresis at p H 3.5. The peptides were stained with ninhydrin and eluted with 6 M HC1, and the * Supplementary data to this article, are deposited with, and can be obtained from, Elsevier Scientific Publishing Co., BBA Data Deposition, P.O. Box 1527, Amsterdam, The Netherlands. References should be made to No. BBA/DD/054/37601/491 (1977) 23. The data are: amino acid analyses of the hydrolysates of the killer whale myoglobin, amino acid analyses and fingerprint patterns of the soluble tryptic peptides, peptic peptides derived from the insoluble tryptic core, and chymotryptic peptides; and amino acid analyses, fingerprint patterns of thermolysin peptides derived from the tryptic peptides (17-31, 79-96, 117-133).

24

1

2

3

4

5

6

7

8

9

lO ii

13

14

16

17

18

19

20

21

22

23

24

25 ~6

27

28

~9

30

31

CCLPt~N Mb

Val Leu Set" Glu Gly GIu Trp CI~ Leu val l~u His val Tr~ Ala Ly~ Val Glu Ala Asp val Ala Gly His Gly GI~ Alp Ile ~

Ile Ar~

KELLER t~qLE Mb

Gly LeU S ~ Asp G1y G1u Trp C~, Leu Val Lea Ae~ vat Trp Gly I.~,* Val Glu Ala A~p Leu Ala Gly Hi~ G1y Gln Asp Ile ~

II~ Azq

Val Glu Ala A~p L~u Aia GIy Hi~ GIy GI~ A ~

Tp Amp Cd.y GluI

Lea V ~ LeulAma V~2. ~

Pe

¢~y ~

PO lle

C.J.y I ~ S~r ~ p Gly Glu TrPlGln Leu V ~ t.eu i G~y lmu Se~ A~p G).y C.~u ~ C ~ G.).y Leu S~" A~lp G~y Glu T ' ~ Gl.n Leu Val LeulA~m Val TrpiCly Lys Val Glu Ala Aep Leu Ala Gly P~i~ Gly Gln Asp Ile L ~ II~ ~ Vnl CO.uAI~ Aspl~eu /~alC..ly X ~ I G l y ClnIAsP Zle LOUIIlO A.~ val C,lu J~ta ~ L~u AlalC,l y Et~ c.,ly G I ~ Ile Leu I l e Arg

Th Th

~

Cd.y

T~

Dou~nq Mb ~LL~

Ile L~/ I~e AD~ l

Glu Ala A ~ Leu Ala I

32 33 ~4 35 35 37 38 39 40 41 42 43 44 45 46 47 46 49 50 51 52 53 54 55 56 57 58 59 60 61 62 l,m~ Ft~ l~s Gly His Pro Glu Thr Leu GIU Ly~ Phe A ~ Lya ~tm Lys Hie I,e. l~s ~ r ~ u ~ ~ ~ ~ ~ ~ ~ u ~ ~ ~

~S~LE Mb

Tp rp C~

~e ~ ~ m~m ~n~ Pro Glu 'IT,r LOUlG.Iu14,m F'~eIA~P L,~ P'nell~/u ~

F'nell4,~ Gly ~

~3

54

65 66

6~

~

59

70 71

72

73

74

75 75

77

78

79

~d~ m = m u ~ ~a r~ I Asp Met /..ys Ala S~r Gl.u Asp r . ~ l L ~

l~uulLys Tt= Glu ~

90

81

62

83

84

85

86

87

88

69

90

91

92

~ L P H ~ Mb ~ 3 L U ~ t~lqLE MO

TIp Tp C~ Ch Tn

14~ Itts Gly A m ~

Val Lmu Ttz" Ala Im~ Gly Ala l i e Lmz Ly~ D ~ LyS Gly Itl~ ~

t,~l

~

~

~

~

~

~

~

~

~

Lysl t , ~ G.ty I1.~ l~ts Aep Ala GlU Lea ra~ Pt-o Lett Ala Cd.n S~-

Ly~ IlllllC-d.yAim Ttur Val l.~al'lhr Ala L ~ Gly Ala I k Leull4rn I ~ l Lyll ~ y ~ Thr ALa L ~ Gly Ala IIo Leu I

~

~

~

~u ~

~ ~ ~1~ ~ ~ l Lys Pro Leu[ Ala C,ln Set

LyS Gly HI~ i41S ASp Ala GlUl LyS Pru LeUt Set Gly KI.~ Ills Aq~ Ala Glut I,ea LysI Leu Ala C.ln1 H~UlAsp Ala Glu I

