Comp. Biochem. Physiol. Vol. 8811, No. 1, pp. 127-132, 1987 0305-0491/87 $3.00+0.00 Printed in Great Britain © 1987 Pergamon Journals Ltd

HEMOCYANIN OF THE CHITON, STENOPLAX CONSPICUA (DALL)

THEODORE T. HERSKOVITS* and MARY G. HAMILTON From the Department of Chemistry, Fordham University, Bronx, New York 10458, and The Division

of Sciences and Mathematics, New York, NY 10023, USA

(Received 30 October, 1987)

Abstract--1. The hemocyanin of the chiton, Stenoplax conspicua, has a molecular weight determined by light-scattering of 4.2 x 106 daltons, (dt) and a sedimentation coefficient of 60 S.

2. The fully dissociated subunits in 6.0 and 8.0 M urea, and at pH 8.9-10 in the absence of divalent ions, have molecular weights of 4.15-4.30 x 105 and 4.17-4.75 x 105 dt, which is close to one-tenth of the molecular weight of the parent hemocyanin assembly.

3. The pH dependence of the molecular weights from pH 4.5 to 11 exldbit bell-shaped transition profiles, best accounted for by a three-species, decamer to dimer to monomer scheme of subunit dissociation, with one acidic and one basic ionizing group per dimer and 5-8 acidic and basic groups per monomer.

4. In the absence of stabilizing divalent ions S. conspicua hemocyanin is relatively unstable. At pH 7.4 in the presence of 0.01 M EDTA, it is predominantly in the dimeric state, characterized by a sedimentation constant of 18 S. It is also more readily dissociated to monomers at high pHs (8-9 and above) than are the C. stelleri and A. granulata hemocyanins.

5. Urea and GdmCl are effective dissociating agents of S. conspicua hemocyanin. The urea dissociation profile obtained at pH 8.5, 0.01 M Mg 2+, 0.01 M Ca 2+, and analyzed by means of the decamer- dimer-monomer scheme of subunit dissociation gave estimates of about 30 amino acid groups (N, pp) at the dimer contacts within the hemocyanin decamers and about 120 groups per monomer within each dimer, suggesting hydrophobic stabilization of hemocyanin assembly.

INTRODUCTION

The respiratory proteins of the Polyplacophora or chitons, found freely dissolved in the circulating hemolymph are hemocyanins (Svedberg and Hede- nius, 1934; Stewart et al., 1952; Redmond, 1962). A number of investigations in recent years have dealt with the structure, subunit organization and oxygen binding properties of the hemocyanins of the gastro- pod molluscs (for recent reviews see van Holde and Miller, 1982; Ellerton et al., 1983; Mangum, 1980). The hemocyanins of the gastropods are large cylindrical particles, with molecular weights of 8-9 x 106 dt organized typically as two discrete sets of decamers held together by non-covalent interactions. Dissociation induced by pH changes or the use of dissociating reagents proceeds through the formation of half-molecules, and dissociated dimers and mono- mers (Svedberg and Pedersen, 1940; Siezen and van Driel, 1974; Engelborghs and Lontie, 1973; Her- skovits et al., 1984, 1985). The hemocyanins of the cephalopods and chitons possess a simpler, decameric structure characteristic of the basic assembly or "half-molecules" of the more common molluscan species. Their molecular weights are found to be correspondingly lower. Molecular weights of 3.3 x 106 to 3.6 x 106 dt have been reported for the hemocyanins of four different species of octopi (Miller and van Holde, 1982; Herskovits and Vil- lanueva, 1986) and somewhat higher values of

Abbreviations used: Tris, tris (hydroxymethyl) amino- methane; GdmCl, guanidinium chloride; PMSF, phenylmethylsulfonyl fluoride; CD, circular dichroism.

4.2 ___ 0.3 x 106dt were obtained in recent work on three polyplacophoran hemocyanins (Ryan et al., 1985; Herskovits and Hamilton, 1986; Herskovits et al., 1986), that have molecular weights close to one-half of the hemocyanins of the more common land and marine gastropods (Wood et al., 1971; Quitter et al., 1978; Herskovits et al., 1985).

