Biochimica et Biophysica Acta, 400 (1975) 387-398

© Elsevier ScientificPublishing Company,Amsterdam- Printed in Tho Netherlands BBA 37110 THE PRIMARY STRUCTURE OF THE MYOGLOBIN OF D I D E L P H I S M A R S U P I A L I S (VIRGINIA OPOSSUM)

A. E.

ROMERO-HERRERAand H. LEHMANN

MRC Abnormal Haemoglobin Unit, Department of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Road, Cambridge CB2 2QR (U.K.)

(Received February 19th, 1975)

SUMMARY Although the primary structures of numerous mammalian myoglobins are known, the only marsupial myoglobin described is that of the kangaroo. It was thought of interest to examine the myoglobin of the opossum. 16 differences were found between the myoglobins of the Australian and the American marsupials.

INTRODUCTION There are several reasons why an investigation of the myoglobin of the American opossum seemed of special interest. This marsupial has been considered a kind of "living fossil" because it has retained its anatomical features for approx. 70 million years [1]. Its haemoglobin is remarkable for its unusual a chain. In all vertebrate haemoglobins so far investigated position aE7 is occupied by the "distal histidyl", yet in the opossum residue aE7 is glutamyl [2]. One would therefore like to know whether the myoglobin shows similar unusual features. Although more than 20 eutherian myoglobins have been analysed, only one metatherian myoglobin is known, that of the kangaroo [3], and a comparison between the myoglobins of a marsupial from both Australia and North America might throw light on their possible relationships. MATERIALS AND METHODS Skeletal muscle from one opossum was used. It had been obtained in the department of Professor R. T. Jones, Portland, Oregon, U.S.A. and sent to Cambridge frozen in solid CO2. Except where a reference is especially given (refs 4-6), all the techniques mentioned subsequently have been fully described with all relevant references in a recently published account of the structure of human myoglobin [7]. The myoglobin was extracted and after (NH4)2SO4 fractionation it was purified by column chromatography on DEAE AS0-Sephadex and paper electrophoresis using a discontinuous buffer system. The myoglobin obtained was precipitated, digested by trypsin, pepsin and chymotrypsin, and the resulting peptides were separated two-

388 dimensionally on paper (high voltage electrophoresis at pH 6.5 and chromatography). When further purification of peptides was necessary reelectrophoresis at pH 3.5 or pH 9.0 was used. The peptides were eluted from the paper, hydrolysed in constant boiling 6 M HCI for 24 and 72 h and the resulting amino acid mixture was analysed automatically ( L O C A R T E analyser). Some peptides were subsequently eluted with NH4OH and digested with subtilisin and thermolysin. Sequential analysis of peptides was achieved by comparing overlapping peptides and when necessary, Sequencing some fragments by dansyl-Edman degradation. Amide groups were established from the electrophoretic mobility of small peptides at pH 6.5 using Offord's equation or by direct identification of the residues in question as phenylthiohydantoin derivatives [4, 5] on polyamide thin-layer chromatography [6]. As an additional improvement the myoglobin was purified, after DEAESephadex chromatography and paper electrophoresis, by CM-cellulose column chromatography (see Fig. 1). After removal of salts by dialysis and freeze-drying the myoglobin was submitted to total amino acid analysis. RESULTS Fig. 1 shows the elution pattern of opossum myoglobin obtained from the CM-cellulose chromatography. Two myoglobin fractions were recovered (peaks 3 3030 z I

3020

3.010

A

~- 60

8G

II

3 f

4

II =

720 EFFLUENT YOLUME

960

'

1:~oo

'

14'~

'

.~,o

ml)

Fig. l. Elution pattern of the myoglobin of Dide, ~hismarsupialis ( Virginia opossum) on CM cellulose (Whatman 23) column chromatography (2.5 cm × 28 cm). Buffers were prepared in 8M urea 2mercaptoethanol (0.010M--0.030M Na2HPO4) and adjusted to pH 6.3 with H3PO4. After the column had been equilibrated with the first buffer the specimen was applied (250 mg) and the column eluted with 800 ml of each buffer using an automatic scanning ultrograd device to create a linear gradient. The effluent was monitored at 280 nm and fractions of 12 ml collected at a flow rate of 60 ml per h. The fractions were pooled as indicated by bars. l and 2, proteins other than myoglobins; 3, deamidated myoglobin (see text); 4, myoglobin.

