Camp. Biachem. PhyMoL, 1975, VoL 50A, pp. 51 to 54. Pergamon ,Press. ,Printed in Great Britain

GENERAL CHARACTERIZATION AND CIRCULAR DICHROISM OF BUCCINUM UNDATUM HAEMOCYANIN J. V. RANNISTER,x W. H. BANNISTER,1 M. BRUNORI,z MARIA R. ROSsI-FANELLIa AND G. ROTILIO2 1Department of Physiology and Biochemistry, Royal University of Malta, Msida, Malta; and 2Institute of Biological Chemistry and Centre for Molecular Biology of the National Research Council, University of Rome, Rome, Italy (Receioed 22 October 1973)

Abstract--1. Bucchutm undatum haemocyanin was found to have a sedimentation coefficient, Sa0,w, of 98 S in the undissociated state. Light-scattering measurements indicated: a weight-average molecular weight of 8.6 x 100 daltons for the undissociated molecule; dissociation into subunits between pH 8 and 9; no dissociation in the presence of 1 M NaCI at neutral pH. 2. Circular dichroism spectra showed the bands characteristic of molluscan oxyhaemocyanins above 300 nm with band maxima at 345,475, 570 and ~ 700 nm and signs ( - , + , - , +). 3. Below 300 nm the spectra showed complex positive contributions between 260 and 300 nm and a positive contribution at 252 nm. The 252 nm band increased in relative magnitude on removal of copper and, more strikingly, on deoxygenation. 4. The contributions between 260 and 300 nm were also sensitive to the presence of copper and oxygen.

INTRODUCTION

0.01 M EDTA. Protein concentrations were determined routinely from the absorbance at 280 nm in this buffer to minimize the effect of light scattering (Heirwegh et aL, 1961). The sedimentation coefficient of the haemocyanin was determined in a Beckman Model E analytical ultracentrifuge at 21,740 rev/min at 10~ The determination was carried out at a protein concentration of 1.88 mg/ml in phosphate buffer, pH 7, of 0.1 ionic strength, and the value of S2o,w was calculated in the usual way. Light-scattering measurements were made with a Brice-Phoenix photometer (Briee et al., 1950) at 436 nm in a 3-ml quadrangular cell temperature-controlled at 20~ No corrections for light absorption were applied to the light-scattering measurements since the absorbance of haemocyanin is near a minimum at 436 nm and dilute solutions were used. Clarified solutions for the measurements were prepared by centrifugation of a concentrated stock solution of the protein, which was then added to Millipore-filtered solvent in the light-scattering cell. The specific refractive index increment of the haemocyanin was assumed to be 0.197 ml/g at 436 nm (Van Holde & Cohen, 1964). On this basis the light-scattering constant H in the Debye equation relating the reciprocal specific turbidity (c/x) to the weight-average molecular weight (Mw) of the solute had the value of 10'6 x 10-6. The measurements for calculating Mw were performed in phosphate buffer, pH 7, of 0.i ionic strength at protein concentrations between 0"29 and 1.88 mg/ml. The dissociation behaviour of the haemocyanin as a function of pH was also followed by light-scattering measurements at 436 nm at a protein concentration of 0'25 or 0.29 mg/ml. Light scattering was followed till the attainment of a steady condition after mixing haemocymain and buffer in the light-scattering cell; pH measurements were made on the final solutions.

THE HAEMOCYANINS 8.re copper proteins of high molecular weight which transport oxygen in molluscs and arthropods (Ghiretti, 1962). The subunit structure and the oxygen-binding properties of these proteins are of considerable interest, and thus various haemoeyanirts have been subjected to investigation. W e report here, for comparative purposes, results on the molecular weight and p H stability of haemocyanin from the marine gastropod B u c c i n u m u n d a t u m (L.), and give circular dichroism ( C O ) spectra which h a v e a bearing on copper and oxygen binding by the haemocyanin. MATERIALS AND METHODS Specimens of B. undatum were obtained from Plymou'th, England. Haemolymph was obtained from the whelks as described previously for M u r e x trunculus (Bannister et al., 1966). Haemocyanin was sedimented from the haemolymph by centrifuging at 120,000 g in a Spinco Model L preparative ultracentrifuge for 3 hr at 4~ The pellet was dissolved in 0"1 M acetate buffer, pH 5.7, and spun down again and redissolved in the same buffer. The haemocyanin was stored at 4~ with toluene as preservative. The haemocyanin was shown to contain 0.26~. copper by means of a Unicam SP 90A atomic absorption spectrophotometer. Apohaemocyanin was prepared by the method of Fern~mdez-MorAn et al. (1966). The haemocyanin and apohaemocyanin were shown on the basis of dry weight estimations to have a specific extinction coefficient, ~m% at 280nm of 15.4 and 16.7, lOm, respectively, in 0.05 M borate buffer, pH 9.2, containing 51