The work reported in this paper on the Californian chiton, Stenoplax conspicua represents a continuation of our physical investigations of the factors and forces which govern the structure and assembly of this group of oxygen-transport proteins. This third polyplacophoran hemocyanin, investigated by light- scattering, ultracentrifugation and spectroscopic techniques, is found to have the same molecular weight of 4 .2+0.3 x l&d t as those of the Acan- thopleura granulata and Cryptochiton stelleri proteins examined in earlier studies (Herskovits et al., 1986; Herskovits and Hamilton, 1986), but is found to be less stable at high pHs and more easily dissociabl¢ to its dimeric form in the absence of stabilizing divalent ions than are the others.

MATERIALS AND METHODS

Live specimens of Stenoplax conspicua were obtained from Pacific Bio-Marine Laboratories, Inc. Venice, CA. The hemolymph was collected, and the hemocyanin was isolated by gel filtration on Bio-Gel A-5m columns in the cold as previously described (Herskovits et aL, 1981, 1986). A fresfily-prepared mixture of protease inhibitors consisting of 0.2% trypsin inhibitor, 0.01 M EDTA, 0.01 M benzamidine, 0.02% sodium axide (Lips et al., 1982) and some added PMSF crystals, was used at the bleeding stages of all the specimens, in order to minimize proteolysis of the hemo-

127

128

Solvent

THEODORE T. HERSKOVITS and MARY G. HAMILTON

Table 1. Macromolecular and spectroscopic parameters of Stenoplax conspicua hemocyanin

(0)22 (0)m,~ 6 E:~ s E ~ M w (deg.cm~/dmol) (dl/g per crn)

pH 7.4, /~ =0.1 Tfis, 0.05MMg 2+, 0.01M Ca 2+ 4.2 x 106 -7300_+300 -32,500_+ 1700 16.3 _+0.2 3.2_+0.2

pH 9.0, /~ =0.1 Tris 4.3 x 105 -7000 -33,000 15.1 3.7

pH 10, /z =0.1 Bicarb/NaOH 4.8 x 105 -6800 -32,400 - - 3.8

2.0 M urea, pH 8.5 0.01 M Mg 2+, 0.01 M Ca :+ 5.8 x l0 s -7700 -36,700 15.9 3.6

8.0 M urea, pH 7.4 0.05 M Mg 2+, 0.01 M Ca 2+ 4.3 x 105 -4000 -34,800 14.4 2.9

1.0 M GdmCl, pH 8.5, 0.01 M Mg 2+, 0.01 M Ca 2+ 7.7 x 10 s -7600 -35,600 15.6 3.7

6.0 M GdmCl, pH 7.4, 0.05 M Mg 2+, 0.01 M Ca 2+ 2.3 x l0 s -1400 -2000 15.3 ~0.5

cyanin. The protein concentrat ion of the hemolymph, mea- sured on four chiton specimens before chromatography was found to be 2.8-3.6%, with the average pH being 7.3.

Protein concentrat ions were measured using a Cary 14 spectrophotometer. The extinction coefficients, E2tT"~ = 16.3 and 14.2 were used at pH 7.4 for the hemocyanin and yellow hemolymph component , respectively. These extinction coefficients were based on refractive index increment mea- surements on dialyzed solutions made in conjunction with absorbance measurements , using the (On/c%)/~ value o f 0.194 g - t . cm 3 (Herskovits et al., 1985). The E27s values used for concentrat ion determination in various solvents are listed in Table 1. The high pH, GdmCI and urea values were based on absorbance measurements on hemocyanin solu- tions diluted from stock solutions o f known protein concen- tration.

Light-scattering and refractive index increment mea- surements were made at 436 n m in a W ood light-scattering photometer as previously described, using both 2.4 x 2.4 cm square cells and cylindrical cells for 90 degree and 35 to 145 degree angular-dependence measurements (Herskovits et al., 1981; Herskovits and Villanueva, 1986).

Circular dichroism measurements were made using a Cary 60 spectropolarimeter equipped with a C D attach- ment.

Sedimentation measurements were made in a Beckman model E analytical ultracentrifuge equipped with both sch- lieren and photoelectric scanner-absorption optics.