389 and 4). Fig. 2 shows the tryptic and peptic fingerprints from peak 4. The patterns obtained from peak 3 were essentially the same, with the exception that the tryptic peptides containing residues 78-96, 79-96 and 80-96, respectively, were slightly less positively charged. Analysis of these peptides yielded the same amino acid composition as those found in the fingerprints of peak 4. Thermolysin digestion and fingerprinting of the tryptic peptides residues 79-96 and 80-96 from peaks 3 and 4, respec-

64i7164\6s,34r,3s64j74

32x34

yl53

146.

97i98

76!96

79’96

w’

--

--

pti

65

ELECTRWHCRESIS _ +

ELECTFiU+FXESIS _

Fig. 2. Dideiphis marsupialis (Virginia opossum) myoglobin. Top, fingerprint of the soluble tryptic peptides. Bottom, fingerprint of the peptic peptides derived from the insoluble core left after tryptic digestion. The staining reactions are indicated. 0 is the point of application. The areas which were cut out for purification by electrophoresis at pH 3.5 and pH 9.0 are boxed.

tively, indicate that in those derived from peak 3 residue 81 asparagine had been deamidated to aspartic acid. This can be seen in Fig. 5 where the negatively charged thermolysin peptides residues 79-85 and 80-85 have one more negative charge than those derived from the equivalent peptides eluted at peak 5 (Fig. 1). This was the result of an artefactual deamidation and not the product of an allelic or duplicated gene. The myoglobin was eluted as a single fraction on DEAE-Sephadex column

390 TO3- 106- - ~

) %

30-33--~

136-1:~~

1- 7

107-113 1 0 4 - 1 0 6 ~ ~ 139-~.~ 98"0039~,~ 90 93MEAT~yR117"123 ~,_ ,t~%,~) 12-29 12-29ox

T

r,..__./ pH63 +

8.11~

ELECTROPHOREsIS

7(~

65-69~ 147- 1 5 1 ~ t47- 153

~J___l.._

47_ 5~5

139-142

~

"116-131 c=.

pH 35 --4--ELEEfE)PHORESIS l

"

@77-

w-

89

77-86--0 (~56- 69

~ (~33-

139- 146ox, 40

pH 3.5 __ ELECTI~PHORESIS Fig. 3. Didelphis marsupialis (Virginia opossum) myoglobin. Top, fingerprint of the chymotryptic peptides. • is the point of application. The middle and bottom patterns show the parts of the chymotryptic fingerprint which were cut out and re-electrophoresed at pH 3.5, The staining reactions are

391 chromatography and on subsequent paper electrophoresis moved as a single band. Thus the deamidation probably occurred during the elution of the myoglobin from the paper with a solution containing 3.1 m M K C N and 0.6 m M KaFe(CN)6, or during the preparation of the apomyglobin (using 1 . 5 ~ HC1 in acetone, v/v), or possibly during the CM-cellulose chromatography. A second batch of myoglobin treated identically, yielded approximately only 2 0 ~ of the deaminated fraction instead of the 4 0 ~ obtained in the first batch, which confirms the artefactual nature of this minor fraction. Table I shows the results from the total amino acid analyses of the whole myoglobin molecule after acid hydrolyses at 24, 48 and 72 h. The number of amino TABLE I AMINO ACID ANALYSES OF THE HYDROLYSATES OF DIDELPHIS MARSUPIAL1S MYOGLOBIN Numbers between parentheses represent the nearest integer. Amino acid

Average values from hydrolysates at 24, 48 and 72 h

Aspartic add Threonine Serine Glutamic acid Proline Glycine Alanine Valine Methionine Isoleucine Leueine Tyrosine Phenylalanine Histidine Lysine Tryptophan Arginine

