Tissue haemoglobins from the gastropod molluscs lunatia heros say and busycon canaliculatum L.

146

Int. J. Biochem., I 97 I,

TISSUE

HAEMOGLOBINS

LIj,lATIA

HEROS

FROM

THE

SAY AND BUSYC0.N JAN

2.

[Scientechnica

GASTROPOD

Ltd.]

MOLLUSCS

CANALICC’LATL-M

L.

P. JOHNSON

Biological Science Center, Boston University, Boston, Massachusetts AND

(Publishers)

KENNETH

R.

022

I 5,

U.S..%

H. READ

Biological Science Center, Boston University, Boston, Massachusetts, 022 15, U.S.-A., New England Aquarium, Boston, Massachusetts 021 IO, U.S.A., and Department of Mollusks, Museum of Comparative Zoology, Cambridge, IMassachusetts, 02 I 38, U.S..4 (Received 12 Feb., 1970) ABSTRACT I. Busycon canaliculatumnerve haemoglobin and heart and radular muscle myoglobins have the same or very similar molecular weights (about 32,000) and absorption spectra; the corresponding globins have identical electrophoretic mobilities in 8 M urea. 2. The native Busyconcanaliculatumhaemoglobins are dimeric and are cleaved to monomeric subunits by reaction p-hydroxymercuribenzoate. 3. Lunatia heros nerve haemoglobin and radular muscle myoglobin have the same or very similar molecular weights (about 15,000) and absorption spectra, and identical electrophoretic mobilities. 4. It is probable that Busyconcanaliculatumand Lunatia heros possess only a single speciesspecific type of haemoglobin in their tissues. IN many species of molluscs haemoglobin occurs in several different tissues (Read, rg66). It has yet to be established whether only one or several different haemoglobins are involved. Manwell (1968, 1964) has stated that myoglobins from Busycon canaliculatum heart and odontophore have the same affinity for oxygen, and that the heart and radular muscle myoglobins of Busycon canaliculatum and Busycon caricum are electrophoretically identical. Wittenberg, Brown, and Wittenberg (I 965) and Wittenberg, Wittenberg, and Valenstein ( 1965) have Stolzberg, established that Aplysia californica nerve and muscle myoglobins have identical (and somewhat unusual) spectra in solution and insitu. Wittenberg, Briehl, and Wittenberg (1965) have found close similarities between absorption spectra and molecular weights of the nerve haemoglobin of Aplysia californica and a muscle haemoglobin (myoglobin) of Aplysia

depilans (Rossi-Fanelli and Antonini, 1957 ; Rossi-Fanelli, An tonini, and Povoledo, I 958). There does, however, appear to be a slight difference in the oxygen affinity of the two pigments. This paper presents evidence that nerve haemoglobin and heart and radular muscle myoglobins in the gastropod Busycon canaliculatum are identical, and that a similar situation exists for nerve and radular muscle pigments in the gastropod Luuatia heros. MATERIALS AND METHODS Busycon canaliculatum L. and Lunatia heros Say were purchased from the Marine Biological Laboratories, Woods Hole, Mass. Radular and heart muscle were ground separately in a mortar with sand and 0.1 A4 sodium phosphate, pH 7.4. The crude extracts were fractionated with ammonium sulphate. The deep red precipitates of myoglobin were redissolved in extraction buffer and chromatographed on a column of Sephadex G-75 (superfine) in equilibrium with the same buffer. Haemoglobin from circumoesophageal ganglia

lg71,2, 146-152

GASTROPOD MOLLUSC HAEMOGLOBINS

and attached nerve was purified in the same way. except that the initial grinding was performed in a glass homogenizer. By way of additional purification the Busycon canaliculatumpreparations, which had been chro-

‘47 RESULTS

ABSORPTION SPECTRA

Table I indicates of the tissue

matographed on Sephadex G-75, were reacted with p-hydroxymercuribenzoate (PMB) as previously described (Johnson, Koppenheffer, and Read, rg7 I). Reaction with PMB is known to split the dimeric radular muscle myoglobin to its monomeric subunits. The PMB-derivatives of the nerve haemoglobin and heart and radular muscle myoglobins were then rechromatographed on Sephadex G-75 to free the pigments from impurities and determine the molecular weight of the derivatives. The removal of PMB from the PMB-derivatives, the subsequent removal of the haem groups, and