Th Th

93

CCLRUN Mb

~

94

95

96

Hi~ AI~ Thr ~

97 98 99 1(30 101 102 103 IO4 105 10~ 107 1 ~ 109 110 ~.11 ].12 113 114 115 116 117 118 119 120 123. £22 123 H ~ Lys Ii~ PTO lle Ly~ "fyr Le~ GI~ Phe Ii~ ~ GIU AI~ I ~ lle ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~

~3LL~ titLE Nb

Tp

Him P'~'o/~.a GI~

X.~ Ala Tnr l,.~|l'd.* 14.* 11.. Pro Xle t,,/~I

Tp Pe

Ch Th Th

DCLPH~ ~ KII//~ t~qlE Mb

~ c ~ Glu ~'wf l l o S~" Gl~Ata l l ~ | lalu Glu PIll Ala l i e Ala II~ ~AI~ Tr~ 14~ H~ILy~ 11o Pro 11o Lya TyClLOU GIU Ptwllle Se~ GIu Ala Ile ~ At~ T ~ ~ m m s I :-llJ f A.I.~ 'l't~r I.,,/m I'L~ Ala TIU" LyaiI

llo lle lie II~

H ~ Vat :-lira Val H£m Val HlslVal

Leu H.t~I Leu H.~ S~" A~JI Leu1 LeUI S ~ Az~ Eia Pro A1a GLn P~e I Hta S ~ Arg e/~ ~zo Ata GIn P~e I K,Ls P.ro ,~.a C..lnlph,0

124 125 126 127 126 129 ).30 131 132 133 134 135 136 137 136 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 G1¥ Ala Aap Ala C,i n GIy A~a ~ A ~ Lys ALto Lmu Glu Lau pt~ Arq I ~ Asp IIQ ~ ~ ~ ~ ~ ~ u ~ ~ y ~ ~ ~ y C.~y Ala Alp Ala Gln C~y Ala M~: Aim Lyl Ala Leo C~u Lej pha A ~ L*IO~lp I ~ ~ ~ ~ ~ ~ ~ u ~ ~ y ~ ~ ~ y

TIp Tp Ch C~ C~

ca~, Axa A~,p~q.~ c.~ ~ , t e a ~,,t] ~ s Ata t ~ u t~a r m l ~ j Asp Ala Gin Gly kla MR|Aa~ Lya Ala Lea Glu LeU Pl~ I ~ t y t Ala ~ . I

Tn Th Tn Tn

~IAIa ~l~la

im~ ~ I

1 ~ p xx* ~aa xxa x,u,q z,/u ~ p x ~ l e a ~q.la t.ym a,y~ z,/~ cau z~a I L ~ Glu Lm~ G1y P t w l ~

Gly

C.L~I~y ALt ~,~ Xm Cys

1 ~ c~. my ~

~

~ . cy, ~m

Fig. 1. Myoglobin of Orcinus orca (Killer Whale) compared with dolphin myoglobin. Tp, tryptic peptides; Pe, peptic peptides; Ch, chymotryptic peptides; Th, thermolysin peptides. ~, enzymic hydrolysis; --~, dansyl-Edman degradation. Dots indicate the amino acid residues which are different in dolphin and killer whale myoglobins. The following residues were arranged by homology: 18-19, 27-28, 35-39, 51-55, 57-60, 70-71, 73-75, 80-84, 94-95, 98-101, 104-1135, 107-108, 110-112, 119-122, 142-144.

25 eluent was hydrolysed at 108 °C for 24 h. The resulting amino acid mixture was analysed in an automated "Locarte" analyser. When dansyl-Edman degradation [4] or further hydrolysis by thermolysin [3] were required the peptides were eluted with 0.5 M NH4OH. The amino acid sequence of the molecule was established by comparing overlapping peptides, and when necessary by dansyl-Edman degradation. Amide and acidic groups were established from electrophoretic mobility. The total amino acid composition of the myoglobin was obtained by hydrolysis for 24, 48 and 72 h at 108 °C, and quantitation of tryptophan was performed by hydrolysis with mercaptoethane-sulphonic acid [5]. RESULTS

The amino acid analysis of the total globin from the myoglobin of the killer whale indicated the presence of 153 amino acid residues. The sequence is illustrated in Fig. 1. Fig. 2 shows the differences among the 5 cetacean myoglobins known so far. There are 6 differences in the sequence between dolphin and killer whale.

O r-4 t~ -H N

,-4 ,-4

14

15

14

sperm whale

6

12

14

dolphin

8

8

killer whale Porpoise

Fig. 2. Matrix showing the number of amino acid differences among the myoglobins of 5 cetaceans.

26 TABLE 1 A M I N O A C I D D I F F E R E N C E S B E T W E E N T H E M Y O G L O B I N S OF T H E C E T A C E A N S Sequential No. : Helical No.