11.8 (12) 3.8 (4)* 6.9 (7)* 22.2 (22) 4.0 (4) 14.9 (15) 14.9 (15) 6.0 (6)** 2.6 (3)* 7.8 (8)** 17.1 (17) 1.0 (1) 8.0 (8) 8.1 (8) 19.2 (19) 1.6 (2)*** 2.0 (2)

* The values for threonine, serine and methionine are those obtained at 24 h. ** The values for valine and isoleucine are those obtained at 72 h. *** Preservation of tryptophan resulted from the use of 4 % thioglycollic acid. acids was 153, the same found in most main chain myoglobins so far investigated. The amino acid sequence of opossum given in Fig. 6 was constructed by overlapping tryptic, peptic and chymotryptic peptides (Figs 2 and 3), from the subtilisin peptides obtained from the chymotryptic peptide residues 15-29 (Fig. 4, top), and the thermolysin peptides derived from the tryptic peptides residues 17-31, 119-133 (Fig. 4, middle), 35-42, 57-63, 148-153 (Fig. 4, bottom), 64-77, 70--77 (Fig. 5, top), 64-69, 64-74, 134-139 (Fig. 5, middle), 78-96, 79-96 and 80-96 (Fig. 5, bottom)*. * Supplementary data to this article are deposited with, and can be obtained from Elsevier Scientific Publishing Co., B.B.A. Data Deposition, P.O. Box 1527, Amsterdam, The Netherlands. Reference should be made to No. BBA/DD/026/37110/400 (1975) pp. 387.

392

I

17-18

0

15-16

‘0 6

19- 27 P ”

c--

pH 65

ELECTROPHOfESIS

_

+

--

pH 63 l

ELECTAOPHORESIS +

Fig. 4. Didelphis marsupialis (Virginia opossum) myoglobin. Top, fingerprint of the subtilisin peptides derived from the chymotryptic peptide residues 15-29. Middle, fingerprint of the thermolysin peptides derived from the tryptic peptides residues 17-31 and 119-133. Bottom, fingerprint of the thermolysin peptides derived from the tryptic peptides 35-39, 57-63 and 148-153. 0 is the point of application. The staining reactions are indicated. Peptides which were separated by re-electrophoresis at nH 3.5 and nH 9.0 are boxed.

393 - 71 70-75 069-71

75 i 77

64-65

76-77

-

pH

3.5 +

ELECTROPHORESIS

134; 137

@l37-I39

0

ARC

’ -

136- 139

--

pH

6.5 .

ELECTRBC%%

-

+

69- 91

Oa HIS

HIS

80788 99-94

92-93

HIS

894-96 79-w PO

0 HIS

HIS

79-95

-m

6.5

+

ELECTKWKRESdS

-

Fig. 5. Didelphis marsupialis (Virginia opossum) myoglobin. Top, fingerprint of the thermolysin peptides derived from the tryptic peptides residues 64-77 and 70-77. Middle, fingerprint of the thermolysin peptides derived from the tryptic peptides 64-69, 6474 and 134-139. Bottom, composite fingerprint showing the thermolysin peptides corresponding to residues 78-96, 79-96 and 80-96 and those derived from the tryptic peptides 79-96 and 80-96 (shown in broken lines) in which residue at position 81 Asn is deamidated to Asp (see text). 0 is the point of application.

Tp Tp Tp Pe Ch Ch Ch ll~ Th Th Th Th Hm,.

Th Th Su Su Su Su Su

Pe Pe Ch Ch Th

Tp Tp Tp Tp

15 Gly Gly Gly

~G1y LystVal G1u~A1aAsp Ile Pro Gly ~G1y Lys Val GlutAla Asp lie Pro Gly ~Val G1u A1a Asp lie Pro Gly tn~ Pro Gly

