Multiple haemoglobins of mandrills, papio sphinx

ht. 3. Bioch.

322

MULTIPLE VINA

~MOGLOBI~S

OF MANDRILLS,

BUETTNER-JANUSCH,

JOHN

AND GEORGE

PAP10 SPlWVX

BUETTNER-JANUSCH,

A. MASON

Departmentsof Anatomy and Zoology, Duke University, Durham, North Carolina, U.S.A. (Rwiwd 13 Oct.,rg6yt ABSTRACT I. Elcctraphorcsis of red cell hacmolysata of mandrills, Pupio sphin*, indicata that two major haemoglobi are present in this spccics. Thcsehaemoglobinsha~~idcntical @ chains and different a chaii, a* and u’. 2. The amino-acid compositions and amino-terminal groups of the a A, a*, and @ chains were determined. Valine is the amino-terminal group of each of the chains. 3. The preunce of two major hacmoglobins in P. sphinx may be a true genetic poiymorphism or may be the result of either gene duplication or ambiguity in transuiption of the genetic code.

MORE than one major haemoglobin component has been detected in red cell hacmolysatcs from individual, supposedly normal, adult

+

-a

w -I

i

-

Origin

Ofiginr

-(I

W

_

_

m

._

+

I

I

2

3

4

I

of cltctrophoretic patterns of human and mandrill haemogiobins. Abont: Intact hacmoglobins after clcctropi;onsis for 4 houn at room temperature, pH g, Tris-borate buffer, 12 V. pa cm. B&w: .Globins after ckctrcphorcsis for 22 hours, 4” C., )JH 8, txrcsveronal buf%r, IO V. per cm. The a chains migrate towards the negative pole, and the B chains towards the positive pole. 1. Human. 2. Pap& sphinx, unhctionated. 3. Papio sphinx HbB, tiom peak B, Fig. 2. 4. Papia sphinx HbA, Tom peak A, Fig. 2. FIG. I .--Div

animals of many species of non-human primates (Buettner-Janusch and BuettnerJanusch, x964; Bamicot, Huehns, and Jolly, 1966; Barnicot and Jolly, 1966; Kitchen, Eaton, and Stengcr, 1968; Nute, Buettnaand Bucttncr-Janusch, r969). Jan=% Among cercopithecids, multiple haemoglobins have been noted in MrrraGn inu (Bamicot and others, rg66), M. n&u (Nutc, I 968)) M. sfkosa (Kitchen and otbcrs, I 968)) and Papio ~g~cqf~halur [ = urtinzu] (BuettnerJanusch, 1963). In some cases, tbc presence of more than one major haemoglobin is a true genetic polymorphism. Other cases may be explained either by postulating ambiguity in transcription of the genetic code or by postulating duplication and subsequent persistence in the population of the cistron controlling synthesis of one or more of the polypcptide chains of baemogiobin. The functional basis for persistence of more than one haemoglobin in a species has not yet been detcrmincd. We report hert the occurrence of two mjor haemoglobins in mandrills, P+W s$&rx. We have isolated these hacmoglobins and separated them into component polypcptide chains, and we present here the amino-acid compositions and the aminoterminal end-groups of the isolated chains.

bfULTIF%E

1970, I, 322-326

HAFAfOGLOBINS

OF bUNDRILLS

323

METHODS AND RESULTS The elccwphoretic and chromatographic methods are in general the same as those described previously (Bradshaw, Rogers, Hill, and Buettner; Buettner-Janusch, Buettncr-

from a single animal were used. This animal was housed for several yean at the Primate Facility, Duke University, and is presently at the Rare Feline Breeding Compound, Center Hill, Florida, :;1;1: 2 animals are aho housed at the facility

Red cell haanolysata from 3 mandrilla were examined by starch-gel elcctrophorcsis at PH g in Tris-borate buffer (Sullivan and Nute, x968).

The two haemoglObii were readily separated by chromatography on IRC 50 resin (Allen, S&roeder, and Balog, x958). The details of the separation are given in the legend to Fig. 2. The

5.0

2v

1

OS

4D-

*

5

0.4

k :: f 4 0.2 0.1

C

Efflumt Volumr(ml)

2.-Typical cbrozaatofrram of a haemolysate from Pupio sf.~hinr. Appkimately 5oo mg. of hacmoglobin were adsorbed on a 2.5 x 52 cm. column of IRC 50 previously equilibrated at 4’ C. with phosphate buffer, developer No. 2, 2s described by Allen and othen ( 1958). Elution was -carried out at 4” C. and 28” C. as indicated. Fractions of T ml. were collectal at the rate of 6 per hour. T&e solid ban indicate the fractions that were pooled. FIG.