the

absorption

maxima

of Busycon canalicu-

haemoglobins

latum and Lunatia heros. Lunatia heros nerve haemoglobin was isolated as a mixture of reduced and oxidized forms. Therefore, for comparison of absorption properties, both the radular myoglobin and nerve haemoglobin of Lunatia heros were completely oxidized with potassium ferricyanide. MOLECULAR WEIGHT Gel

filtration

of partially

canaliculatum haemoglobins

Busycon

purified

on Sephadex

G-7 5

Table Z.-ABSORPTION PEAKS PEAK (nm.) HAEMOGLOBINSOURCE

FORM a

-_ Busyconcanaliculatum Radula Heart Nerve Radula Heart Nerve

Lunatia heros Radula Nerve

oxy

5%

ox,

577-576 577-576 577-576

537 537 537-538 541-542 541-542 541-542

-

507-508

396

-

505-510

396

5% 5%

OXY Carbonmonoxy Carbonmonoxy Carbonmonoxy

Ferricvanide oxidized Ferricvanide oxidized

electrophoresis of the resulting globins in 8 M urea were accomplished as previously described (Johnson and others, 1971). Lunatia heros nerve haemoglobin and radular muscle myoglobin, purified by ammonium sulphate fractionation and chromatography on no additional purificaSephadex G-75, required tion to give a single band on gel electrophoresis. The heart of Lunatia heros contained too little myoglobin for isolation by these methods. Prior to electrophoresis Lunatia heros haemoglobin and myoglobin were converted to the met form by addition of a j-fold molar excess (ferricyanide to haem) of 0.01 M K,Fe(CN),. Electrophoresis was performed in a system similar to that of Davies (rg64), but was modified for our particular proteins along the lines suggested by Williams and Reisfeld (1964). Beta-alanine was substituted for glycine as the trailing ion, and the initial pH values of the sample and running gels were raised to 7.1 and 9.2, respectively.

(Fig. heart

Soret

P

418 418 418 4’4-415 414-4’5 414-4’5

I) shows that nerve haemoglobin and

molecular

radular

muscle

weights of about 32,000.

of these preparations molecules

into

the molecular i.e., about

with

subunits

weight

16,000

PMB of

have

Reaction cleaves

about

the

one-half

of the native pigments,

(Fig. 2).

Lunatia heros, whose radular globin

is a monomer

I 4,800

(Read,

liger, and Read,

and

myoglobins

muscle

of molecular

1968 ; Koppenheffer,

myoweight

Terwil-

I 97 I ) , has nerve haemoglobin

of similar molecular

weight

(Fig. 3).

ELECTROPHORESIS

Busycon canaliculatum Globins from the various Busycon canaliculaturn haemoglobins give only a single band

ht. J. Biochem.

JOHNSON AND READ

148

DISCUSSION

when electrophoresed in 8 M urea at PH 2.7, either separately or when all three are combined in equimolar amounts (Fig. 4A).

The similar spectra, molecular weights, and behaviour on reaction with PMB of the

Monomeric PMB derwative of radular Mb Burycon radular

Mb dime

Buryton radular

Mb dlmer.

0.4

g 0.3 z 2 0.2 B b B u 0.1

Volume

of column

effluenr

(ml.)

FIG. I .-Sephadex G-75 chromatography, in 0.1 M sodium phosphate, pH 7.4, of Busgcon canalicudatumhaemoglobins. A, Nerve haemoglobin, sample volume 15 ml. B, Heart myoglobin, sample volume 15 ml. Note that the elution volume ‘of radular muscle myoglobin is presented as a reference point. Column dimensions 1.9 x g8 cm. Flow-rate IO ml. per hour. Lunatia heros After oxidation to the met form, Lunatia heros nerve haemoglobin and radular muscle myoglobin show identical electrophoretic mobilities, and give only a single benzidinepositive band when run together in equimolar amounts. The gels in Fig. 46 are stained with Amido schwarz. The minor bands are benzidine-negative and indicative of nonhaemoglobin-containing impurities.

nerve, heart, and radular muscle haemoglobins of Busycon canaliculatum indicate a certain likeness among these proteins, as do the spectra and molecular weights of nerve and radular muscle haemoglobin from Lunatia heros. The similarities must be extremely close, for electrophoresis of equimolar quantities of combined Lunatia heros nerve and muscle haemoglobin, or equimolar mixtures of Busycon canaliculatum nerve, heart, and

GASTROPOD

‘97’3 2

MOLLUSC

radular muscle globins (in 8 lZ1 urea) gives only a single band in each case. Although it is tempting to conclude that each species has only a single type of haemoglobin, Braunitzer, Buse, and Braig ( 1969) have reported that a crystallizable haemoglobin fraction from

HAEMOGLOBINS

‘49

the midge Chihironomus thummi, previously thought to be homogeneous on the basis of ionexchange chromatography and acrylamidegel electrophoresis, actually consists of at least z very similar haemoglobins which differ by 2 uncharged amino-acid residues.