~Val

tmy m. t 67 Thr Thr Thr

68 Val Val Val

tGly His Pro Glu

tAla

69 70 71 72 73 74 75 76 Leu Thor Ala Leu Gly Asn ]le Leu Leu~Thr Ala Leu Gly Ash Ile Leu Leu Thr Ala Leu Gly Asn~Ile Leu tThr Ala Leu Gly Asn Ile Leu Thr Leu Glu Lys PhetAsp Lys Phe~Lys His LeutLysset Glu Asp Glu ~ t Lys Ala ~ r Glu Asp Leu~Lys Lys HistGly Ala Thr Val Leu~Thr Ala Leu Gly ~n Ile Leu~ Thr LeutGlu Lys Phet tLys His Leu Lys ~ r G1u Asp G1u /~t tys Ala Ser G1u Asp LeutLys tys His G1y Ala Thr Val LeuI ~Lys His Leu Lys ~ r Glu Asp Glu ~t~Lys Ala Ser Glu Asp Leu Lys Lys His Gly Ala Thr Val Leut ThrtLeu GIu Lyst tale Set GIu Asp~teu Lys LyslHis GlYtA1.aThrtVa] LeutThr AlatLeu G]y ~ n t l l e Leu ~Ala Thr ValtLeu Thr Alat Thr Val Leuf tA1a Leu~ ~Val Leu Thr Alat tThr Ala Leu G1y Ash lletLeu

66 Ala Ala Ala

His Gly Gln~Glu Val Leut His Gly Gln GlutVal Leu~ His Gly Gln~ His Gly Glnt

tG,y ~n G~u v,~ Leut

Gly Gln Glu~Yal Leut]le Argt Gly Gln Glu Val Leut Gly Gln Glu~

Gly Gln Glu Val Leu~lle Arg Leu Phe)Lys Gly His Pro Glu

39 40 41 42 43 44 45 46 47 ---,448 49 50 51 52 .--~53~54 55 56 57 58 59 60 61 62 63 64 65 Thr Leu Glu Lys~Phe Asp LystPhe Lys~His Leu Lys~Ser Glu Asp Glu Met Lys~Ala Ser Glu Asp Leu Lys Lys~His Gly Thr Leu Glu LystPhe Asp Lys Phe Lys~ ~A)a Set G1u Asp Leu Lys~Lys~His Gly ~His Gly

tAsn

G1y Gln Glu Val LeutIle Argt

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 LystVal Glu Ala Asp lle Pro Gly His Gly Gin Glu Val Leu lle ArgtLeu Phe Lys~Gly His Pro Glu Lys~Val Glu A1a Asp lie Pro G1y His GIy Gin G1u Val Leu~ ~Leu Phe Lys Gly His Pro Glu Lys~

tG1yLysi

14 Trp Trp Trp

G1y Leu ~ r Asp G1y Glu Trp~Gln Leu...~._..ValLeu~AsnAla Trp Gly Lys Val Glu Ala Asp lle Pro G1y His GIy Leu ~ r Asp Gly G1u Trp G1n~LeuVal Leu~AsnAla Trp G1y Lys~ GI~ Leu SewrAs._pG1y Glu~ ~Trp Gly Lyst GIy Leu ~ r Asp G1y Glu Trp~G?n Leu Val LeutAsn A1a Trp~Gl¥ Lys Val G1u Ala Asp lie Pro Gly His A!~ Trp G!y Lys ,~ib Val Glu AIm A~p .-,Jb ....Ib ...,ib lle ,..,ab G1u A1a Asp lie Pro Gly His tVal Glu Ala ~p lie Pro Gly His ~Val GlutAla Asp lie Pro Gly His

1 ? 3 4 5 6 7 8 9 10 ll 12 13 Gly Leu Ser Asp Gly Glu Trp Gln Leu Val Leu Asn Ala Gly Leu ~ r Asp G/y Glu Trp Gln Leu Val LeufASn Ala ~Ala

Glu Glu Glu Glu

Aia Ala Ala Ala

Glu Glu Glu Glu

Leu Leu Leu Leu

Us Us Us L,ys

Pro Pro Pro Pro

Leu Ala Leu Ala keu Ala LeutAla

Gln Gln Gln Gln

Set Set Set Set

Hts His His His

Ala Ala Ala Ale

Thr UstHts I.yst Thr Lyst Thr Lyst Thr UStHts

t

._. _ ~

tLeu A1a Gin set ,is Alat

lit

lle Gln Val lle GlntVal lle Gin Val lle Gin Val lle GlntVal

lle Gin Ile Gln lie Cdn lle G1n lle Gin tGln

Asp Phet

tAla Ale Met Gly Us]

