The myoglobin of the gastropod mollusc Uttorina littorea L

ht. 3. Bioch.,

1970, I, 457-464

457

THE MYOGLOBIN MOLLUSC TOM

OF THE GASTROPOD

LITTORLh’-A

L. KOPPENHEFF’ER

WTTOREA

AND KENNETH

L.

R. H. READ

Biological Science Center, Barton University, Roston, Massachusetts 01215, U.S.A., and New England Aquarium, Centi Wha$ Boston, Massachusetts 021 IO, U.S.A. (Raciocd

IO

NOD.,1963)

ABSTRACT I. The radular muscle myoglobin of L&a&a littorcohas bun purified. 2. The myoglobin has an apparent molecular weight of 30,000 and is cleaved into monomeric subunita by phydroxymercuribenzoate (PMB). 3. The monomeric derivative is &ctrophoretically homogeneous on acrylatnide gels. 4 Removal of PMR from the monomer rmulu in partial dimerization.. 5. The amino-add composition of the monomer is Lys,, His,, Arg,, Asp,,, Thr,, Ser,,, Glul,, Pro,, Glym AL +GR, V&s Ma,, na,, km Tyru fie,,s Trp,. 6. The myoglobinofthe congeneric speciesL&o&a Mylifar has an apparent molecular weight of 30,000. 7. Li#minclliuwaomyoglobin is compared with other gastropod myoglobins.

THE raduiar muscle myoglobins of the pro&ranch gastropod& Bwycon (Read, 1g66, unaktum (Terwiuigcr 1967, 196% Band Read, xg6g), and Fusitriw oregonutsis (Tcrwilliger and Read, Ig7ob), arc dimeric and have molccuiar weights ranging from 3 1,400 to 35,4oo. Littork littweu myoglobin has an apparent molecular weight of 30,ouo; this value decreases upon dilution of the protein, suggesting that the molecule is dimeric and capable of dkociation into monomeric subunits (Read, 1968). This paper reports the purification and amino-acid composition of these subunits. MATERIALS AND METHODS G2NItR.U The prosobranch mesogastropod L&o&a litbrsoL~collectedalongthenorthemcoaatof Mamachusetts in twelve doaen lota and transportedtoRostonwheretheradularmuacle5were excised and placed under carbon monoxide at O’C. Thcmyoglobinwaa ahaaedbvprindine themusdaiiti mortar with sand and-a smail amount ofbH 7-o buffer 0’2 M in NaCl contatnina ~rxg.N~~,.H,O~o.t52g.N~,.t~O perlitre. The macuatedti86uewascentri&galat 17,000 rev. per minute and the r&due dincarded. The myoglobin-comaining supematant was

treated with carbon monoxide and succekvdy broughtto4opercentand6opercentsaturation with ammonium sulphatc. At each stage the colourlas non-myoglobin-amtaining precipitate wasspundownanddiscardal. Matofthemyoglobin precipitated between 60 aud 7o per cent immorhn -aulphate saturation and. w-as spun down and redissolvedin ,H 71’0buffer. Gel filtration wad peifomiedon a column of Sephadex c-75 in equilibrium withhpH7.0 buffer and calibrated with Bugcon m myoglobin (mol. wt. 3x,400), chymotqkogen A (mol. wt 25, I cm), and sperm whale metmyoglobin (mol. wt. I 7,816). Ion-exchange chromatography wasperhonnedoncolumnsofDEAE(dicthylaminoethyl)-Sephadat A-50 in e&librium with oo8 M ammo&m bicarbonate -and CM (carboxymethvlkdlulose eouilibrated with 0’0 I M sodium phosphate buffer at’pH 6.0. fitkkna a&t$ra Lamarck was collected near La Parguera, Puerto Rico, and flown to Roston where the myoglobin was exuxted and fractionated in the manner ducribed above. Gel filtration dthe myoglobin was performed on a cohtmn of %nhadcr G-75 L%merfb~. Ali opcrati& ‘dckribed’ above, except for removaloftheraduhumuscles,werecarriedout at 4” C. Amino-acid analyses were puformed according to the method of Moore, Spa&man, and Stein (1958) with a Reckman Spinco Modd I 2oC amino-acid analyser. Duplicate I-mg. samplea of lyophiliaed protein were hydroiysed in sealed

