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Immunochemical properties of the haemocyanins from Buccinum undatum (L.) and Neptunea antiqua (L.)
Comp. Biochem. Physiol., 1975, Vol. 52B, pp. 219 to 225. Pergamon Press. Printed in Great Britain
IMMUNOCHEMICAL PROPERTIES OF THE HAEMOCYANINS FROM BUCCINUM UNDATUM (L.) A N D NEPTUNEA ANTIQUA (L.) E. J. WOOD Department of Biochemistry, University of Leeds, 9 Hyde Terrace, Leeds LS2 9LS, England (Received 8 July 1974)
Abstract--1. Rabbit antiserum to Buccinum undatum haemocyanin cross-reacted with Neptunea antiqua haemocyanin and vice versa, the reaction in each case being one of partial identity. Antiserum to B. undaturn haemocyanin also cross-reacted weakly with Colus gracilis haemocyanin, and very weakly with Murex trunculus haemocyanin. 2. Depending upon the pH and the Ca 2+ or Mg 2÷ concentrations, the reaction of the haemocyanins with their rabbit antisera resulted in the formation of single or multiple precipitin lines in agar and different equivalence points in the quantitative precipitin assay. 3. Analytical ultracentrifugation and electrophoretic analysis showed that this phenomenon was caused by pH-induced dissociation of the haemocyanins, both whole molecules and their dissociation products being capable of reacting with antisera. 4. An interpretation of the patterns observed in immunoelectrophoresis is given based on the time taken for haemocyanin molecules to dissociate at certain pH values, and the electrophoretic mobilities and diffusion coefficients of the dissociation products.
INTRODUCTION
HAEMOCYANINS from arthropods and molluscs have frequently been used in immunological studies. Some workers have investigated phylogenic relationships by means of cross-reactions (Boyd, 1937; Denuc6 & Cushing, 1963; Malley etal., 1965), while others have been concerned with studying the nature of the immune response (Weigle, 1964; Dixon et al., 1966). In the latter case haemocyanins were used because of their high antigenic potency and because they are easily available in comparatively large quantities. Also it is possible to study the primary immune response since laboratory animals are unlikely to have encountered the antigens previously. Because haemocyanins are very large multimeric protein molecules, great caution is needed in interpreting the results of experiments in which they have been allowed to react with antisera against them. The size of the molecules in relation to that of immunoglobulin molecules means that a small weight of antibody protein can precipitate a very large weight of antigen protein (Weigle, 1964). The fact that raising the pH slightly causes the haemocyanin molecules to dissociate partly or wholly into smaller sub-units having the same or additional antigenic determinants (Bartel & Campbell, 1959), can lead to the situation where a single pure antigen produces several precitin lines in agar against its antiserum (Tournabene & Bartel, 1962; Weigle, 1964). This situation is complicated by other factors. The extent of dissociation is influenced not only by pH but also by calcium and magnesium ion concentrations. Some agars contain considerable amounts of these ions (Wood et al., 1968). The rate of dissociation of haemocyanins may be slow (Wood, 1973). Under some conditions dissociation requires days to come to completion, so that experiments performed immediately after preparing haemocyanin solutions
may give different results to experiments performed with the same solutions 2-3 days later. Finally, the mobilities of the products of dissociation are different so that patterns obtained in immunoelectrophoresis can be very complex. Clearly the interpretation of the immunological reactions of haemocyanins with antisera demand a full understanding of the physicochemical properties of the haemocyanins. In the present work the reactions between the haemocyanins from Buceinum undatum (L.) and Neptunea antiqua (L.) and their rabbit antisera have been investigated. METHODS
H aemoc yanins
Haemolymph was obtained from Buceinum undatum, Neptunea antiqua, and other whelks, and usually the haemolymph from several animals was pooled. The haemocyanin was purified and concentrated by preparative ultracentrifugation (Wood & Peacocke, 1973). The purity of the preparations was checked in the analytical ultracentrifuge, and haemocyanin concentrations were determined from their absorbance at 278nm (Wood, 1973). Preparation of antisera Antisera were raised in rabbits by injecting sterilized haemocyanin solution in 0'85~o saline intradermally into the back. Usually six injections were given at 2-3 day intervals, of either 1 mg or 10 mg haemocyanin. The saline was at about physiological pH and therefore the haemocyanin would be in the undissociated state at the time of injection. Test bleedings were taken from the marginal ear vein after each injection except the first. Two to three weeks after the sixth injection the rabbits were killed and exsahguinated. The serum was separated in the normal way.