)Leu Glu Leut

UstAI--)LeT GIu ~..tP,.~gf ~ystAlaL~. GlutL.uP,. ~ t

tAla Met Gly Us t

~His Pro Gly AsPtPhe G1y Gly ASp Ala GlntAla A1a MettG1y tGly Gly ASp Ala GlntAla Ala)~t Gly

Pro ~

_~ ._.

tGI..t.u GIYtP,.GI. Gly tL~" Glyt

t Us ~ L~_tut

Us t L~ t Us t Us His Pro Gly Asp Phe ~ --~ --~GIy ASp .-~ Ale Gln Ala A1a Met Gly Us t Lys His Pro Gly ASp PhetGly Gly ASp Ala Gin Ala Ala ~ttGly Lys Ale LeutGlu Leu PhetArg Asn Asp Met Ala Ala I.ys TyrtUs Glu Leu Gly Phe Gln Gly Us His Pro Gly ASp ~e Gly Gly ASp Ala Gin Ala Ala MettGly Us Ala Leu Glu Leu Phe~Arg Asn Asp VettAla Ala Us TyrtLys Glu Leu Gly Phe~

tser t.,ys ~

Set Set Set Ser Ser Set

tHis Pro G1y Asp Phe GIy GIy Asp A1a Gin A1a A1a Met GIy UstA1a Leu G1u Leu Phe ArgtAsn Asp feet A1a A1a UstT,yr UstG1" Leu Gly Phe Gin Gly tTyr Us Glu Leu Gly Phe Gln Gly

115 fig 117 118 .119 IZO 121 122 123 124 12G 12G 127 128 129 130 131 132 133 134 135 13G 137 138 139 140 141 14Z 143 144 145 1445147 148 149 150 151 15Z 153 -=~

Us t

t~s Gly AS...nHis GIu Ala Glut tGly Asn His Glu Ala GIut t~s Gly Asn His GlutAla Glut

.-~ .--~ .-~ ..~ tSer His Ala Thr Us His@Us Ile Set- Val Gln Phe Leut LystUs Us Gly As, ,is Glu Ala GlutLeu Us ProtLeu Ala Glntser HJsjAla Thr Lyst

Ust

Us lle Ser Val Gln PhetLeuGIu PheiIle Set GIu Ala¢lle tlle Ser Val Gln Phe Leut tlle Set GlutAla lle tAla lle tlle Us Us Lys Gly ASh His Glu Ala Glu l.eu Lys Pro L~tAla GIn Ser HlstAla Thr Us H1stL,ys ll..~eSe._~rVa.) Glntehe Leu Glu Phetlle Set GI- Ala lle tUs Us Lys Gly ASn His Glu Ale Glu LeutUs Pro LeutAlaGln ~r His Ala ~r L,ys.istuslle Ser Val Gln e~tteu Glu ~e t

Asn His ASh His Ash His Asn His

77 78 79 80 81 82 83 84 85 8G 87 88 89 90 91 9Z 93 94 9G 9G 97 98 99 100 101 lOZ 103 104 105 106 107 108 109 110 111 11Z 113 114

UstLys Lys Gly UstUstLys Gly UstUs UstGly tUs Gly

Fig. 6. Myoglobin sequence of Didelphis marsupialis (Virginia opossum). Tp, tryptic peptides; Pe.. peptic peptides; Ch, chymotryptic peptides; Pe, peptic peptides; Th, thermolysin peptides; Su~ subtilisin peptides; ~ enzymic hydrolysis. --~ dansyl-Edman degradation.