458

KOPPENHRFFER

evacuated glass tubes with 6 N hydrochloric acid for 24, 48, and 72 hours at I 10’ C. Following hydrolysis the HQ was removed by rotary evaporation and the samples redissolved in 3-5 ml. ofpH 2-2 citrate buffer. Cysteinc was determined as cysteic acid (Hirs, I 956) and the tryptophan content obtained by the spectrophotometric method of Goodwin and Morton (xg46). Prior to tryptophan determination haem was removed from the rxotcin by the method of Teale (1959).

a

Vduma

AND

READ

Znt. 3. Biochem.

of Raemeyer and Huehns (x967). The PMB derivative of the myoglobin was obtained by treating the protein with an approximate lofold excess of PMB and reacting it for 12 hours at 4” C. PMB was removed from the myoglobin by a methodsimilartothatofTyuma,Benuch,and Ben& ( xg66). Twenty ml. of a 2-mcrcaptoethanol solution (1.6 ml. n-mercaptoethanol brought to 20 ml. with fiH 7.4, WI M phosphate bu&r)wereappliazltoax-8xgocm.columnof

of column afXwnr

(ml.)

FIG. I .-Chromatography of ammonium sulphate fractionated myoglobin on a column of Sephadex (0) andat54onm. G-75. Cobnnn volume 224 cc; flow-rate 18 ml. per hour. Abmrbanceat28onm.

(A). Disk-gclcl~wascMiedoutaccord-

ing to the procedure ofDavis (1964). The myo-

globii was made 25 per cent in bufkrcd sucrose Wore layering it on the top3 of the gels. G& were stained with Amid0 black. hC!‘lON

wn’xi

~-HYDRO~YYE~OA~~

A soiution of sodium p-hydroaymcnsuriantoate (PMB) was prqrcd aWniing to the procedure

ScpbadaG25intxpilibriumwitha~ phosphate (O-I M) aminoimin~

acid(oqM)bt&Tardjuacdto#H7q. The b&rwarprrvioulyxrubbedwith~~~d treedwithcarbonxnon~ F tion ofthe 2-lneruptoeth8Qol the--nEz; PMB wasaddcdandthecolumndevdopedslowlyina cnvironmcp+ ThedluWionofthe -ted nmwlsabout 14hours.

1970, 1

MYOGLOBW

OF GASTROPOD

RESULTS

k3CBDUBE Following ammoniupl sulphate fiactiona-

hJlUFICATION

tion Littorina iittor6a myoglobin was chromatographed on a column of Sephadcx G-75. The cluting carbonmonoxymyoglobin exhibited maxima at 420, 538, and 570 nm. The &action cofiesponding to the bar in Fig. I was concentrated by dialysis against solid suaose and dialysed against 0~18 M ammonium bicarbonate. This material was

Volume

459

MOLLUSC

gave riseto a cbromatogram similar to that in Fig. 2. Accordingly, in fkther purikations chromatography on CM-cellulose was Omittd CIAAVAF~I~~REGENERATION

OFTIIE

uncool

Material which had been cbromatographed on DEAE-Scphadex was reacted with PMB and chromatographed on a a&mm of Sephadcx G-75. The myoglobin &ted as a large peak in a position corresponding to a molecular weight of about

of column

dlucnr

(ml.)

FIG. 2.-Chromatography of material corresponding to the bar in Fig. I on a column of DEAEScphadcx A-50. Column volume 6o c.c.; Aow-rate M ml. per hour; bufIkr 0.08 M ammonium bicarbonate. Absorbance at 280 nm. (0) and at 540 run. (A) ; 0, Ratio between absorbancu at 280 and 540-m chromatographed on a column of DEAESephadex and appears in Fig. 2 as a single peak In pr&&ary expuiments the myoglobin was then chromatographed on a column of CM-cellulose in equilibrium with pH 6.0, o-ox M sodium phosphate butkr. Under these conditions the myoglobin was not retarded and the eluting material had a ratio of absorbances at 280 and 540 run. similar to that obtained after chromatography on DEAE-Sephadex. Rechromatography of the myoglobin on DEAE-Sephadcx

17,ooc+x8,ooo and a very small peak in the 3o,ooo-35,000 molecular weight range (Fig. 3). Electrophoresis of the monomeric derivative at pH 8.8 resulted in the appearance of a single component. Regeneration of the dimer was attempted by removing the PMB from a portion of the monomeric derivative. The reagent-f& material was chromatographed on a column of Sephadex G-75 (Fig. 4). Although some material elutes in the range of the monomer, Fig. 4 resembles the elution pattern of native

460

KOPPRNHEFFER

myoglobin in Fig. I, suggesting that much matuial has been reconverted to the dimeric state. hdINO-ACID

hALYSIS

The monomeric derivative was prepared for amino-acid analysis by removing the

ht. J. Biochm.