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E.J. WOOD
Immunological reactions Radial diffusion analysis was performed in 0-75 or 1~o agar gel (Oxoid Ltd.) made up in a suitable buffe~'. The choice of buffer in all cases was governed by the state in which it was desired to have the haemocyanin e.g. whole molecules (sedimentation coefficient, 100S), partially dissociated molecules (wholes, halves, and tenths), or completely dissociated molecules (tenths, sedimentation coefficient, ~ 16S) (see Wood & Peacocke, 1973; Wood, 1973). Several arrangements of wells were tried out in order to obtain clear precipitin arcs so that cross-reactions could be seen easily. Because of the great difference in size of antibody molecule and antigen molecule, the precipitin arcs often fell very close to the perimeter of the antigen well. This is because the rate of movement of proteins through the gel depends upon the diffusion coefficients of the proteins (see Allison & Humphrey, 1960). Immunoelectrophoresis was performed in 0'75 or l'0yo agar on microscope slides, as described previously (Wood et at., 1968).
Quantitative precipitin reaction The quantitative precipitin reaction was performed by mixing equal volumes (normally 0'5 ml.) of diluted antiserum and haemocyanin solution, all dilutions being done in a buffer at the appropriate pH so that the haemocyanin was in the associated or dissociated state as desired. (Weigle (1964) reported that performing the quantitative precipitation assay at different pH values in the range pH 6-%8.8 had little effect on the results obtained with the system BSA-anti BSA). The tubes were allowed to stand for 2 hr at 37 ° after which they were placed in the refrigerator overnight. The precipitates were then spun down, washed, and taken up in 0"5 N NaOH. The total protein in the precipitate was measured by the absorbance at 280 nm. The amount of haemocyanin in the precipitate was in some cases estimated from the copper content determined by atomic absorption spectrophotometry.
Analytical ultracentrifugation Analytical ultracentrifugation (sedimentation velocity) was performed with a Beckman model E instrument equipped with a schlieren optical system and a rotor temperature indicating and control system (see Wood, 1973).
Electrophoresis Electrophoresis on cellulose acetate membranes was performed in the usual way using Oxoid strips and a Shandon-Kohn apparatus. The Tris-EDTA borate buffer used was prepared by mixing appropriate quantities of two solutions: A" 0.14 M boric acid containing 0"02 M EDTA and B: 0"14 M Tris containing 0"02 M EDTA, to give the desired pH. Electrophoresis was performed for up to 1 hr at 2 mA per strip and then the strips were fixed and stained in 0"2~ (w/v) Ponceau S containing 3~o (w/v) trichloroacetic acid. After drying, the strips were cleared with Shell Ondina 17 oil and scanned in a densitometer. RESULTS
The state of the haemocyanin The dissociation state of the haemocyanin was determined by analytical ultracentrifugation from the
number of boundaries appearing and their sedimentation coefficients. Whole or native molecules (100S, molecular weight 9 x 106), one-half molecules (60S), and one-tenth molecules (16S) were the main species encountered (Wood, 1973). A similar picture emerged from cellulose acetate membrane electrophoresis and it seemed likely that under the conditions used the larger molecules had the higher charges, and moved faster.
Production of antibodies The injection into rabbits of repeated doses of 10 mg ofB. undatum or N. antiqua haemocyanin proved to be a powerful antigenic stimulus. Antibodies to haemocyanin were detected in the serum by agar diffusion after the third injection. Injection of repeated doses of 1 mg. of the haemocyanin also led to the production of antibodies. The antibody titre (equivalence point) of the final bleeding in the quantitative precipitin test was in this case considerably less than when 10 mg. doses of haemocyanin had been injected (see below, and Fig. 5).
Cross-reactions between different haemocyanins A rabbit antiserum to B. undatum haemocyanin cross-reacted in agar with N. antiqua haemocyanin, and to a much lesser extent with Colus gracilis haemocyanin. Similarly the antiserum to N. antiqua haemocyanin cross-reacted strongly with B. undatum haemocyanin and weakly with C. gracilis haemocyanin (Fig. la). At pH 7.3 where there is no dissociation of the haemocyanins, single precipitin arcs were observed. The cross-reactions appeared to be those of partial identity. At a higher pH value (pH 8'3, Fig. lb) the same crossreactions occurred, but the precipitin lines were closer to the centre-well and were not so well defined. Ultracentrifugal analysis showed that at this pH the whole haemocyanin molecules were completely dissociated into one-tenth molecules. These have higher diffusion coefficients and therefore move closer to the centrewell before meeting antibody molecules (see Allison & Humphrey, 1960). It was also observed at this pH that some of the lines appeared to be double (Fig. lb). No cross-reaction of either B. undatum or N. antiqua haemocyanin antiserum was observed with the haemocyanins of Caliostoma zizyphimum, Littorina saxatilis, or Nassarius reticulatus (in some cases untreated haemolymph was used because of the small amounts of material available), but a weak cross-reaction was observed with the haemocyanin of Murex trunculus (Fig. ld). Similarly an antiserum to M. trunculus haemocyanin reacted very weakly with B. undatum and N. antiqua haemocyanins, showing that these haemocyanins have some antigenic determinants in common. Figure l(c) shows double precipitin lines more clearly and these were obtained by raising the pH until both whole and half molecules co-existed. Indeed both double and single precipitin arcs were obtained in the same experiment by controlling not only the pH but also the calcium ion concentration, since the latter represses dissociation of whole haemocyanin molecules. In Fig. lc the pH of the buffer in which the agar was made up was pH 8'0, and the buffer contained 0.005 M EDTA. The centre-well contained antiserum and the haemocyanins (except well C) were diluted in the same buffer and were allowed to stand at room temperature for 24 hr before being placed in the wells.