RESULTS

Light-scattering molecular weights and character- ization o f S t e n o p l a x c o n s p i c u a hemocyanin

T h e m o l e c u l a r w e i g h t o f t he n a t i v e h e m o c y a n i n a n d t he ye l low h e m o l y m p h c o m p o n e n t were in- v e s t i g a t e d a t p H 7 . 4 in 0.1 M Tr i s buffer , 0.05 M M g ~+, 0.01 M C a 2+. T h e h e m o c y a n i n w a s a l so s t ud - ied a t a lka l ine p H s a n d in t he p r e s e n c e o f d i s s o c i a t i n g a n d d e n a t u r i n g so lven t s , 6 - 8 M u r e a a n d 6 M G d m C l . T a b l e 2 p r e s e n t s t h e m o l e c u l a r we igh t s , Mw a n d t he s e c o n d vir ial coeff ic ient B de r ived f r o m the c o n c e n t r a t i o n d e p e n d e n c e o f t he l i g h t - s c a t t e r i n g da t a . I t is a p p a r e n t f r o m the m o l e c u l a r w e i g h t d a t a t h a t in n o n - d e n a t u r i n g so lven t s a t p H 8.9 a n d above , a n d in 6 a n d 8 M u r e a S. eonspicua h e m o c y a n i n is ful ly d i s soc ia t ed , w i th m o l e c u l a r w e i g h t s c lose to o n e - t e n t h o f t h e p a r e n t h e m o c y a n i n a s s e m b l y . T h e a b s o r b a n c e a n d c i r cu la r d i c h r o i s m b a n d s a t 346 a n d 222 n m a s s o c i a t e d w i t h o x y g e n b i n d i n g a n d t he in- t egr i ty o f t he fo lded d o m a i n s o f t he h e m o c y a n i n c h a i n s a re la rge ly p r e s e r v e d in t he se so lven t s (Tab l e 1 a n d Fig. 1). T h e h e m o c y a n i n c h a i n s a re fu l ly

u n f o l d e d in 6 M G d m C l as s u g g e s t e d by t he dec rease in a m p l i t u d e o f t he 222 n m p e p t i d e b a n d as well a s

Table 2. Light scattering molecular weight data of Stenoplax conspicua hemocyanin and its yellow hemolymph component

Protein concentration (~n/Oc)~ B a

Solvent range (g/L) (cm3/g) M w (L/tool per g2)

Hemocyanin pH 7.4,/z =0.1 Tris,

0.05M Mg 3+, 0.05M Ca 2+ 0.5-5.1 0.194 4.2+0.30 × 106'b 6 .9 -40 x 10 -t°'b

pH 8.9, # = 0.1 Tris, 0.01M EDTA 0.09-0.56 0.200 4.17 x 105 - 3 x 10 -7

pH 9.0,/z = 0.1 Tris 0.25-1.0 0.200 4.29 + 0.12 x 105 - 1 x 10 -7

pH 10, ~ =0.1 Bicarb/NaOH 0.09-1.0 0.204 4.75 + 0.03 x 105 - 2 x 10 -7

6.0 M urea, pH 7.4, 0.05M Mg 3+, 0.01M Ca 2+ 0.05-0.6 0.161 4.15_+0.13 x 105 - 1 x 10 -6

8.0 M urea, pH 7.4, 0.05 M Mg 3+, 0.01 M Ca 2+ 0.09-1.0 0.141 4.30 ± 0.30 x 105,c - 1 × 10 -6,~

6.0 M GdmCl, pH 7.4, 0.05M Mg ~+, 0.0M Ca 2+ 0.2-t.2 0.141 2.3 x 105 ~0

Yellow Hemolymph Component pH 7.4,/~ = 0.1 Tris,

0.05M Mg 2+, 0.01 M Ca 2+ 0.2-1.2 0.194 4.3 × 105 2 × 10 -9

~Error estimates of mol. wts based on two or three independent determinations. ~02 bParameters based on the data fit with K, , ,~ = 5 x 10-" M ( (Herskovits and Hamilton, 1986)

CParameters based on fit of the linear e = 0.05 to 0.3 g/L range of the K'c/R o vs c of the data.

Stenoplax conspicuus hemocyanin 129

u)

'Q

*a s^

1 cl < Urea I -13.0

2.5

2.0

1.5

1.0

,^

-0.5 h 3.0 2

-7

-6 2.5

-5 2.0

-4 1.5

-3

-2 1.0

-I 0.5

0 I 2 3 4 5 6

Reagent cone. WI

Fig. 1. Comparison of the effects of subunit dissociation (M,) and unfolding [(O),,] of S. conspicua hemocyanin produced by increasing urea (A) and GdmCl (B) concen- tration, reflected by the changes in light-scattering mol. wts and C.D. spectra at 222 nm. pH and ionic conditions given

in Table 1.

the 346 nm copper absorbance band. In addition there is a decrease in molecular weight to 2.3 x 10’ dt, suggesting some degradation or breaks in the folded polypeptide chains, similar to that of C. stelleri and A. granulata hemocyanins (Herskovits et al., 1986; Herskovits and Hamilton, 1986).