6 0.12 -

0.10 6usycooradulardimer I I

0.04 -

0.02 -

& 50

I 100 IS0 Volume of column effluent (ml.)

200

FIG. I.-Sephadex G-75 chromatography, in 0. I M sodium phosphate, @H 7.4, of Busyconcanaliculatum haemoglobins from Fig. I after reaction with PMB in the same buffer. A: Nerve haemoglobin. Reaction concentrations were 0.019 mM haem, 123 mM PMB, in a final volume of 14 ml. 6, Heart myoglobin. Reaction concentrations were 0.014 mM haem, 1.4 mM PMB, in a final volume of 18 ml. Column dimensions I ‘9 x g8 cm. Flow-rate IO ml. per hour.

ijo

JOHNSON

AND

The presence of only a single band on electrophoresis of the Busycon canaliculatum giobins in 8 .Gf urea demonstrates that the native nerve and heart haemoglobins are true

READ

Itit.

3.

B&hem.

of haemo~lobin. The more usual case is exemplified in insects by the multiple, genetically determined haemoglobins found in individual larvae of various species of

0. A 0.

i E

2 9

0.

,” s

0”

a0, 0.

Volume

of column

effluent

(ml.)

Fro. 3.--Sephadex. G-75 chromatography, in 0.1 M sodium phosphate, pH 7.4, of partially purified Lunatia hems haemoglobin. A, Radula myoglabin. B. Nerve haemogiobin. Sample volumes 5 ml. Column dimensions I .g x 98 cm. Flow-rate IO ml. per hour. dimers composed of identical subunits (subject to the above reservation), as has been reported for the raduIar myoglobin (Johnson and others, 1971). Busycon canaliculatum and Lunatics heros are unusual in probably having only a single type

Chironomus (Thompson and English, I g66), and in the bivalve molluscs by the 2 or 3 major haemogtobins found in the blood of individual specimens of Anadara trapezia (Nicol and Ogower, 1967). OR the other hand, the radular muscle myoglobin of the

1971,

GASTROPOD MOLLUSC HAEMOGLOBINS

2

gastropod A&A (limacina ?) consists of only 1 component (Tentori, Vivaldi, Carta, 4ntonini, and Brunori, 1968). Other results which indicate that the nerve haemoglobin and radular muscle myoglobin of Aplysta are extremely similar (Wittenberg, Briehl, and W’ittenberg, 1965; Wittenberg, Wittenberg, and others, 1965; Wittenberg, Brown, and Wittenberg, 1965) make it appear probable that Aplysia also contains only a single type of tissue haemoglobin.

found globins

effective (Benesch

‘5’ with vertebrate haemoand Benesch, 1969).

ACKNOWLEDGEMENT This

work

Research Grant

was supported No. HE 10565

by U.S.P.H.S. (HEM).

REFERENCES

RENESCH, R.. and BENESCH, R. E. (I 969)) ‘ Intracellular

organic

phosphates

as

regulators

of

B FIG. 4.-A, Acrylamide-gel electrophoresis. at pH 2.7, in 8 M urea, of Busycon canaliculatum globins: 1, 20 l.tg. radular globin. 3, 20 ug. heart globin. 5, 20 ug. nerve globin. 7, 20 pg. each of radular, heart:

B, Acrylamide-gel electrophoresis at pH 9.2 of and nerve globins. Stained with Amido schwarz. Luruztia heros methemoblogins. 2, IO pg. radular myoglobin. 4, IO pg. nerve haemoglobin. 5, 5 pg. radular myoglobin plus 5 pg. nerve haemoglobin. Stained with Amido schwarz.