AND READ

Littohna anguiajka Ammonium sulphate fractionated myoglobin from L&nina angulifcr was chromatographed on a c&mm of Sephadex G-75 (Superfine) (Fig. 5). The apparent molecular weight of about 30,ooo is slightly less than that of Burgcon cum&d&m myoglobin.

H

aBusycon dimrric myogiobin (Mol. Wt. 3LSoo)

Sperm whale mcrmyogiobm (Mol. Wt. 17.816)

0

Volum* of column ttluenc (ml.)

3.-Chromatography of myoglobin on a column of Sephadex G-75 following treatment with PMB, Eqerirnental conditions were ickntieal to those described in Fig. I. Absorbance at ~&ML (0) d at 540 -. (A). FIG.

PMB fkom the protein. After reaction under nitrogen with an excess of 2-mercaptoethanol for several hours at room tenqerature, the protein was dialysed exhaLUtively against O-qM ammonium bicarbonate bef’ lyophilization. The amino-acid composition of themonomcr,calculatedonthebasisoftwo residues of hi&dine per peptide chain, is presented in Tab& I.

DISCUSSION GFJVERAL L&urinu li#orca myoglobin was judged pure on the basis of a constant ratio of absorbance3 at 280 and 590 nm. obtained after chromatography on DEALSephadu A-50, as well as by the appearance of the monomeric de&ative as a single band on dkk g& The apvce of a single compqwnt suggese

MYOOLOBIN OF GASTROPOD

1970, 1

461

MOLLUSC Sperm whale metmyglobin (Mol. Wt. 17.816)

0.02 -

E’ r 7 8

0.01 -

x ‘0 5

\ I 140 Volume

of column effluent

I 160

760

(ml.)

FIG. ~-Chromatography of the monomkc derivative on a column of Sephadcx C-75 afb? removal of PMB from the protein. Expcrimmtal conditions were identical to thw described in Fig. I. V , A~bance at 418 IUII. Burpn 020

conollculotum

Sperm whale mnmyoglobin

myoglobin

I 0.15

% % H

0.10

d b i; <

0.05

2 < 6

I

o-02-

&h,

40-

80

60 Volume

of column

loo

120

I40

+Rhnt (ml.)

FIG. 5.-Chromatography of ammonium nalphate hctionated myaglobia from Litrorino ungui$h on a column ofSephadci G-75 (Supertine). Cal umn volume I7ac.c.; flow-mc7ml. pcrhour. Aborbanceat&onm.(0)andat~mn. (A).

Int.3. Biochtm.

KOPPEN?%EFFzRANDnJ%AD

462

that the. dimer is composed of identical subUIlitS. The myoglobin of Littoriau uagulifctcrhas an apparent molecular weight of 30,000 and is probably also a dim=. THE ROLE OF SULPH-HYDRYLGROUPS The dimeric myoglobin of Littkna littma, myoglobins (Johnson, xg6g; Tcrwilligcr and Read, xg6g), is cleaved into subunits by the

as well as that of other prosobranch

cant because it indicates that the subunits are not held together by disulphide bonds. This is in contrast to the dimeric myoglobin of the amphine~ran K&&au ttrnicarawhich can be cleaved by ~Vzthylmaleimidc only after prior treatment with 2-~crcaptoethanol (Tetilligcr and Read, xg7oa). &lBUNfT IDENTITY Mammalian haemoglobin is a tetramer composed of two a and two p chains and

l-able I.-AWNO-ACID comm6nl ONOF

&ttOhl

lit&T&l

~YOGLoBlN

No. OFI&~IDUPS No. OF NEAREJT INTEGER PER 2 bSIDt7RS DETBRyMATIoNs HlsTmNR* PERMONOKER 6 s

9’26fo’24

9

2 7 2t 14 8

7.4: *o-34 x.71fo.04 ‘gz do.9 . + 12-o*:

I2

17’0 io.3 4’95**I9 19’3 f@4 x5.8 f~3 :*8993$i . 4’r2*cPox

16.7+~3 0.91 fVo6 12.xfW3 I.17 II

I7 5 *9 16 6 : 17 I I2

0

I

‘57 Molecularweight

I

f 724747

lFkrorscxprcsscdasitandard-oftbemean.

t Allowing for go pa cent yield after S&ram, Moore, and Bigwaod (1954).