52

J.V. BANNISTER,W, H. BANNISTER,M. BRUNORI, MARIAR. RO~I-FANELLIAND O. ROTILIO

Circular dichroism spectra were recorded at 20~ by means of a Cary 60 spectropolarimeter equipped with a 6001 CD attachment. The calibration of the CD attachment was checked with a solution of d-10-camphorsulphonic acid in water. Measurements were made in cells of l-era light path. Molecular ellipttcity was calculated on the basis of a copper unit of 25,000 molecular weight.

Table 1. Visible and near-ultraviolet circular dichroism bands of B, tmdatum oxyhanmocyantn* i

i

L

iiii

)11

inll iiii

.Ill

I

iiiiiiii

ii) 1

Band maximum ~700 570 475 345

RESULTS

General The haemocyanin sedimented as a single symmetrical peak in the ultracentrifuge with a sedimentation coefficient, Sl0,w, of 98 S. The values of Heir for a number of haemocyanin concentrations in lightscattering experiments gave the straight-line plot shown in Fig. 1. The value of He/r for infinite dilution indicated a weight-average molecular weight of 8.6x10 6 daltons. Light scattering indicated stability of whole molecules up to pI-I 7.6-8.0, Full dissociation into subunits occurred above p H 8 and no whole molecules were present at pH 9 (Fig, 2). The kinetics of pI-I-induced dissociation appears to be complex. A large fraction of the dissociation process was too fast to be followed by ordinary mixing methods; however, under some conditions it was observed that complete dissociation was achieved only after many minutes (approximately 30 mini, Dissociation of the haemocyanin was not observed in the presence of 1 M NaC1 at neutral pH, In this respect the haemocyanin resembles the fl-haemo. cyanin of Helix pomatta (Lontie &Wttters, 1966),

iii

Molecular ollipttclty (10a dee emI drool -x) pH 7

pH 9.2

+2,3 - 1.1 + 3.1 -51.8

-- 1.0 + 3.4 -30,3

* Protein concentrations were 0,55 mg/ml (pH 7) and 0.38 mg/ml (pH 9.2) for the near-ultraviolet and 33.8 mg/ml (pH 7) and 23.0 mg/ml (oH 9.2) for th0 visible measurements. Buffers were0.1 M Trls--HCI, pH 7, and 0.05 M borate, pH 9.2, 8 ,oc ~. d ~ r'~ -~ ~o g ~ 2~

7'0

7'5

80

Circular dichroism

fl 5

9'0

pH

Similarly to other molluscan oxyhaemocyanins (Nickerson & Van Holde, 1971; Bannister & Wood, 1972), the CD spectrum in the visible and near ultraviolet showed bands at ~ 700, 570, 475 and 345 nm (Table 1).

~'0

I

' 0 X

Fig, 2. The pH.stability B, undatum haernocyanin as followed by light-scattering measurements. Protein concentration 0.25 mg/ml in Tris-HCl buffer, 0.1 ionic strength (A) or 0,05 M borate buffer (A); protein concentration 0.29 mg/ml in phosphate buffer, 0'1 ionic strength ( 9 or 0.05 M borate buffer (Q).

I

0

,0-----0

I'

..

,0

~

O--

I'0

0 v

I o,5 Protein

I .... I.o

concentration,

t t.5

. . . . . . 2.0

r'ng/rnl

Fig. 1. Plot of the function Hc/-~against protein concentration for light scattering measurements on B. undatumhaemoeyanin.

Buccinum haemo~yanin

Details of ultraviolet CD spectra are given in Figs. 3-5. A complex spectrum was observed in the 260-300 nm region with prominent bands positioned at about 273, 282 and 290 am. Band positions did not vary with p H and, therefore, with. the state of aggregation of the haemocyanin; however, changes in the relative magnitude of the bands were observed on changing the p H (Fig. 3).