Immunochemical properties of haemocyanins
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Fig. 1. Double diffusion in 0"75~oagar of haemocyanins against rabbit anti-haemocyanin. (a) The centrewell contained rabbit anti-N, antiqua serum, and the outer wells contained: A: N. antiqua haemocyanin, 10 mg/ml; B: B. undatum haemocyanin, 10 mg/ml; C: B. undatum haemocyanin, 5 mg/ml; D: N. antiqua haemocyanin, 5 mg/ml; F: B. undatum haemocyanin, 10 mg/ml; E: C. gracilis haemocyanin, i0 mg/ml. Buffer: Tris-EDTA-borate, pH 7.3. (b) As for Fig. l(a) except that the buffer had a pH of 8-3, and the haemocyanins were diluted in this buffer. (c) Tris-EDTA-borate buffer, pH 8"0, EDTA concentration, 0.005 M. Centre-well: neat rabbit anti-N, antiqua haemocyanin serum, The haemocyanins (except well C) were diluted into the above buffer and stood 24 hr before being placed in the wells. The haemocyanin in well C was diluted into buffer containing 0-05 M CaC12immediatelybefore being placed in the well. All haemocyanins 10 mg/ml. Well A: C. gracilis haemocyanin; wells B and C: N. antiqua haemocyanin; well D: B. undatum haemocyanin. (d) Normal Tris-EDTA borate buffer, pH 7.3 and haemocyanins (except wells A and D) diluted in the same. Centre-well: neat rabbit anti-N, antiqua haemocyanin serum. All haemocyanin 10 mg/ml. Haemocyanin in wells A and D diluted in Tris-EDTA borate buffer, pH 8-0. Wells A, B, and D: N. antiqua haemocyanin; wells C and F: M. trunculus haemocyanin; well E: C. gracilis haemocyanin. The haemocyanin in well C was diluted in buffer containing 0"05 M CaClz and the solution was placed in the well immediately after dilution. This haemocyanin therefore did not dissociate, while the others dissociated to yield a mixture of half and whole molecules, each of which produced their own precipitin line. Figure l(d) shows the same phenomenon in a slightly different way. Here the agar was made up in TEB buffer pH 7-3, and the haemocyanins, except those in wells A and D, were diluted in the same buffer. Single lines resulted at this pH since the haemocyanins did not dissociate. The haemocyanins in wells A and D were diluted in TEB buffer, pH 8.0. Dissociation into a mixture of whole and half molecules resulted, and reassociation did not take place in the EDTA-containing agar since re-association only takes place in the pres-
ence of calcium or magnesium ions under these conditions (Wood, 1973). These experiments illustrate the importance of controlling closely the conditions when antibody-antigen reactions are being performed with haemocyanins. Similar results were obtained with haemocyanin solutions in which one-half and one-tenth molecules co-existed, but here the precipitin lines due to the one-tenth molecules were much more diffuse. It is clear that some of the antigenic determinants of whole molecules and of sub-units are common, but there findings do not rule out the exposure of new determinants when dissociation takes place. lmmunoelectrophoresis
In immunoelectrophoresis a number of precipitin lines were observed and in some cases the patterns
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E.J. WOOD
were very confusing. Often 3 lines were observed and these had different degrees of curvature and some of them appeared to extend from the starting well to beyond the furthest point to which protein travelled during electrophoresis. Figure 2 illustrates some of the patterns obtained, and Fig. 3 offers, in diagrammatic form, an explanation for the patterns in terms of the various factors involved, namely differences in mobility between whole molecules and sub-units, differences in diffusion coefficients, and differences in the rates of dissociation. Cellulose acetate membrane electrophoresis had shown that dissociation products moved more slowly than whole molecules. It therefore seems likely that the sharp, inner precipitin lines due to reaction with the fastest-moving species correspond to the precipitin
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Co •Fig. 3. Diagrammatic explanation for patterns observed in immunoelectrophoresis. The well initially contains haemocyanin, the agar is made up in a buffer of pH at which the haemocyanin would dissociate partially into one-half and one-tenth molecules,and the troughs contain antiserum. A: Electrophoresis performed after dissociation is complete, i.e. three molecular species present; B: electrophoresis performed immediately after dilution into the buffer, i.e. little dissociation during the electrophoresis, dissociation takes place during diffusion in agar; C: composite situation in which some dissociation takes place during electrophoresis and further dissociation of whole molecules takes place during diffusionafter electrophoresis has stopped.