$ Obtained by extrapolation to zero time of hydrolysis. 3 Obtained from values for p-hour hydrolysate. 11Obtained by the method of Goodwin and Morton (1946). 7 Obtained from amino-acid composition; molecular weight of one haan without a ligand is included in this value. sulph-hyd.+attacking reagent PMB; these subunits undergo pa&al dimerization when thcmcrcuTyisrullovuL ThissUggW.sthat the integrity of the sulph-hydryl groups is involved in the maintenance of the normal quattmary str~cturc of Lithnamyoglobin. The fact that the d@neric myoglobins arc cleaved by reaction with PM3 aloneis signifi-

bin& oxygen in a co-operative C&ion. It is thought that the co4qzativity is a result of interactions bclWccn the chains which occur dm+ng the oxygenation process. This is relktcd in the diht cod-Gonal states assumed by oxygenated and dcoxygcnatcd hacmoglobin. The abnormal human haanoglobin, Hb H, consists of 4 identical

/3 chains, kils to change conformation when oxygenated (Puutz and Mazzarclla, I g63), and does not bind oxygen in a co-operative manner (Bencsch, Ranney, Benuch, and Furthermor c,isolatedachains Smith, @I). of human haemoglobin exist as partially dissociated dimcrs which do not bind oxygen co-op~ratively (Tyuma and others, I 966). Co lYWBnON OF &ttO7iM Tabk II.-A~No-AcID littom ARD Fuditon mgonensisMyoa~o~ms AMINO-ACID

463

bfYOGLOBlN OF GASTROPOD MOLLUSC

1970, 1

FUSitTbl -* 16.5 3 3 I 17 x : ‘4’5 2.5 6-5 5 ‘4’5 2’5 ‘3’5 Rcaetlt

Total residues

158.5

Molecular weight

I 7,670

Liuwina littma 9

2

7

2

14 8 I2

17 5 19 16 6 : 17 1 I2 I

I57 * 7474

l No. of residues to nearest haIf integer. Data from Tekvilliger and Read (xg7ob).

In this respect the dimuic gastropod myoglobins are unusual because those investigated appear to consist of identical subunits (Bcucbrum r&u&z, Tcxwilligcr and Read, 1g6g ; and Littorina litforea), yet bind oxygen according to a sigmoid dkociation curve (Tawilligcr, 1g6g). The question arises as to whether non-identity of subunits is an absolute req uircment for co-opcrativity in haemoglobins and myoglobins. At this point the answer is unclear; perhaps more detailed study will show that the dimuic gastropod myoglobins consist of non-identical subunits.

AMINO-ACID

~MPOSI7’ION

determined amino-acid compositions of prosobranch myoglobins have been tabulated and compared elsewhere (Tcrwill&r and Read, Ig7ob). The myoglobin of the mcsogastropod F&ton oregoncnsisis somewhat different from the myoglobins of the nwgastropods Bugam and Buccirmm. Only 3 histidine residues occur pa subunit in myoglobins FWitritOn,while the neogas~pod contain 5-7 residues. There are, of course, other diffkrcnces, but several simikitiu arc striking. All prosobranch myoglobins analyscd prior to this study are rich in lysine (16-5-18 x&dues per chain) and low in arginine (2 or 3 residues). All contain more acid, than glutamic acid aspal-& more alanine than glycine, considerably more leucine than isoleucine, and abdut the same amount of cystcine. Table I.. compares the ‘&no-acid composition of Fushiton orcgoneasis and Littorina &or.. myoglobins. Littorina myogiobin contains g lysine and 7 arginine residues whereas Previously