Do3

sP~'~ ~ DOU~m~

:

D3

KILiZ~

1 NAI

15

21

28

45

54

66

A2 AIO All AI3

4

B2

B9 BI6 CD3

D4

E9 El7 E~6 ~ 8

VAL ( ~

12

13

35

HIS VAL ALA VAL II~ S ~

74

83

85 121 122 129 144 151 152 (~3 (~4

H6 H21 HC2 HC3

A~G G~J3 VAL ALA (~// (~.;JG~Y ASP GLY AIA TYR GLN

VAL GLU HIS VAL ALA VAL ILE GLY LYS ASP ASP ALA ASP ~

AIA GIxN ~ Y

G~3Y ASP ASN VAL GLY L~J ILE GLY LYS ASP ~

AIA G~N GLY ALA PHE HIS

ALA ASP ~

ALA PHE HIS

GLY (~JJ ASN VAL (~Y L~J VAL (~Y LYS GLU ASN (~Y GILl A~q AIA GLU ~LY THR PHE HIS A.R. DOLPH]~ D 2 ]

GLY ASP ASN ILE GLY LEU VAL GLY LYS GLU ASN (~Y (~U GILl"GLY ASP ALA ALA PHE HIS

Fig. 3. View of the myoglobin molecule, showing the specific area of mutations for 5 cetaceans known so far. Shaded areas represent the region of mutations at the front of this view of the molecule. Hatched lines represent the region of mutations at the rear.

27 DISCUSSION It is well known that the amount of myoglobin found in the muscles of the order cetacea is very high [6]. If one assumes that the shape of the killer whale myoglobin resembles that of the sperm whale which is a globular protein of dimensions 42 A x 35 A × 23 A [7], one can notice that the residues in which the cetaceans differ from each other involve specific regions of helices A, B, E, EF, G, GH, H and HC (Table I and Fig. 3).

Fig. 4. View of the myoglobin molecule, showing the specific area of mutations for 6 ungulates known so far. Shaded area represents the region of mutations at the front of this view of the molecule. Hatched lines represent the region of mutations at the rear.

28 It seems that different groups of animals mutate at different areas of the myoglobin molecule. This could be due to different physiological requirement, or specific selection. A similar example is shown for ungulates in which the mutations in 6 species (horse, zebra, ox, sheep, red deer, pig) differ in helices, A, B, E, F, FG, G, GH, H and HC (Fig. 4). Whilst making these observations we have referred to the possible ancestral therian myoglobin chain of Romero-Herrera et al. [8] and to the observations of Margoliash et al. [9] on cytochrome c. It is hoped to expand these observations on localisation of mutations in different groups of animals. ACKNOWLEDGEMENTS We are grateful to Professor R. J. Harrison, F.R.S. of the Department of Anatomy, University of Cambridge for the killer whale muscle. We want to thank Dr. R. Dickerson for his permission to utilise his diagram of the myoglobin molecule. This work was supported by the Muscular Dynstrophy Group of Great Britain, O.C. is supported by a research grant from CONICIT, Government of Venezuela. REFERENCES 1 2 3 4 5 6 7 8

Kluh, I. and Bakardjieva, A. (1971) FEBS Lett. 17, 31-34 Romero-Herrera, A. E., Lehmann, H., Castillo, O. (1976) Biochim. Biophys. Acta 420, 387-396 Romero-Herrera, A. E. and Lehmann, H. (1974) Proc. R. Soc. Lond. Ser. B. 186, 249-279 Hartley, B. S. (1970) Biochem. J. 119, 805-822 Penke, B., Ferenczi, R. and Kovais, K. (1974) Anal. Biochem. 60, 45-50 Scholander, P. F. (1963) Harvey Lectures 57, 93-110 Kendrew, J. C. (1959) Fed. Proc. 18, 740-751 Romero-Herrera, A. E., Lehmann, H., Joysey, K. A. and Friday, A. E. (1973) Nature 246, 389-395 9 Margoliash, E., Fitch, W. M., Markowitz, E. and Dickerson, R. E. (1972) in Structure and Function of Oxidation-Reduction Enzymes, (Ehrenberg, A., ed.), pp. 5-17, Stockholm: Wirksel 10 Edmundson, A. B. and Hirs, C. H. W. (1961) Nature 190, 663-670 11 Bradshaw, R. A. and Gurd, F. R. N. (1969) J. Biol. Chem. 224, 2167-2181 12 Dwulet, F. E., Bogardt, R. A., Jones, B. N., Lehman, L. D. and Gurd, F. R. N. (1975) Biochemistry 14, 5336-5343