53

found in the present work. Above p r i g . 5 the haemocyanin showed a small quantity of 63.8 S species and then dissociated completely into inhomogeneous material with a sedimentation coefficient around 11 S. This is reflected in the sharp profile o f the pH stability diagram revealed by light scattering (Fig. 2). r.._ o. 4 0

I

E I

-

I

I

I

J

g~ \

-~ ~o

~

20

%

~'

;\/ o

4a

.~ o

'~ "~

20

~. -2o2~ ~

I

.......

270

t

-io .ll 250

I sTo Wavelength,

t ,,, 290 nm

I

290

Wavelengfh,

310

nrn

Fig. 4. Ultraviolet circular dichroism spectra of B. ta~datum haemocyanin. Protein concentration was 0.44 mg/ml in 0.1 M acetate buffer, pH 5'7 (the apohaemocyanin buffer contained 2 mM CaC12. Oxyhaemocyanin, ~; apohaemocyanin, - - -. 31o

I

[

I

I

I

I

Fig. 3. Ultraviolet circular dichroism spectra of B. undatum oxyhaemocyantn. Protein concentration was 0.55 mg/ml in 0.1 M Tris-HCl buffer, pH 7 (- ), and 0.38 mg/ml in 0.05 M borate buffer, pH 9.2 (- - -). The various bands were differently sensitive to the binding of copper and oxygen. The 290 nm band was least sensitive to deoxygenation (Fig. 5) and removal of the copper (Fig. 4). The 282 nm band was also insensitive to deoxygenation (Fig. 5) but it increased in relative magnitude in the apohaemocyanin (Fig. 4). The 273 nm band was marked in the deoxyhaemocyanin spectrum (Fig. 5) and appeared as a pronounced shoulder in the apohaemocyanin spectrum (rig. 4). Below the 260-300 nm region the oxyhaemocyanin showed a prominent positive band at about 252 nm. The band increased in relative magnitude in the apohaemocyanin (Fig. 4) and on deoxygenation (Fig. 5), the change in the deoxyhaemocyanin being more marked. DISCUSSION The haemocyanin of B. undatum was investigated by Eriksson-Quensel & Svedberg (1936). They found a single component between pH 5 and 9.5 with an average sedimentation coefficient, S~0,~,, of 102.1 S, which compares with the value of 98 S

~

% 20

~"

\ \

o

Ie' "" s"

..........

=______ _-__.=

o

N -20

~50

I 270

I 290

I 3~0

I 3:50

I 350

I 370

390

Wavelength, nm Fig. 5. Ultraviolet circular dichroism speetra of B. undatum haemocyanin. Protein concentration was 0"86 mg/ml in 0.1 M borate buffer, pH 9.2, containing 0.01 M EDTA. Oxyhaemoeyanin, ....; deoxyhaemocysmin, - - -, containing less than 20% oxyhaemocyanin. The molecular weight of undissociated molluscan haemoeyanins is considered to be near 9 million daltons (Wood et aL, 1971). The value of 8-6• 108 daltons found by light scattering for Buccinum

