The primary structure of the myoglobin of rabbit (Oryctolagus cuniculus)

Biochimica et Biophysica Acta, 439 (1976) 51-54

© Elsevier ScientificPublishing Company, Amsterdam-- Printed in The Netherlands BBA 37376 THE PRIMARY STRUCTURE OF THE MYOGLOBIN OF RABBIT ( O R Y C TOLAGUS CUNICULUS)

ROMERO-HERRERA, H. LEHMANN and O. CASTILLO University Department of Clinical Biochemistry, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QR (U.K.) (Received December 31st, 1975) A. E.

SUMMARY The amino acid sequence of rabbit myoglobin was determined. It contains 153 amino acid residues and differs in three of them from all known mammalian myoglobins: Ala 5, His 34 and Thr 35.

INTRODUCTION We have established the amino acid sequence of the myoglobin of the rabbit (Oryctolagus cuniculus) in an attempt to elucidate the phylogenetic relationships of

this lagomorph. As five antigenic regions of the sperm whale myoglobin molecule have been determined by Atassi [1], we considered it of special interest to study the rabbit myoglobin sequence, because some of the antisera to the sperm whale molecule were prepared in this animal. A detailed account of our phylogenetic and immunological deductions will be published elsewhere. The myoglobin of the rabbit contains 153 amino acid residues, as in all the major fractions of the mammalian myoglobins studied so far, and differs from the sequence of man and sperm whale by 16 and 22 residues, respectively. MATERIALS AND METHODS 500 g of rabbit skeletal muscle were stripped of fat and connective tissue and minced in a blender. After aqueous extraction the myoglobin was purified, as routinely done in our laboratory, by (NH4)zSO4 precipitation [2], DEAE-Sephadex column chromatography [2] and paper electrophoresis [3, 4]. Removal of the haem was achieved by acid acetone [5] and the resulting apomyoglobin was then submitted to CM-cellulose column chromatography [2]. After hydrolysis of the myoglobin in 6 M HC1 for 24, 48 and 72 h at 108 °C, the amino acid composition of the molecule was established in an automatic analyser (Locarte). Quantitation of tryptophan'was performed by hydrolysis with mercaptoethane sulphonic acid [6]. The myoglobin was digested with trypsin [3], pepsin [3] (the insoluble core left after tryptic digestion) and chymotrypsin [3]. The resulting enzymic peptides were separated by fingerprinting (two-dimensional high voltage electrophoresis and

52 1 P-AB~.,'T Mb

2

3

4

5

6

7

8

9

10

11

Gly Leu Set Asp ~/a Glu Trp Gln Leu Va/ ~

12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 ASh Val ~ Gly Lla~ Val Glu Ala A~p Ieu Ala Gly HIS Gly
Pe Pe

c.tyu~, ~

c~*-, ~

~,p m a GZu ~

~ u [ ~ n Va~ ~

G~y Ly, va~ G~u m a Alp ~ur

~ax ~ . z ~ kg[

Pe Pe Q~

Gly le~ Set Asp A~a Glu Trp~In 1~u Val Leu|Asn Val Trp|Gly I~s Val GIu Ala Asp Lm/ Ala Gly His Gly Gln Glu Val 1~u|Ile Arg

Ch

Ala Asp|Leu A1a Gly His Gly Gln |Ala Gly H~E Gly Gin GIM[ ~Gln Glu val L e ~

Th Th

~A~elT Mb 'l'p

32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Le~ Phe Iris T~/ Hi~ Pro Glu Thr Le~ GIu Lys Phe A~p Lys Phe Lys His I~u Ly~ Se~ GIu Asp GIu Met Lys A/a S ~ (~u Amp

cZu Amp

A~ lma set GZU ~,~

[~h, A ~ ~ , ~ e ~'l Q1

Le~ I'Ve[His %'1at His Pro GIu Tnr Leu Glu Lys Phe[Asp Lys Phe[Lys HLS Lm/|

[G~u ~, me[

Ch C~ Q1

IVAE~ITMb

61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 1~u LMs Dis H£s Gly Ash ~%r Va~ L~/ ~nr Ala 1~a Gly Ala Ile Le~ L~S Lys 14~ Gly His His Glu Ala Glu lle Lys Pro I~/ Ala Val Lm/ ~nr A/a ieu Gly A~a Ile Le~ l~s|Lys|Lys Gly His K~s GI~ Ala Glu 11e Lys Pro Le~ Ala ~LyS Lys|Gly His His Clu Ala GIu Ile Lys Pro ieu Ala

LMS|~

Pe

GIU Asp

|I~s His Leu Lys Se~ Glu Asp Glu Met LyS A]a Set Glu Asp

.,rip Pe

fly, ~ , ~e. ~ s s ~ GIn Asp G~u ~LyS His leu Lys Se~ GIu Asp GIU Met I~S Ala ~

I~Lts Gly A ~ ~ r

Val Le~lTnr Ala Leul [Tnr A l a Leu GIy Ala I Le~Lys Lys H/S Gly Asn Tnr Val l~a|Tnr A l a Leu Gly Ala I l e Leu~Lys l y s Lys Gly His His Glu Ala GlU I l e Lye Pro Leu~Ala

C~

~als Glu|Ala Glu~Ile Lys Pro Leu~A/a His His Glu~ ~ Ala

Th "/'c,

91

92

93

94

95

96

97

98

99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

~%~BIT Mb C4n Set His A/a ~ z Lys|Kts Ly~ Ile Pro Val Lys[

~His Pro

|Ala Tnr L y s | Pe Pe Pe

Pe Pe

'In

[Leu Glu P h e r l l e Set Glu A l a r I l e ~Ala Xle rile rAla Xle Gln Se~ His Ala ~ Lys H~s|Lys Ile Pre Va] Lys TyrrLeu Clu Phe Ile Sex Glu A/a Ile rAla ~%r L~s H~S| [Leu Glu Phe[Ile Se~ Glu Ala lle