The ekctrophorctic pattems from each animal were essentially identical, and each showed two maior haanotzlobin bands (Fip. I ). We label the phorcsis of globii in a urea-veronal b&r system (chcrnoff and Petit, 1964) indicates that the difkrencca in the haunwlobins are in the a chains and that the B chains a&identical (Fig. I). Thus, ~h?l*'~W"--__Hba,A~, -*VI.

For additional

and

~Bh?~~M*lli=

studies hacmolysata

Effluent Volume (ml) FIG. 3.-Typical chromatogram of globm prypared fkom HbA of Popi sphinx Approximately 5oo mg. of globin were chromatographcd on a 2.5 x55 cm. column of IRC 9, essentially aa dcacribed by Wilson and Smith ( xg5g). Elution warstartcdwith2Murca,~Hr~g. Alinear gradicntto8Murca,pHx~g,wasstartcdatthe point indicated by the f%st arrow. The second arrow indicates the point at which 8 M urea, PH I ‘9, was introduced. Fractions of approximately r2 ml. were collected at the rate of 6-8 per hour. The solid bars indicate the fractions that were pooled.

ratio

of total absorbancy of peak A to total absorbancy of peak B is 55 : 45. This ratio was the same on four chromatograms. Hence the two haanoglobii are present in approximately equal amounts. Starch-gel elcctrophoruis in Tris-borate bufkofaliquots of the material from the two major peaks shown in Fig. 2 and clcctrophorcsis of the globins in urea-veronal buffer indicate that peak A contains Hbcr,*& and B contains HbalB& (Fig. 1). Each of the isolated hacmoglobins was conccntrated (Buettncr-Janus& and othus, I 969)) globin was prepared (Anson and Mirsky, I 930; BuetmerJanusch and others, 1g69), and a and B chains

BUETTNER-JAKUSCH

324

were separated (Wilson and Smith, 1959). A typical chromatogram is shown in Fig. 3. The amino-acid compositions and aminoterminal groups are given in Tabfcs I and II. These values were determined as previously described (S&ram, Moore, and Bigwood, 1954; Bencze and Schmid, x957; Spa&man, Stein, and Moore, 1958; Stark and Smyth, 1963 ; BuettnerJanusch and others, rg6g). The comparable data for Papio cynoccphalucand human haemoglobins are

ET

u* and aa chains of Pa@ sphinr, the data here suggest that a* chain contains one more histidyl residue and one less alanyi residue than a* chain (labb I). The relative chromatoqaphic and clcctrophoretic mobilides of the two intact haemoglobins and the two isolated a chains also indicate that a* is more negatively chaqed than aB. This leads us to speculate that one or more aspartyl or glutamyl residues in the a* chain may be replaced by asparaginyl or glutaminyl residues in

Pajio sphinx a* AMNo-ACID

LySitle HistidiIIC Aqinine Aspanic acid Threonine SCZiIIe Glutamic acid Proline Glycine Alanine ValiXE Methionine IsOlCtIciIIC Lcucine Tyroainc Phcnylalanine Half-cystine Tryptophan

Int. j’. Biochem.

AL..

Papio sphinx a”

lo-Horn hydrolyI& 12.8 10.6 2.8 12’2 7.6 IO’0 5’9 ;:; 16.1 12.5 * ‘5 0 19.0 ;:; -

70-Hour hydrolysis

Average (nearest integer) l

zo-Hour hydrolysis

12.9

*3

12-g

3 12 8 II 6

X3 12’2

z 16 13 2 0 19

;:A 16.8 12.5 1’3 0 19-o

7.8 17.2 x2.8 I.2 0 19.0

2:; -

;:; -

IO.7 3’2 12-O 5’1 6.9 7.8 16.6 12.8 I.2 0

x9.0 ;:“,

I

II

i(7) I

;:; 6.0

70-Hot11 hydrolysis

Average (nearest integer) *

13.0 9.8 3.1 X2.0

13

8:: 8:;

I

Papio _

Homo sajtien.9:

C$ELS+

3 12 8

13 II 3 12 8

II IO 3 12 9

II

II

II

7 7 7 16 13 2 0 19 3 7 I I

5 7 7 21 13 2 0 18 3 7

14*

141

IO

6 ;3 17 13 2 0 19 i(7) I I

I I

-I-

Total NH&=%-teit . . Residues recovered

Iqo (14

140 (141)

Valine

ValinC

0.72

040

Valine

Valine

* Data are expressed as residues per molecule. Integral values for thrc~nine and serine were obtained by extrapolation to zero time of hydrolysis. Integral values for valine arc based on 7o-hour value. t Data from Buettncr-Janusch ( x969). $ Data from Hill, Konigsberg, Guidotti, and Graig (x 962). also presented. In Table II the data reported are for mandrill 8 chain from Hba,a8, . The aminoacid composition of mandrill 8 chain from Hba,*8, is essentially identical. Valine is the amino-tumid residue of each chain. Although detailed structural studies will be ~.tccessary in order to specify homologies between

a*. On the basis of composition alone, haemoglobins from the two species of Papio appear to resemble each other more than either ruanblcs the haemoglobii of man (Tables I, I.). Noteworthy are the amotmts of lysinc and alanine in a chains and the amounts of lysine, argininc, and aspartic acid in 8 chains.

MULTIPLE HAEMOGLOBINS

197% 1 DISCUSSION

Determination of the amino-acid sequences of the polypeptidc chains of mandrill haemoglobins may enable us to decide between the alternatives of gene duplication and ambiguous transcription of the genetic Table II.-A~XNO-ACID Comoam

ON8 AND

OF

MANDRILLS

325

present in all members of the species. In addition to elucidation of the amino-acid sequences of the polypeptide chains, extensive population surveys ofthis species are certainly necessary in order to determine whether Pa+ S$J&LIT is polymorphic at the haemoglobin loci. RESIDUES

N&TERypw.

OF

p cz%uNs

O? HAEMocaosxNs

Papio sphin* Atdmo-ACID no-hour Hydrolysis LySiIX Histidine &inine Aspartic acid ThreoIline Serine Glutamic acid Proline Glycine Alanine V&e Methionine ISOlcucine LeudnC TyrOSiIlc Phenylalanine Halfcystine Trvptophan Total

I I ‘9 8.7

2-o 15.1

6.8 5’3

II’2

6-2

12’2

13’5 15.8 o-5 0 18.0 3’1 7’3

7o-hour Hydrolysis II.9 8.7 2-2 14.6 6.3 4’2 IO-4 6.0 12.1 14’1 17-2 0.6 0 18.0 2.8 7’2 -

Horn0 sa$&ns$

Average (nearest integer)* II 9 2

‘5 : II

6

I2

14 =7 I 0 18 3 7 2 2

13 9 2 15 5 6 I2 6 12 (13) *5 17 I 0 18 3 8 2 2

II 9

3 13 3 ‘I I 7 13 15 18 I 0 x8 8 2 2

144

146 (147)

146

NH,-taminal amino-acid

VaiiIIe

ValiDc

ValiIlc

Residua recovered

0.78

*Data as TableI. t Data from Buettner-Janus& ( x969). $ Data from Hill and 0th~~ (I 962). code as explanations for the presence of two major haemoglobins in Papio sphinx. The data presented here, based as they are on only 3 animals, do not permit us to decide whether these haemoglobins represent a genuine genetic polymorphism in Papio sphinx or whether these haemoglobins are

ACKNOWLZDGE~

The work was supported in pan+ by grants GM 13222 and FR 00388 fmm the U.S. Public Health Service. One of the authors (J. B.-J.)

holds a U.S. Public Health Service Research Career Development Award. We thank Dr. J. A. Bergeron, Duke University, and Mr. R. E. Baudy, Center Hill, Florida, for providing samples.

REFERENCES ALLEN, D. W,, Sotmo~~mt, W. A., and BALES, J.

abnormalities of the polypcptide chains of hcmoglobm ‘, Bw yb 750-756. Hu, R. J., KONIO~ERG, W., Gumorrr, G., and CRUG, I... C. (xg62), ‘ The structure of human hanogiobin. I. The sep2ration of the a and 3 chaixs and their amino acid compcaition ‘, -7.