Fig. 2. Electrophoresis and immunoelectrophoresis of haemocyanin in agar. Electrophoresis of B. undatum haemocyanin run immediately (a) and 1 week (b) after dilution into buffer, pH 8.1. The "tail" of dissociation products can be seen in b, c and d immunoelectrophoresis with similar agar gels: anti-B, undatum haemocyanin serum in trough. (e): more pronounced effect of dissociation during and after electrophoresis (see Fig. 3). Anti-B. undatum haemocyanin serum in top trough, anti-N, antiqua haemocyanin serum in bottom trough.
reaction with whole molecules (Fig. 2), and the outer, more diffuse lines to the reaction with one-tenth molecules. The fact that the inner lines were sharp and close to the antigen zone confirms that the inner lines are due to associated haemocyanin i.e. having a low diffusion coefficient, and a high mobility. The time required for dissociation to come to completion is also important. Thus if haemocyanin is freshly diluted into buffer and electrophoresis takes place immediately, a complicated situation may arise. During electrophoresis (2 hr max.) the whole molecules continue to dissociate and since the dissociation products move slower than the wholes, they form a "tail" (Fig. 2b). When electrophoresis stops all the species continue to diffuse in a radial direction (12-24 hr), but the wholes and possibly the halves also, continue to dissociate. The dissociation products now move faster (radially) than the wholes. Eventually the different molecules meet the antiserum (which reacts with wholes, halves and tenths) and form immobile precipitin bands. If on the other hand dissociation has been allowed to come to completion before electrophoresis, a completely different pattern would be expected. In this case up to 3 lines should be seen and where these approach one another they may or may not show reactions of identity depending on whether the antigenic determinants of wholes are completely similar to those of tenths or not. An even more complex situation may result if the antibodies to the haemocyanin are present as IgG and IgM, which have different diffusion coefficients and are perhaps directed to different determinants.
Immunochemical properties of haemocyanins
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Fig. 4. Quantitative precipitin test with Buccinum undatum haemocyanin. Serum diluted 1:4 in buffer of the appropriate pH and mixed with an equal volume (0-5 ml) of buffer containing the stated amount of haemocyanin. Finally precipitate taken up in 0.5M NaOH and absorbance at 280 nm recorded. (O) associated haemocyanin molecules (reaction at pH 7.0 wholes); (e) dissociated haemocyanin molecules (reaction at pH 9.0 tenths). The arrows show the first tube with antigen excess. Inset: Precipitation of B. u,datum haemocyanin by its rabbit antiserum. - - ( 3 - - and - - ~ - - refer to the antibody reaction with associated and dissociated haemocyanin respectively (left abscissa), and - - A - - and - - & - - refer to the antibody-antigen ratios with associated and dissociated haemocyanins respectively (right abscissa).
The quantitative precipitin test The results of the quantitative precipitin reaction between B. undatum haemocyanin and its rabbit antiserum (raised by injection 6 x 10 mg. of the haemocyanin) are shown in Fig. 4. Different equivalence points were obtained with the associated (whole molecules) haemocyanin as compared with the dissociated (one-tenth molecules) haemocyanin. (Similar results were obtained for N. antiqua haemocyanin and its antiserum). This observation may be explained by the great difference in size between antigen and antibody molecules. Assuming a mol. wt of about 150,000 for the antibody molecule, a molecular ratio of antibody molecules to antigen molecules of 1:1 would corre-
spond to a weight ratio of about 1:56 for associated haemocyanin, and for dissociated haemocyanin about 1:5.6. Thus depending on the actual combining ratio, very small weights of antibody can precipitate large weights of whole haemocyanin. In fact for whole haemocyanin molecules the equivalence ratio (on a weight basis) was about 0.25 while the figure for tenth molecules was about 2.2 (Fig. 4). Figure 5 shows the precipitin curve for dissociated haemocyanins in more detail, and also shows quantitatively the cross-reaction between B. undatum and N. antiqua haemocyanins. For example with antiserum to B. undatum haemocyanin, maximal precipitation took place at about 1.7 mg haemocyanin/tube, whereas that
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Fig. 5. Quantitative precipitin reaction with haemocyanins. In each case the antiserum was to B. undatum haemocyanin, and the conditions, except for the buffer pH, were the same as those in Fig. 4. O: associated B. undatum haemocyanin; A: dissociated B. undatum haemocyanin; O: associated N. antiqua haemocyanin; &: dissociated N. antiqua haemocyanin; El: associated B. undatum haemocyanin with the antiserum obtained by immunization with doses of I mg/ml haemocyanin instead of 10 mg/ml.