It is probable that a given species of molecule will perform essentially the same function whatever its location in the body. Thus nerve and muscle haemo2lobins nrobably both perform the same function in gastropods. However, it seems quite possible that the oxygen equilibrium and associationdissociation kinetics could be modified to suit 1

differing

requirements

ent tissues through

the

slightly

interactions

molecules

functionally

analogous

of differ-

with to

small those

oxygen release by haemoglobin ‘, Jlr’ature, Lond., 221, 618-622. BRAUNITZER,G., BUSE, G., and BRAIG, S. (1g6g), ’ Polygene Structur der Insekten-Himoglobine ‘, Naturwisscnschaften, ~6. 2 I =,. DAVIES, B. J. - (Ii&, ‘-Disc electrophoresis, part II ‘, Ann. N.?‘“. Acad. &i., 121, 404-427. JOHNSON, J. P., KOPPENHEFFER,T. L., and READ, K. R. H. (1971), ‘ Identical subunits of dimeric myoglobin from the eastropod mollusc Busvcon canal&datum L. ‘, Znt. 3. B&hem., 2, 65-71, _ KOPPENHEFFER,T. L., TERWILLIGER,R. C., and READ,K. R.H. (1g71), ’ Myoglobinofthegastropod mollusc Lunatia heros Sayc’, Ibid., 2, I I I--I 16.

JOHNSONAND

152

MAXWELL, C. (rg6o), ‘ Histological specificity of respiratory pigments. I. Comparison of the coelom and muscle haemoglobins of the polychaete worm Trauisia pupa and the echiuroid worm Arhynchite pugettensis ‘, Comp. B&hem. Physiol., 1. 267-276. -L (1<63j, ‘The chemistry and biology of haemoplobin in some marine clams. I. Distribution ofThe pigment and properties of the oxygen equilibrium ‘, Ibid., 8, 209-2 18. - - (rg64), ‘ Chemistry, genetics, and function of invertebrate respiratory pigments--configurational changes and allosteric effects ‘, in Oxygen in the Animal Organism (ed. DICKENS,F., and NEIL, E.), pp. 49-119. New York: Macmillan (Pergamon)

.

NICOL,P. I., and OGOWER,A. K. (1g67), ‘ Haemoglobin variation in Anadara trapezia ‘, .Nature, Lond., 2x6, 684.

READ, K. R. H. (rg66), ‘ Molluscan haemoglobin and myoglobin ’ , in Physiology of Mollusca (ed. WILBUR, K. M., and YONGE, C. M.), vol. II, pp. 209-232. New York: Academic Press. - - (1g68), ‘ The myoglobins of the gastropod molluscs Busycon contra&urn Conrad, Lunatia heros Say, Littorina littorea L., and Siphonaria gigas Sowerby ‘, Camp. B&hem. Physiol., 25,

molluscs ‘, in Symposium on Protein Structure (ed. A.), pp. 14.4-147. London : Methuen. TENTORI.L.. VIVALDI. G.. CARTA. S.. ANTONINI. E., and BRUNORI,‘ M. ‘ ( 1968); ‘ Amino-acid composition of Aplysia myoglobin ‘, Nature, Lond., NEUBERGER,

228,

487.

THOMPSON,P. E., and ENGLISH,D. S. (1966), ‘ Multiplicity of haemoglobins in the genus Chironomus ( Tendipes) ‘, Science, N. 2”., 152, 75-76. D. E., and REISFELD,R. A. ( 1g64), ‘ Disc electrophoresis in polyacrylamide gels:

WILLIAMS,

extension to new conditions of pH and buffer ‘, Ann. J’v’.Y. Acad. Sci., 121, 373-381. WITTENBERG. B. A.. BRIEHL.R. W.. and WITTENBERG, J. B: (IgS;), ‘ Hae’moglobins of invertebrate tissues: nerve haemoglobins of Aphrodite, Aplysia, and Halosydna ‘, Biochem. j., g&363-37 I. WITTENBERG,J. B., STOLZBERG,S., and VALENSTEIN,E. (rg63), ‘ A novel reaction of haemoglobin in invertebrate tissues. II. Observations on molluscan muscle ‘, Biochim. biophys. Acta, 2%

530-535.

J. B., BROWN,P. K., and WITTENBERG,B. A. (Ig65), ‘ A novel reaction of haemoglobin in invertebrate nerves, I. Observations on annelid and molluscan nerves ‘, Biochim.

WITTENBERG,

biophys. Acta, 128, 5 18-529.

81-94.

ROSSI-FANELLI, A., and ANTONINI,E. (rg37), ‘ A new tyne of mvoalobin isolated and crystallized from ‘ ihe m&&s of ApEysiae ‘, Biochemistry, Leningrad, 22, 3 I 2-32 I. - - - - and POVOLEDO,D. (I g58), ‘ Further study on myoglobin. II. Chemical and biochemical properties of a new type of myoglobin in

READ

Key Word Index: Haemoglobin, molecular weight protein, Busycon canaliculatum, myoglobin, Lunatia heros, mollusc haemoglobins, nerve haemo-

of

globins.