Reversibility from the fully dissociated state at high pH or in 8 M urea was found to be extensive but incomplete. From initial values of molecular weights of 4.20 x 10’ to 4.75 x 10’ dt obtained at pH 8.9 and 0.01 M EDTA, pH 10, and in 8.0 M urea (Table 2), substantially higher values of the molecular weights of 2.52 x 106, 2.70 x 106, and 2.71 x 106dt were ob- tained (c = 0.27, 0.32 and 0.27 g. L-l), following the removal of the dissociating solvents by dialysis against pH 8.5 Tris buffer containing 0.05 M Mg*+ and 0.01 M Ca’+.

Effects of pH, urea and other dissociating reagents on the subunit structure

Figures 2 and 3 show the influence of pH and urea on the stability of the S. conspicua hemocyanin assembly, followed by light-scattering molecular weight measurements M, at low hemocyanin concen- tration of 0.1 or 0.2 g. L-l. The effects of pH and urea concentration were analyzed using the molecular weight expression

M, = 4.2 x lo6 (1.0 - 0.8 a2 - 0.1 ala*) (1)

together with the pairs of equations 2, 3 and 4, 5, giving estimates of the weight fractions, a2 and a, of hemocyanin decamers which dissociate to dimers and the fraction of dimers which form monomers (Her-

skovits and Villanueva, 1986; Herskovits et al., 1986)

x [l + a,/10-pK1]5NI.[1 + 10-pK2/aH]5N*

(2)

4 -= (1 - 4

.[l + aH/10-PK3]2N3

x [l + 10-P”/a,]2Nb (3)

and

a: ~ = 3.2 x 1O-4 (M,,)4 Kfvq.ipp

(1 - a2) c4(1 - a$

(4)

These equations were derived for fitting a three- species sequential scheme of decamer to dimer to monomer dissociation. In equations 2-4, K~~~pp and K?,‘,pp are the apparent dissociation constants of the hemocyanin decamers dissociating to dimers and the dimeric intermediates dissociating to monomers. The four sets of pKi and Ni terms refer to the pK and the apparent number of ionizing or protonating groups on the acidic and alkaline sides of the dissociation

‘f Q * i

2.5

’ I 1 I I I I I 5 6 7 6 9 IO

PH

Fig. 2. Effects of pH on the mol. wt (M,) of S. conspicua hemocyanin investigated in the presence of 0.01 M Mg*+ (a); the absence of stabilizing divalent ions (b); and the presence of 0.01 M EDTA (c). Solid lines drawn through the data points represent fitting based on the decamer- dimer-monomer scheme of subunit dissociation given by eqs. l-3; the dashed curve represents the decamer to mono- mer scheme of dissociation based on eqs. 5 and 6 of Herskovits et 01. (1986) Curve a, K$&,= 4 x 10enM4, Kt;.‘,,=l x 10-‘OM, pK,=4.5, pK,=9.4, pK,=3.5, pK,= 10.2, N, =N, = 1, N,=N,=8; curve b, Kz&= 4 x 1O-28 M4, K& = 1 x lo-*M, pK, =4.0, pK,= 8.0, pK, = 3.5, pK, = 9.0, N, = N, = 1, N, = N4 = 8; curve c, a, = 1 .O, K$, = 1 x lo-’ M, pK, = 3.5; pK, = 9.0, N, = N, = 8; curve d, K$&, = 1 x lo-” M9, pK = 4.5, pK = 9.4, m = 10, N, = N2 = 1. For all the calculations M,, = 4.2 x lo6 was employed; for curves a and d, c = 0.2 g/L, while for c~rws b and c, c = 0.1 g/L, buffers p = 0.1 (Herskovits et

al., 1985a).

C.S.P.(B) 88,1--1

130 "I't-mODOge T. HERSKOVlTS and MAgV G. HAMILTON

o K

3.5 ~ (0) 3.0

2.5-- ~ e

l I b"~, ' . . /~ 2.O ~.5-_ "--. o,,,,,

1.0 - - * ' ~ a

0 O" 0..5 1,0--

0 8 Hcl ~

0.4

0.2

o 0.5 I.o I.s z.o z.,~ Urea cone. (Col

Fig. 3. Effects of urea concentration on the molecular weight, Mw(A) and the species distribution (B) of S. con- spicua hemocyanin, at pH 8.5, 0.01 M Mg 2+ and 0.01 M Ca 2+. The dissociation data were fitted using eqs. 1, 4 and 5 of the text and eqs. 5 to 8 of Herskovits and Villanueva (1986). (A) Curve a, decamer to dimer dissociation,