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for N. antiqua haemocyanin against the same antiserum was about 0.2 mg haemocyanin/tube. The corresponding figures for dissociated haemocyanins were about 0-1 and 0.04 mg haemocyanin/tube. It may be observed here that the precipitin curves for the dissociated haemocyanins appear to be somewhat irregular. A possible explanation for ,this is that at pH 9'2 it is highly likely that the material is not in fact homogeneous one-tenth molecules, but that some one-twentieth molecules may also be present. In support of this is may be mentioned that in the analytical ultracentrifuge, the material at pH 9.2 does not sediment as a symmetrical peak (see Wood & Peacocke, 1973). Also shown in Fig. 5 is the curve obtained with the rabbit antiserum raised by the injection of 6 doses of 1 mg haemocyanin instead of 6 doses of 10mg. Maximum precipitation for associated haemocyanin then occurred at about 0.12mg haemocyanin/tube, compared with about 1.7 mg haemocyanin per tube. DISCUSSION It has been shown repeatedly that haemocyanins are powerful antigens This may be related to their relatively high content of carbohydrate or to the nature of the carbohydrate (see below), but it is also true to say that haemocyanins are distinctly "foreign" proteins as far as mammals are concerned (Malley et al., 1965; Dixon et al., 1966; Wood et al., 1968). Haemocyanins have therefore been used as challenge antigens in the investigation of the immunological response (Mannick et al., 1962; Dixon et al., 1966). Mannick et al. (1962) found that keyhole limpet (Megathura crenulata) haemocyanin given to dogs either intravenously, subcutaneously or intradermally, always resulted in the production of precipitating antibodies, and they considered haemocyanin superior to any other common antigen. Haemocyanins have an added advantage that mammals will almost certainly never have encountered them before, and therefore the primary immunological response can be observed; the same is difficult to guarantee to be true for bacterial antigens. It may be noted that Dixon et al. (1966) showed that 13 li_labelled haemocyanin injected into rabbits disappeared very rapidly from the circulation. Thirty min after injection of 2mg, 24~o of the 1311 was present in precipitable form in the total serum volume and after 6 hr, only 1"5~o. In contrast, Aisen et al. (1964) had shown that the half-life of coeruloplasmin in rabbits was 56 hr and in rats 27 hr. This was of course homologous coeruloplasmin. However Ashwell's group (van den Hamer et al., 1970) have more recently shown that if some of the sialic acid residues are removed from coeruloplasmin, the modified material has a half-life of minutes. It therefore seems that the terminal sialic acid residues of glycoproteins somehow protect them against catabolism and it is interesting to recall that purified haemocyanins contain virtually no sialic acid (Dijk et al., 1970; Albergoni et al., 1972). It seems likely that haemocyanins are rapidly catabolised in mammals but that a small fragment or fragments are retained which serve as the immunological stimulus (see Campbell & Garvey, 1961). In the present work it has been confirmed that mollusc haemocyanins are l~ighly antigenic in rabbits, even in doses of 6 x 1 mg. Dixon et al. (1966) mention that with keyhole limpet haemocyanin in rabbits maximal
primary responses were elicited by 2 mg. haemocyanin. From both the immunological and the biological viewpoints a thorough understanding of the mechanism of the reaction between antibody and haemocyanin molecule is essential in evaluating results from the study of immune responses and biological cross-reactions. Denuc6 & Cushing (1963) studied many cross-reactions between crustacean haemocyanins but had difficulty in distinguishing between a haemocyanin that contained several antigens and a single haemocyanin that dissociated and produced several precipitin lines. The present work has confirmed the work of Tournabene & Bartel (1962), and of Weigle (1964), both of whom used M. crenulata haemocyanin. A pure antigen may indeed give rise to more than one precipitin line if it can dissociate partially and if the products of dissociation are active immunologically. This is especially true where the dissociation is from a very large molecule (having a low diffusion coefficient) to a small molecule (having, comparatively, a high diffusion coefficient). The position occupied by a precipitin line in agar has been shown to be determined by the diffusion coefficient both of the antibody and of the antigen (Allison & Humphrey, 1960). Furthermore the equivalence point can vary, as in the present case, depending