(x959), ‘0bservati0rls on the chromatogmphic heterogeneity of normal adult and fetal human haemoglobin: a study ofthe effccu ofcrystalhaation and chromatography on the heterogeneity bidchem., !q& I~g-r5$&. . and isolcucine content ‘, 3. Am. them. SOL, KITCIIEN, H.. BATON. T. W.. and S?ENGER.V. G. 80, 1626-1634. ( 1968); ‘ Hemoglob& typ& of adult, fet& and ANsoN, M. L., and M~RSIKY, A. B. (rg3o), ‘Protein ucwborn subhumau primates: Mucucctfi&ti ‘, cuagdation and its rcvcmal. The preparation of A&c/U &o&n. B&$&s*, X2% 227-234. insoluble globin, soluble globin and hcmc ‘, Nrrra, P. E. (xg68), ’ Gctwic and evolutiotuuy J. gnr. P&ioL, 1% .&g-~~& studies of hemoglobins and tran&rins in the BARNICOT,N. A., HUIWNS, E. R,, and Jou;ut C. J. primates’, Doctoral diawrtatiorl, Duke uni(x966), ‘ Biochemical studies on haemoglobin versity. variants of the irus macaque ‘, Proc. R. Sk. B, - - BuwrmwJ.wusui, V., and BUET~NERx65,224-244. JANXIXZ* J, (rg69), ‘ Genetic and biochemical - - and JOLLY, C. J. (rg66), ‘ Naemogkhin studies of transfcrrins and hemoglobins of poiymorphism in the orangutan and au animal CUago ‘, Fob primrrt., ray 2+2a7. with four major haemogiobius ‘, Natire, &n&., SU-IRAM, E;, MOORE, S., and B~cwooo, E. J. 210,64o-642. Chromatographic determination of BEN~ZE, W. L., and Scxutu, R. { 19571, ‘ Deter(‘9541, cyst& as cyst&c acid ‘, Biocfta. J., 57, 33-37. mination of tymsine and tryptophan in pro&‘ACZICMAN, D. H., STEIN, W. H., and MOORE, S. teins ‘, Analyt. Chm., 29, I rg3-x ~6. (x958), ‘ Automatic recording apparatus for use ~IUDSXUW, R. A., ROOEIZS,I.. A., HXU, R. L., in the chromatography of amino acids ‘, An&i. and BtmTrtm2$wuscx, J. (1965)~ ‘ The clwrlt.,34 I rgo-t2o6. amino acid composition and the amino-terminal G. R, and SYYTH, D. G. f&3), ‘The end groups of the a- and $&aim of four lcmur STuse of cyanate for the determination of NH,hemoglobins ‘, AT& Bin&m. Biqhys., xog, 571terminal r&dues in ptoteirx *, J. 6ioI. C&n., 576. @, 214-226. Buarrrm~-JAN~J~CH, J. (rg63), ‘ Hemoglobins and SULLIVAN,B., and NUTE, P. E. ( rg68), ‘ Structural transfcrrinsof baboons ‘, Folk Primat., I, 73-87, and functional proper& of polymorphic - - and BUE~R-JANUBCX, V. (19641, ‘ Hun+ hemoglobins from orangutans ‘, G&s~ Princeglobius of primates *, in Euoi~~ and Genetic km, Ss, 213-124 Bioioe of Primates (ed. Bur.rrmm-Jwcx, J.f, WIISON, s., aud SMrrx, D. B. (Ig5g), ‘ Separation vol. II. D. 7%. New York: Academic Press. of the vaiyi-leucy1 and vaiyl-glutamyi-polypcp- - Bts%-&&-J~mxox, V., and MAsoN, G, A. tide chains of borsc globin by fractional precipi(rgSg), ‘ Amino acid compositions and amiuotation and cohmm chromatography ‘, Can, 3. terminal end groups of a and 6 chains from Biochcm. Physial., 37, 405-416. polymorphic hemoglobins of Pongo #I‘, dnhs &c&m. B&p&., IS 164-170. B~~~T-~NEz-JANusM, V. (tgfjg), unpublished data. C~ZRNOFF,A. I., and PETIT, N. hf., jun. ( t 9641, Key Ward Mex: Haemoglobin, mandrill haemu’ The amino acid composition of hemoglobin, giohius, Papi0 spbk hacmoglobins, primate III. A oualitativc method for idcntif%nn u haemaglobin, multipie hacmoglobins. *