10 2 - 3 1 5 10 2 . K ~ . ~ = 5 x 10 M, N;~; =30, curve b, decamer to 0 l 66 9 101 monomer dissociation, l ~ v p = 5 × 10- M, N,, =30;

O 2 curve c, decamer--dimer--rnonomer dissociation, ~.~ '~pp = --31 4 2 1 - 1 3 1 0 2 2 1 ' 5 × 10 M , K~.a~ v = 1 × 10 M, Na,i; = 27, Nazi, = 120.

For all calculations Mi0 =4.2 x 106, K s = 0.032 M -I and c = 0.1 g/L were used. (B) Species distribution of decamers (He10), dimers (He2) and monomers (He0 were calculated as previously described (Herskovits and Russell, 1984;

Herskovits et al., 1984).

curves (see Fig. 2), Ml0 is the molecular weight of the hemocyanin decamer, here 4.2 x 106dt, N 1°'2 and " ' a p p

N2~ are the apparent numbers of urea binding sites at contact areas of each dimer which become exposed to solvent as a result of dissociation, and the number of monomer binding sites in each dimer, respectively; co is the urea concentration, and KB is the binding or interaction constant of the urea with the average amino acid group at the contact areas of the subunits taken as 0.032M -1 (Herskovits et al., 1978). The legends of Figs 2 and 3 list the best fit parameters used to generate the different curves drawn through the experimental data, together with some further information required. Curves based on the three- species scheme of subunit dissociation are drawn with solid lines while those based on the two-species decamer to monomer or deeamer to dimer schemes are drawn with dashed or dotted lines.

The sedimentation data of Figs 4 and 5 indicate that 20 S dimeric species are present in the transition regions produced by both pH changes and the pres- ence of urea. A shift from 20 S to 11 S has been interpreted by van Holde and Cohen (1964) in terms of rapidly equilibrating species of dimers and mono- mers shifting to predominantly monomeric species at high pH. Dissociation at high pH or the presence of 1-2 M urea or GdmCl is not accompanied by any

significant changes in the folded domains of the hemocyanin chains housing the oxygen binding cop- per atoms. This is suggested by the absence of any pronounced changes in the CD spectra at 222 nm and 346 nm (Fig. 1, Table 1).

DISCUSSION

The hemocyanins of the four Polyplacophora that have been investigated to date, including that of the chiton, S. conspicua of the present study, are found to have the same molecular weight of 4.2-t-0.3 x 106dt (Ryan et al., 1985; Herskovits and Hamilton, 1986; Herskovits et al., 1986). These he- mocyanins of different species represent four families of chitons (Abbott, 1974; Smith and Mackenzie, 1948) designated as Acanthochitonidae (C. stelleri), Chitonidae ( A. granulata ), Ischnochitonidae ( S. con- spicua), and Mopalididae (K. tunicata). Light- scattering measurements on the fully dissociated sub- units in 6-8 M urea and in alkaline solutions give similar molecular weights of about one-tenth of the parent hemocyanins. For S. conspicua hemocyanin the molecular weights listed in Table 2 range from 4.15x105 to 4 .30x105dt in 6 -8M urea and 4 .17x l05 to 4.75 x 105dt at pH's 8.9-10.0. It is apparent from these measurements that the parent hemocyanin assemblies are decamers.

Another common feature of the chiton hemo- cyanin decamers shared by the S. conspicua, C. stelleri and A. granulata proteins investigated by light-scattering is that the titration of a single amino acid group per dimer or monomer is capable of destabilizing the decameric assembly, thus causing dissociation of the subunits. In the presence of 0.01 M Mg 2÷ ions, the apparent pK-values of these groups, based on the three-species, decamer to dimer to monomer scheme of subunit dissociation, are 4.5 and 9.4 for S. conspicua hemocyanin, while for the hemo- cyanins of C. stelleri and A. granulata the corre- sponding pK values are 5.5 and 8.8 and 5.5 and 9.9, respectively (Herskovits and Hamilton, 1986; Her- skovits et al., 1986). The three-species scheme of pH dissociation requires the further ionization or de- protonation of 5-8 groups at the acidic and alkaline ends of the pH transitions with pK-values ranging from 3.5 to 5.0 and from 10 to 10.2 (see Fig. 2).