Fusititon myoglobii

as well as the neogastro-

pod myoglobins listed in TuwiUgcr and Read (Ig7ob), possess many more lysine and fewer arginine residues. Littorina myoglobin also has more glutamic acid than aspartic acid and more glycine than alanine, which again is in contrast to other prosobranch myoglobins. All dimuic gastropod myoglobins, including Littorina, &I far analyzed have at least 3 times as much leucine as isoleucine. Apart fkom this the amino-acid composition of Lit& myoglobin bears little resemblance to other prosobranch myoglobins. ACKNO-EMENTS We thank Dr. Maxim0 Ccramc Vivas and his staff of the Department of Marine sciof the University ofILrt0 Rico for their a&stance. This work was supported by U.S.P.H.S. Rucar~h Grant No. HE-10565 (HEM). REFERENCES BENEEH, R. E., WY, H. M., Bm, R., and SMITH, G. M. (@I), ‘ The chemistry of

the Bohr effect. II. Some propcrdes of hcmoglobin H ‘, 3. biol. Chmz., zz#, sgdi+gctg.

464

KOPPRNHEFFER

B. (~$4, ‘ Disc elecwphoruis II: Method and application to human serum proteins ‘, Ann N.2.-.Acad. Sci., IPI, 4.04-427. GOOD-, T. W., and MORTON,R. A. ( 1946)) ‘The spcctrophotomeuic determination of tvrtkne 2nd trwtonhan in Droteios’. Biochm. ,.

DA-,

AND READ

SCHIUX, E., Mooa~, S., and BIOWOOD.E. J. !$g$s)&lc :~z!z~b&f&~~

T%, F. W. J. (x959), ‘ Cleavage of the haemprotein link by acid mcthylethylk2tone’, Bkhk bk@&. Act29u, 543. I?&?? !??~1g56), ‘ The oxidation of rhoTa~wnuok~, R. (1g6g), unpublished data. nuckmc with perfoxmic acid ‘, 3. biol.Chcar., --andR.w,K.RH.(@g),‘Qurrternary oxg, 611-621. structure of the raduku muscle myoglobin of JO-N, J. P. (rg6g), unpublish&d&ta. Buccinw~ mdatum L.‘, Gmp. Biochm. PhJsial., MOORE,S., SPACXMAN,D. H., and STEIN,W. H. 3x,554(19702), ‘The radul2r muscle myo(x958), ‘ chmmatography of amino aci& on ---__ sulfoluitai DolYstyrene resins. An improved globim of the aDiba mollm K& system ‘, An& &m., 82, I 185-r 190. e Wood, oypbkhitcn sklhn’ Middendorf PERUTZ,M. F., and d, and Mojalia mwaxa Gould ‘, Int. 3. Biocbm., I, L. (x963), ‘ A pmlimimq X-ray analysisof hauno8lobin H ‘, 28x-zgt. Nature,Land.,xgg, 639. - - - - ( Ig7ob), ‘ The zulular muscle myom. K. R. H. CI ~661. ‘ The characterization of globins of the gaatropod mollm, Amaea testuradharmuscle‘~yo~obll from the gastmpcd din& scntwn Erwlxholtz, Ftibiton kamtschatkam mollusc, Bupwn canalindatwn L.‘, Gmp. Bit&m. Jones, l-q&z fibmlis Arlamr, FunXttm ongone& Redfield and l%ak lumellasa Gmelln ‘, Plrlsiol., vr 375-1-390. - - (1g67), ‘ The radul2r muscle myo$3b&of Cmnj~. Bindwm. Ph/siol., in the paws. the gastropod mollusc Buycon car&m * ‘, TYUMA, I., BENESCR, R. E., 2nd BEN==, R. (@6), ‘The prcpaation 2nd proputia of -‘f[~%;-‘~ke myoglobins of the gastxopod isolated a and 8 subunits of hemoglobin A ‘, molluw kyam c&mri2m Omrad, &tat&z Bkhmi.s@, 5,2g57--1g62. kvs Say, fittarka &tore2L. 2nd Siphawrio&as Sow&y ‘, Ibid., 2% 81% Ryz 2 A., “d HU-NS, E. F. (967), Iizy Word Zn&x: Radul2r myoglobin, molhmc, ech2msm of the *aOon of Littotinalittma. haemoglobin ‘, 3. n&k Bill., 25, 253-273. L

.