upon the pH at which the precipitin reactions are performed. This phenomenon is explainable in terms of our knowledge, from ultracentrifugal and electrophoretic analysis, of the behaviour of haemocyanin at different pH values. The immunoelectrophoretic patterns obtained in the present work were very similar to those obtained by Masseyeff et al. (1963) with Cymbium neptuni haemocyanin and by Denuc6 & Cushing (1963) with a variety of haemocyanins. Three or more lines were observed in immunoelectrophoresis at pH 8.6. The patterns consisted of short inner lines and much longer more diffuse outer lines extending almost from the origin well. It seems likely that the short inner lines correspond to the precipitin reaction with whole molecules and the outer diffuse lines to that with one-tenth molecules (Fig. 3). Bartel & Campbell (1959) and Weigle (1964) were able to show that in the process of dissociation of whole molecules, some new antigenic determinants were revealed. Weigle also emphasised the lack of precision inherent in a precipitin reaction where antibody and antigen molecules differed markedly in size. A small error in measurement of the total precipitiate results in a large error in the calculated value for antibody. The finding of a cross-reaction between haemocyanins from B. undatum and N. antiqua, and to a lesser extent with that from C. gracilis, is not surprising in view of the close similarities of the animals. Haemocyanins from other whelks that were obtainable did not cross-react, with the exception of that from Murex trunculus. It may be noted that the haemocyanins of M. trunculus and M. brandaris partially cross-reacted when tested with anti-M, trunculus serum (Wood et al., 1968). The results from the quantitative precipitin assay are in agreement with those of Weigle (1964) where at equivalence the antibody-antigen weight ratio was about 0"22 for whole molecules and 2.0-2-5 for dissociated molecules. These latter are probably one-tenth molecules (Wood, 1973) and it may be significant that
Immunochemical properties of haemocyanins
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BARTELA. H. & CAMPBELLD. H. (1959) Some immunochemical differences between associated and dissociated haemocyanin. Archs Biochem. Biophys. 82~ 232-234. BOYD W. C. (1937) Cross-reactivity of various haemocyanins with special reference to the blood protein of the black widow spider. Biol. Bull. 73, 181-183. CAMPBELLD. H. & GARVEYJ. S. (1961) The fate of foreign antigen and speculations as to its role in immune mechanisms. Lab. Invest. 10, 1126-1150. DENUCr~J. M. & CUSHINGJ. E. (1963) Comparative serology of crustaceans and molluscs of the coast of South California. Protides of the Biological Fluids ll, 146-149. D1JK J., BROUWERM., COERT A. & GRUBER M. (1970) Structure and function of haemocyanins. VII. Biohim. biophys. Acta 221,467-479. DIXON F. J., JACOT-GUILLARMODH. & MCCONAHEYP. J. (1966) Antibody responses of rabbits and rats to haemocyanin. J. Immunology 97, 350-355. HEIRWEGHK. & LONT1ER. (1960) Decrease of the copper bands of Helix pomatia haemocyanin. Nature, Lond. 185, 854-855. MALLEYA., SAHAA. & HALL1OAYW. J. (1965) Immunochemical studies ofhaemocyanin from the giant keyhole limpet (Megathura crenulata) and the horseshoe crab (Limulus polyhemus). J. Immunology 95, 141-147. MANNICK J. A., LEE H. M. & EDGAHLR. H. (1962) Effect of 6-mercaptopurine on the immunological reactivity of dogs to haemocyanin and kidney homotransplants. Ann. N.Y. Acad. Sci., U.S. 99, 762-767. MASSEYEFFR., GOMBERTJ., TANGUYU. & NEUZILE. (1963) Contribution a l'6tude biochimique de l'h6mocyanine de Cymbium neptuni II l~tude 61ectrophor6tique et immuno61ectrophor6tique. Bull. Soc. Chim. Biol. 45, 1133-1144. SEIZENR. J. & VAN DRIELR. (1973) Structure and properties of haemocyanins. VIII--Microheterogeneity of ~haemocyanin of Helix pomatia. Biochim. biophys. Acta 295, 131-139. TOURNABENET. & BARTELA. H. (1962) Antigen dissociation as a factor in the immunodiffusion analysis of haemocyanin. Tex. Rep. Biol. Med. 20, 683-685. VAN DEN HAMERC. J. A., MORELLA. G., SCHEINBERGI. H., HXCKMANJ. & ASHWELLG. (1970) Physical and chemical studies on ceruloplasmin. IX. J. Biol. Chem. 245, 439% 4402. WEETALLH. H. & WELIKYN. (1965) Immunoabsorbent for Acknowledgements--I am indebted to Mrs. J. Waterhouse the isolation of purine-specific antibodies. Science, N. Y for collaboration in the early part of this study, and to Miss 148, 1235-1237. Lindsey Mosby for skilled technical assistance. My thanks are due to the staff of the Wellcome Marine Station, Robin WEIGLE W. O. (1964) Immunochemical properties of haemocyanins. Immunochemistry 1,295-302. Hood's Bay, U.K., for supplying the whelks and to Dr. A. Graham, University of Reading, Reading, U.K., for help WOODE. J. (1973) Gastropod haemocyanins: dissociation of haemocyanins from Buccinum undatum, Neptunea antiqua, with their identification. and Colus gracilis in the region pH 7.5-9.2. Biochim. biophys. Acta 328, 101 106. Abbreviations used: EDTA--ethylenediaminetetracetic WOOD E. J., BANNISTERW. H., OLIVER C. J., LONTIE R. & WITrERS R. (1971) Diffusion coefficients, sedimentation acid; TEB buffer--Tri~EDTA-borate buffer. coefficients, and molecular weights of some gastropod haemocyanins. Comp. Biochem. Physiol. 