Stenoplax conspicua hemocyanin is the least stable of the three chiton hemocyanins we have examined so far. The sedimentation constant of 18 S obtained at pH 7.4 in the presence of 0.01 M EDTA (Fig. 4, tracing b) signifies dissociation of the decameric assembly. The molecular weights of 4.17 x 105 and 4.29 x 105 dt obtained at pH 8.9 and 9.0 (Table 2) suggest also an instability of the dimeric inter- mediates at relatively low alkalinity, reminiscent of the behavior of some of the octopi hemocyanins (Miller and van Holde, 1982; Herskovits and Vil- lanueva, 1986). In contrast, stable decamers were observed in the pH 5.0 to 8.5 region with C. stelleri hemocyanin in divalent ion-free solvents in both the presence and absence of EDTA. For the most stable hemocyanin of A. granulata, complete dissociation to monomers was found to be incomplete even at pH 10, with an observed molecular weight of 6.3 x 105 dt. For this hemocyanin a higher pH of 10.7 and 0.01 M

Stenoplax conspicuus hemocyanin 131

(a)

b

C

d

b)

I A Fig. 4. Effects of pH, urea and Mg 2+ ion concentration on the sedimentation patterns of S. conspicua hemocyanin. (A) Effects ofpH and Mg2+: (a) pH 7.4; (b) pH 7.4, 0.01 M EDTA; (c) pH 8.5, 3 x 10 -3 M Mg2+; (d) pH 8.5. (B) Effects of 0-2.0 M urea, pH 8.5, 0.01 M Mg 2+, 0.01 M CaZ+: (e) 0 M urea; (f) 1.2 M urea; (g) 1.6 M urea; (h) 2.0 M urea. Rotor speed 34,000 rpm; hemocyanin concentration, 3-4 g/L,

different solvent conditions attained by dialysis.

60

50

40

30

20

I0

0

d

60

50

40

30

20

I0

0

_- , - . -%-~ , .] ( a

i - " ~ u ~ " e ~ e ~ "

I I 6 7

I 0.5

I I I 1 8 9 I0 pH

22 L I i 1 i

t .o 15 2 .0 2 .5 3.0 Urea conc.(M )

Fig. 5. Diagram of the sedimentation coefficients (s20.,) vs pH (A) and urea concentration (B) of S. conspicua hemo- cyanin. Data points given by solid symbols represent the major sedimenting component. O, 0 - data in absence of divalent ions; A--0.01 M EDTA; <>, O-1.3 x 10 -3 M Mg2+; D, 1-0-2.0 M urea, pH 8.5, 0.01M Mg 2+, 0.01M

Ca 2+"

EDTA was required to produce complete dis- sociation to the monomer with a molecular weight of 4.6 x 10 sdt (Herskovits et al., 1986).

Both our sedimentation and light-scattering data suggest that the dissociation reaction is a two-step process. Dimeric species with sedimentation con- stants close to 20 S are observed in the ultracentrifuge in the presence of urea and the absence of stabilizing divalent ions (see Fig. 4, tracings b, d and g-h and Fig. 5). In more quantitative terms the dissociation of the decameric assembly of S. conspicua is best de- scribed in terms of the two-step, three-species scheme of subunit dissociation given by equations 1-5, used to characterize the dissociation behavior of both the octopi and two other chiton hemocyanins (Her- skovits and Villanueva, 1986; Herskovits and Ham- ilton, 1986; Herskovits et al., 1986). The common theme of relatively few ionizing or proton binding groups and the much larger number of urea binding sites at the contact areas of the subunits is perhaps our most significant observation on the poly- placophoran, A. granulata, C. stelleri and the present, S. conspicua hemocyanins. Hydrophobic stabilization of both the dimer to dimer contacts of the subunits in the decamers, and the monomer to monomer contacts within the constituent dimers are clearly implicated among both the cephalopdan and poly- placophoran hemocyanin assemblies. This has also

132 THEODORE T. HERSKOVFrS and MARY G. HAMILTON

been suggested for the stabilization o f the side-to-side contacts within the decamers o f the more complex di-decameric and tri-decameric assemblies o f the he- mocyanins of the land and marine gastropods, Helix pomatia, Littorina littorea and Lunatia heros (Her- skovits and Russell, 1984; Herskovits et al., 1985).

Acknowledgements--Supported in part by a Faculty Re- search Grant from Fordham University and Grant RR- 07150 from the National Institutes of Health, U.S. Public Health Service.

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