Ile Ile Ile Ile Ile lle

His Val His Val His Val HXS Val His[Val His[

Leu His Set I~s| Leu HlS~ leur leu~ LeurHis Sex Lys HIS Pro

Sl.n|S~ ~ , . Ala[ C1. ~ Uis A/a[

121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 RAESIT Mb

~n Ch O'. Th 'l'h Th It

czy Amp Phe GZy ~

Asp AXa CXn AZa A ~ Met Set ~ s . ~ a .t,eu czu . t ~ v,",e ~,~ . ~ n .'~p n e A]~ ~ a G ~ T~r L ~ ~ ~ ~ ~ G ~ CZy Leu Phe Arg~Asn Asp I l e Ala Ala Gln Tyr Lys~Glu Leu Gly Phm Gin Gly

Gly Amp Phe Gly Ala Asp A/a Gin Ala Ala ~ t | S e r Lys Ala Leu Glu Leu Phe~Arg Ash Asp I l e Ala Ala Gln ~'r~Lys GlU Leu Gly Phe Gin Gly rraa mar r ~ Lys Ala LeurGlu ~ Phe[ II~,; GIU Lea Sly PhmrGln Gly

,~la G ~ T z r ~ s l ~Gly Ala Asp ; d a Cln~Ala Ala Met Sex Lys[

|A~n~Asp lie Ala /~3.aCln[

Fig. l. The myoglobin primary structure of the Oryctolagus cunieulus (rabbit). Tp, tryptic peptides; Pe, peptic peptides; Ch, chymotryptic peptides; Th, thermolysin peptides; [", enzymic peptides; ~ , dansyl-Edman degradation.

53 chromatography) on Whatman 3MM paper. The fingerprints were subsequently developed with ninhydrin and stained for specific amino acids as described elsewhere [3]. The tryptic peptides, containing residues 17-31, 79-96, 80-96, 119-133 and 140-147, were in addition, eluted from preparative fingerprints and digested by thermolysin [3]. 15 mg of globin were aminoethylated [7], digested by trypsin and fingerprinted as described above. All enzymic peptides were eluted from paper using 6 M HCI, hydrolysed at 108 °C for 24 h, and their composition estimated in an automatic amino acid analyser. When necessary peptides were sequenced by stepwise dansyl-Edman degradation [8]. Amide groups were assigned from the electrophoretic mobility of small peptides at pH 6.5 and by utilising Offord's formula [9]*. RESULTS Compared with other mammalian skeletal muscles which have been investigated in our laboratory, that of the rabbit yielded an unusually low amount of myoglobin. From 500 g of tissue only 40 mg of myoglobin was obtained, which is about one-tenth of our usual yield. The amino acid analyses from the whole myoglobin molecule accounted for 153 amino acid residues, including two residues of tryptophan. The comparison of the tryptic fingerprints, obtained from aminoethylated and non-aminoethylated globin, showed no difference between them, indicated the absence of cysteine. Fig. 1 shows the amino acid sequence of rabbit myoglobin. It was obtained from overlapping enzymic peptides, by dansylation, and combined dansyl-Edman degradation. All amide and acidic groups were established, with the exception of Glx 26 and Glx 27 which were assigned by homology as found in most of the known myoglobins. When the rabbit myoglobin was aligned against the 29 known mammalian myoglobins it was found that residues Ala 5, His 34 and Thr 35 are exclusive to this animal. The residues at position 5 and 34 have no apparent function in the myoglobin of sperm whale [10, 11], however, residue Ser 35, in the sperm whale is hydrogen bonded to the main chain carbonyl group of residue 31, helping to stabilise the corner BC (corner between helices B and C), it is likely that residue Thr 35 in the rabbit assumes the same function. ACKNOWLEDGEMENT This work was supported by a grant from the Muscular Dystrophy Group of Great Britain.

* 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/040/37376/439 (1976) 51. The data deposited contain the following material: amino acid analyses of the hydrolysates of the whole rabbit myoglobin. Fingerprint patterns and amino acid analyses of tryptic peptides, peptic peptides (derived from the insoluble core left after tryptic digestion), chymotryptic peptides and thermolysin peptides derived from the tryptic peptides residues 17-31, 119-133, 79-96, 80-96 and 14(I-147.

54 REFERENCES i Atassi, M. Z. (1972) in Specific Receptors of Antibodies, Antigens and Cells. 3rd Int. Convoc. Imm. pp. 118-135, Buffalo, N.Y. 2 Romero-Herrera, A. E., Lehmann, H. and Fossey, D. (1975) Biochim. Biophys. Acta 393, 383-388 3 Romero-Herrera, A. E. and Lehmann, H. (1974) Proc. R. Soc. Lond., Set. B, 186, 249-279 4 Graham, J. L. and Grunbaum, B. W. (1963) Am. J. Clin. Pathol. 39, 567-578 5 Ingram, V. M. and Stretton, A. O. W. (1962) Biochim. Biophys. Acta 62, 456-474 6 Penke, B., Ferenczi, R. and Kovacs, K. (1974) Anal. Biochem. 60, 45-50 7 Clegg, J. B., Naughton, M. A. and Weatherall, D. J. (1966) J. Mol. Biol. 19, 91-108 8 Hartley, B. S. (1970) Biochem. J 119, 805-822 9 0 f f o r d , R. E. (1966) Nature 211,591-593 l0 Kendrew, J. C. (1962) Brookhaven Syrup. Biol. 15, 216-228 l l Watson, H. C. (1969) Prog. Stereochem. 4, 299-333