40B, 19-24. WOODE. J. & PEACOCr,E A. R. (1973). Murex trunculus haeREFERENCES mocyanin. Physical properties and pH-induced dissociaAISEN P., MORELLA. G., ALPERTS., ~¢. STERNLIEBI. (1964) tion Eur. J. Biochem. 35, 410-420. Bilary excretion of coeruloplasmin copper. Nature, Lond. WOODE. J., SALISBURYC. M., FORMOSAN. & BANNISTERW. 203, 873-874. H. (1968) An electrophoretic and immunologic study of ALBERGONIV., CASSINIA. • SALVATOB. (1972) The carboMurex trunculus haemocyanin. Comp. Biochem. Physiol. hydrate portion of haemocyanin from Octopus vulgaris. 26, 345-351. Comp. Biochem. Physiol. 41B, 445-431. ALLISONA. C. & HUMPHREYJ. H. (1960) A theoretical and experimental analysis of double diffusion precipitin reactions in gels, and its application to characterization of Key Word lndex--Haemocyanin; Buccinum undatum; antigens. Immunology 3, 95-106. Immunochemistry; Neptunea antiqua. the weight ratios differ by a factor of about 10. Working with M. crenulata haemocyanin that had been modified by coupling to it 6-trichloromethylpurine, Weetall & Weliky 0965) found a weight ratio of antibody to antigen of 0-34. For disSOciated keyhole limpet haemocyanin, Dixon et al. (1966) found an antibodyantigen nitrogen ratio of 2'5-3'0. In the quantitative precipitin assay it was observed that in the region of antibody excess, the amount of antibody precipitated by a given amount of dissociated haemocyanin was greater than by a similar amount of associated haemocyanin. In order to precipitate the maximum amount of antibody protein much larger amounts of associated haemocyanin were needed than dissociated haemocyanin. The antibody-antigen ratio at equivalence was about l0 times higher with dissociated haemocyanin than with associated. Both curves showed classical behaviour in that in the antigen excess region addition of more antigen resulted in inhibition of precipitation. In contrast, Weigle (1964) found it difficult to establish the equivalence point with the system antiserum-associated haemocyanin. Malley et al. (1965) found that the amount of rabbit antibody produced to Limulus polyhemus was 2"7-4-0 mg. antibody protein/ml, serum. A value of about 2-4 mg./ml may be calculated from the present work where B. undatum haemocyanin was used as antigen in doses of 6 x 10mg. The present work has yielded no information about the proposed microheterogeneity of haemocyanin (see Siezen & van Driel, 1973), These workers showed by a number of techniques that an apparently homogeneous preparation of H. pomatia ~-haemocyanin consisted of a family of closely similar but not identical molecules. It may be that the combination of immunological techniques together with such methods as isoelectric focussing may in the future be able to throw some light on this problem.
IMMUNOCHEMICAL PROPERTIES OF THE HAEMOCYANINS FROM BUCCINUM UNDATUM (L.) A N D NEPTUNEA ANTIQUA (L.) E. J. WOOD Department of Biochemistry, University of Leeds, 9 Hyde Terrace, Leeds LS2 9LS, England (Received 8 July 1974)
Abstract--1. Rabbit antiserum to Buccinum undatum haemocyanin cross-reacted with Neptunea antiqua haemocyanin and vice versa, the reaction in each case being one of partial identity. Antiserum to B. undaturn haemocyanin also cross-reacted weakly with Colus gracilis haemocyanin, and very weakly with Murex trunculus haemocyanin. 2. Depending upon the pH and the Ca 2+ or Mg 2÷ concentrations, the reaction of the haemocyanins with their rabbit antisera resulted in the formation of single or multiple precipitin lines in agar and different equivalence points in the quantitative precipitin assay. 3. Analytical ultracentrifugation and electrophoretic analysis showed that this phenomenon was caused by pH-induced dissociation of the haemocyanins, both whole molecules and their dissociation products being capable of reacting with antisera. 4. An interpretation of the patterns observed in immunoelectrophoresis is given based on the time taken for haemocyanin molecules to dissociate at certain pH values, and the electrophoretic mobilities and diffusion coefficients of the dissociation products.