54

J, V. BANNISTER,W. H. BANNISTER,M. BRUNORI,MARIA R. RO$SI-FANI~LLIAND G, ROTIL10

undatum haemocyanin compares with the value of 8.76 x 106 daltons determined by the same technique for Plla leopoldvillensis haemocyauin by Elliott & Van Baelen (1965). Visible CD studies have amply confirmed the asymmetry of the copper site in oxyhaemocyanin (Van Holde, 1967; Takesada & Hamagnchi, 1968; l',lickerson & Van Holde, 1971 ; Bannister & Wood, 1972) and have revealed significant differences between molluscan and arthropod haemocyanins (Nickerson & Van Holde, 1971). Multiple transitions (see, for example, Table 1) are expected from a copper site of lower-than-cubic symmetry (Bannister & Wood, 1972). The CD in the 260-300 nm region is attributable to transitions of aromatic residues and cystine (Nickerson & Van Holde, 1971; Wood & Dalgleish, 1973). From the evidence of CD spectra conformational changes involving these residues occur on removal of oxygen (Fig. 5) or of copper (Fig, 4). However, it is not possible to say whether the conformational changes occur at or outside the copper and oxygen binding site. A more dramatic change on removal of oxygen or copper is signalled by the CD band at 252 nm (Fig. 4 and 5) which increases in magnitude. This band has been tentatively attributed to histidine residues by Nickerson & Van Holde (1971). It is not a lowlying copper band since it persists and, in fact, increases in relative magnitude on removal of copper. A more striking increase is observed on removal of oxygen (Fig. 5), which suggests a stronger perturbation of the transition by the copper-oxygen complex than by the copper alone. In addition it was found that approximately the same change in CD (at 252 nm) was observed both at neutral pH, where the hemocyanin is undissociated, and at pH above 9, where the molecule is dissociated. This finding suggests that the oxygen-dependent CD change reflects a local structural change, and is in no way related to co-operative effects in the binding of oxygen by hemocyanins. REFERENCES

BANNISTERW. H., BANNISTER.1. V. & MICALLEFH. (1966") Purification of haemocyanin from hemolymph by absorption to calcium phosphate. Experientia 22, 626--627.

BANNISTERW, H. & WOODE. J. (1972) Gausstan analysis of the visible and near-ultraviolet absorption and circular dlchroism spectra of haemocyanin from Murex trtmcuhts. Comp. Biochem, Physiol. 43B, 1033-1037. BRICE B. A., HALWERM. & SPEtSER R. (1950) Photoelectric light-scattering photometer for determining high molecular weights. J. opt. See. Am. 40, 768-778, ELLIOTTF. G. & VANBAELENH. (1965) Poids mol6eulaire et zone de stabilit6 de l'h6moeyanine de Plla leopold. villensis. Bull. Soc. Chim. biol. 47, 1979-1986. ERmSSON-QUENSEL L-B. & SVEDBERO T. (1936) The molecular weights and pH-stability regions of the haemocyanins. Biol. Bull. mar. biol. Lab., Woods Hole 71,498-547. FERN~NDBZ-MOR,/~N I-[., VAN BRLIOOENE. F. J. & Orl~tnr M, (1966) Macromolecular organization of hemoeyanins and apohemocyanins as revealed by electron mierosooypy, d. molec. Biol. 16, 191-207. GmRm'rl F, (1962) Hemerythrin and hemocyanin. In Oxygenases (Edited by HAYAISm O.), pp. 517-553, Academic Press, New York. HEmW~OH M., BOROINON H. & LoN'rm R. (1961) Separation and absorption spectra of cx- and 8haemocyanins of lfellx pomatla. Blochhn. biophys. Aota 48, 517-526. Lorcrm R, & WITr~.RS R. (1966) Helix pomatla homo. eyanins. In The Biochemistry of Copper (Edited by PEISAeHJ., AmENP, & BLUMB~ROW. E.), pp, 455--462. Academic Press, New York. NICKERSONK. W. & VAN HeLen K. E. (1971) A comparison of molluscan and arthropod hemocyanin--I, Circular dlchrolsm and absorption spectra, Comp, Blochem, Physlol, 39B, 855-872. TAK~ADAH. & HAMAOUCmK. (1968) Circular dichroism ofhemocyanln, d. Blochem,, Tokyo 63, 725-729, VAN HELVE K, E. (1967) Physical studies of hemoeyanins--III. Circular dlehrolsm and absorption spectra. Biochemistry 6, 93-99. VAN HOLDEK. E. & COHENL. B. (1964) Physieal studies of laemocyanins--I. Characterization and subunit structure of Lollgo pealet hemoeyanin. Biochemistry 3, 1803-1808. WOOD E. J., BANNISTERW. H., OLIVERC. J., LomaE R. & WrlaXRS R. (1971) Diffusion coefficients, sedimentation coefficients and molecular weights of some gastropod haemocyanins. Comp. Biochem. Physiol. 40B, 19-24. WOOD E. J. & DALGLEISHD. G. (1973) Murex trunculus haemocyanin--2. The oxygenation reaction and circular dichroism. Fur. d. Blochem. 35, 421-427. Key Word Index--Haemocyanin; Buccinum ,mdatum; Mollusca; sedimentation coefficient; molecular weight; pH stability; circular dichroism.