Th Th Th Th

Ch Ch Ch

Tp Pe Pe Pe Pe

Tp

Ch Ch Ch Th Th lh Th Th

Tp Xp Tp Tp Pe Pe Pe

396 To complete the amino sequence 70 residues were identified by dansylation or combined dansyl-Edman degradation (Fig. 6). Table II shows the assignment of the side chain amide and acidic groups. TABLE lI A S S I G N M E N T OF SIDE C H A I N A M I D E A N D ACIDIC GROUPS IN OPOSSUM MYOGLOBIN These groups were established from the electrophoretic mobility of small peptides at pH 6.5 by using Offord's equation: mobility is proportioned to charge and to tool. wt -H3, and by direct identification of phenylthiohydantoin (PTH) derivatives. , negatively charged; t, positively charged; N, neutral; Pe, peptic; Ch, chymotryptic; Tp, tryptic; Su, subtilisin ; Th, thermolysin. The sequential numbers of these peptides are given in parentheses. Sequential No. 4 6 8 12 18 20 26 27 38 41 44 52 53 54 59 60 74 81 83 85 91 102 105 109 113 116 122 126 128 136 140 141 148 152

Residue Asp Glu j Gin Asn Glu Asp Gin Glu Glu Glu Asp Glu Asp Glu Glu Asp Asn Asn Glu Glu Gln Gin Glu Glu Gin Gln Asp Asp Gin Glu Asn Asp Glu Gln

(

)Pe (1-6, 1 - 7 ) ; ( - ) C h ( 1 - 7 )

iN) (N) (--) (N) iN) (-) iN) (N) (N) I ~

/

] J

Pe and Ch (8-11) Ch(12-14); (-4) Tp and Pe (12-16) Th and Su (17-18) Su (19-26) Su (25-26) Su (27-29) Th (35-.39) Ch(41-43); (N) Th (4~42) Tp (43~15); (N) Ch (44-46)

(-)Tp(51-56) ( ) Tp (57-62) (N) Th (72-74, 70-75) PTH derivative ( ) Th (80-85) ( ) Th (84-85) (N) Th (89-91) (N) Pe (99-104); ( ÷ ) Ch (98-102) i ) Ch(103-106) ( ) Pe (107-109, 107-110) (N) Pe (110-113) ( t ) P e (114-118) ( ) Th (119-122) PTH derivative ( - ) Th (124-128) ( )Th(134-136, 134-137) PTH derivative PTH derivative ( - ) Th (148-150) (N) Th (151-153)

DISCUSSION

The finding of an artefactual deamidation, which has changed residue 8l asparagine into aspartic acid, during the process of purification of the opossum

397 myoglobin is important because it has been reported that an asparagine followed by glycine in a protein sequence is very susceptible to deamidation [8-10], a Gly-Asn sequence, however, is not so readily deamidated [10-13]. We want to emphasize that deamidation was only detected when the protein was submitted to CM-cellulose column chromatography and not during the previous steps of purification (DEAESephadex chromatography and paper electrophoresis). It has been established in our laboratory [14] that residue 122 of horse and sperm whale myoglobin is aspartic acid and not asparagine as previously reported [15, 16]. Titration of the carboxyl groups of sperm whale and horse myoglobins performed by Jansen [17] and Jansen et al. [18] accounted for the number of carboxyl groups, which would be expected from this allocation of an aspartic acid rather than an asparagine to position 122. In both species the sequence 121-122 is Gly-Asp, and no traces of asparagine were found in the crude myoglobin extracts or at any stage of their purification, in contrast to the findings for the sequence 80-81 (Gly-Asn) of the opossum myoglobin. Although the line of descent of the opossum is not completely known, there were creatures remarkably similar to opossum living during the late Cretaceous (approx. 70 million years ago). If these animals were direct ancestors of the modern opossum, it has been suggested that this species represents a kind of "living fossil" or "immortal" and therefore is a good example to illustrate bradytelic (slow rate) evolution [1]. The interest in the present study was to investigate whether or not the bradytelic evolution found at phenotypic level was paralleled by a correspondingly small number of changes at the molecular level. We were aware of the fact that this is not so when the ~ and fl chains of the opossum haemoglobin are compared with the and fl chains of other extant species which diverged almost at the same time, for example those of human haemoglobin (ref. 2 and Jones, R. T., personal communication). In the case of the myoglobin molecule 15 is the average number of amino acid differences found when the myoglobins of hominoids and cercopithecoids are compared with that of the opossum. This number is smaller than the average found when cebids, prosimians, cetaceans, ungulates and carnivores are compared with the opossum (18.5, 24, 30, 28 and 24, respectively). We know that the average number of differences found between the myoglobins of lineages which diverged during the late Cretaceous is approx. 24. Thus when hominoids and cercopithecoids are compared with the opossum 15 amino acid differences reflect a slow rate of evolution, but when the other lineages are compared this phenomenon disappears. To investigate which of the two lineages, the one for instance leading to man or the one leading to the opossum, was responsible for a slow rate of evolution, a cladogram was constructed and an ancestral therian myoglobin deduced, using all myoglobin sequences available. By this means 22 single point mutations were allocated to the branch leading to man and 16 to that leading to the opossum, since they diverged from their common ancestor. The average values found for the prosimians, cetaceans, ungulates and carnivores were 24.25, 26.33, 28.33 and 20.75, respectively. It might be concluded that the branch leading to the opossum, at least for the evolution of its myoglobin, is indeed bradytelic, a characteristic also shared by the kangaroo. However, we want to point out that the smallest number of single point mutations along a particular lineage of descent was 11, scored by the treeshrew.

398 C o m m o n ancestry between k a n g a r o o a n d o p o s s u m is s u p p o r t e d by the shared residues 74 Ser, 102 G l n a n d 103 Phe. In the m y o g l o b i n s so far investigated these three residues are only f o u n d in the two marsupials. ACKNOWLEDGEMENTS W e are grateful to Professor R. T. Jones o f the Biochemistry D e p a r t m e n t o f the M e d i c a l School, University o f Oregon, Portland, U.S.A. for p r o v i d i n g us with muscle specimens o f o p o s s u m . This w o r k was s u p p o r t e d by a g r a n t from the M u s c u l a r D y s t r o p h y G r o u p o f G r e a t Britain. REFERENCES 1 Simpson, G. G. (1967) The Meaning of Evolution pp, 100-102, Yale University Press, New Haven and London 2 Stenzel, P. (1974) Nature 252, 62-63 3 Air, G. M. and Thompson, E. O. P. (1971) Aust. J. Biol. Sci. 24, 75-95 4 Blomb~ck, B., Blomb~ick, M., Edman, P. and Hessel, B. (1966) Biochim. Biophys. Acta 115, 371-396 5 Edman, P. and Begg, G. (1967) Eur. J. Biochem. 1, 80-91 6 Summers, M. R., Smythers, G. W. and Oroszlan, S. (1973) Anal. Biochem. 53, 624-628 7 Romero-Herrera, A. E. and Lehmann, H. (1974) Proc. R. Soc. London, Ser. B, 186, 249-279 8 Lang, A., Lehmann, H., McCurdy, P. R. and Pierce, L. (1972) Biochim. Biophys. Acta 278, 57-61 9 Ambler, R. P. (1963) Biochem. J. 89, 349-378 10 Shotton, D. M. and Hartley, B. S. (1970) Nature 225, 802-806 11 Braunitzer, G., Gehring-Miiller, R., Hilschmann, N., Hilse, K., Hobom, G., Rudloff, V. and Wittmann-Liebold, B. (1961) Z. Physiol. Chem. 325, 283-286 12 Schroeder, W. A., Shelton, J. R., Shelton, J. B., Cormick, J. and Jones, R. T. (1963) Biochemistry 2, 992-1008 13 Schroeder, W. A., Shelton, J. R., Shelton, J. B., Robertson, B. and Babin, D. R. (1967) Arch. Biochem. Biophys. 120, 1-14 14 Romero-Herrera, A. E. and Lehmann, H. (1974) Biochim. Biophys. Acta 336, 318-323 15 Boulanger, Y., Dautrevaux, M., Han, K. and Biserte, G. (1968) Bull. Soc. Chim. Biol. 50, 16511669 16 Edmundson, A. B. (1965) Nature 205, 883-887 17 Jansen, L. H. M. (1970) Ph.D. thesis, Nijmegen University 18 Jansen, L. H. M., De Bruin, S. H. and Van Os, G. A. J. (1972) Int. J. Peptide Protein Res. 4, 339-342