INTRODUCTION
HAEMOCYANINS from arthropods and molluscs have frequently been used in immunological studies. Some workers have investigated phylogenic relationships by means of cross-reactions (Boyd, 1937; Denuc6 & Cushing, 1963; Malley etal., 1965), while others have been concerned with studying the nature of the immune response (Weigle, 1964; Dixon et al., 1966). In the latter case haemocyanins were used because of their high antigenic potency and because they are easily available in comparatively large quantities. Also it is possible to study the primary immune response since laboratory animals are unlikely to have encountered the antigens previously. Because haemocyanins are very large multimeric protein molecules, great caution is needed in interpreting the results of experiments in which they have been allowed to react with antisera against them. The size of the molecules in relation to that of immunoglobulin molecules means that a small weight of antibody protein can precipitate a very large weight of antigen protein (Weigle, 1964). The fact that raising the pH slightly causes the haemocyanin molecules to dissociate partly or wholly into smaller sub-units having the same or additional antigenic determinants (Bartel & Campbell, 1959), can lead to the situation where a single pure antigen produces several precitin lines in agar against its antiserum (Tournabene & Bartel, 1962; Weigle, 1964). This situation is complicated by other factors. The extent of dissociation is influenced not only by pH but also by calcium and magnesium ion concentrations. Some agars contain considerable amounts of these ions (Wood et al., 1968). The rate of dissociation of haemocyanins may be slow (Wood, 1973). Under some conditions dissociation requires days to come to completion, so that experiments performed immediately after preparing haemocyanin solutions
may give different results to experiments performed with the same solutions 2-3 days later. Finally, the mobilities of the products of dissociation are different so that patterns obtained in immunoelectrophoresis can be very complex. Clearly the interpretation of the immunological reactions of haemocyanins with antisera demand a full understanding of the physicochemical properties of the haemocyanins. In the present work the reactions between the haemocyanins from Buceinum undatum (L.) and Neptunea antiqua (L.) and their rabbit antisera have been investigated. METHODS
H aemoc yanins
Haemolymph was obtained from Buceinum undatum, Neptunea antiqua, and other whelks, and usually the haemolymph from several animals was pooled. The haemocyanin was purified and concentrated by preparative ultracentrifugation (Wood & Peacocke, 1973). The purity of the preparations was checked in the analytical ultracentrifuge, and haemocyanin concentrations were determined from their absorbance at 278nm (Wood, 1973). Preparation of antisera Antisera were raised in rabbits by injecting sterilized haemocyanin solution in 0'85~o saline intradermally into the back. Usually six injections were given at 2-3 day intervals, of either 1 mg or 10 mg haemocyanin. The saline was at about physiological pH and therefore the haemocyanin would be in the undissociated state at the time of injection. Test bleedings were taken from the marginal ear vein after each injection except the first. Two to three weeks after the sixth injection the rabbits were killed and exsahguinated. The serum was separated in the normal way.
219
220
E.J. WOOD
Immunological reactions Radial diffusion analysis was performed in 0-75 or 1~o agar gel (Oxoid Ltd.) made up in a suitable buffe~'. The choice of buffer in all cases was governed by the state in which it was desired to have the haemocyanin e.g. whole molecules (sedimentation coefficient, 100S), partially dissociated molecules (wholes, halves, and tenths), or completely dissociated molecules (tenths, sedimentation coefficient, ~ 16S) (see Wood & Peacocke, 1973; Wood, 1973). Several arrangements of wells were tried out in order to obtain clear precipitin arcs so that cross-reactions could be seen easily. Because of the great difference in size of antibody molecule and antigen molecule, the precipitin arcs often fell very close to the perimeter of the antigen well. This is because the rate of movement of proteins through the gel depends upon the diffusion coefficients of the proteins (see Allison & Humphrey, 1960). Immunoelectrophoresis was performed in 0'75 or l'0yo agar on microscope slides, as described previously (Wood et at., 1968).
Quantitative precipitin reaction The quantitative precipitin reaction was performed by mixing equal volumes (normally 0'5 ml.) of diluted antiserum and haemocyanin solution, all dilutions being done in a buffer at the appropriate pH so that the haemocyanin was in the associated or dissociated state as desired. (Weigle (1964) reported that performing the quantitative precipitation assay at different pH values in the range pH 6-%8.8 had little effect on the results obtained with the system BSA-anti BSA). The tubes were allowed to stand for 2 hr at 37 ° after which they were placed in the refrigerator overnight. The precipitates were then spun down, washed, and taken up in 0"5 N NaOH. The total protein in the precipitate was measured by the absorbance at 280 nm. The amount of haemocyanin in the precipitate was in some cases estimated from the copper content determined by atomic absorption spectrophotometry.
Analytical ultracentrifugation Analytical ultracentrifugation (sedimentation velocity) was performed with a Beckman model E instrument equipped with a schlieren optical system and a rotor temperature indicating and control system (see Wood, 1973).
Electrophoresis Electrophoresis on cellulose acetate membranes was performed in the usual way using Oxoid strips and a Shandon-Kohn apparatus. The Tris-EDTA borate buffer used was prepared by mixing appropriate quantities of two solutions: A" 0.14 M boric acid containing 0"02 M EDTA and B: 0"14 M Tris containing 0"02 M EDTA, to give the desired pH. Electrophoresis was performed for up to 1 hr at 2 mA per strip and then the strips were fixed and stained in 0"2~ (w/v) Ponceau S containing 3~o (w/v) trichloroacetic acid. After drying, the strips were cleared with Shell Ondina 17 oil and scanned in a densitometer. RESULTS
The state of the haemocyanin The dissociation state of the haemocyanin was determined by analytical ultracentrifugation from the
number of boundaries appearing and their sedimentation coefficients. Whole or native molecules (100S, molecular weight 9 x 106), one-half molecules (60S), and one-tenth molecules (16S) were the main species encountered (Wood, 1973). A similar picture emerged from cellulose acetate membrane electrophoresis and it seemed likely that under the conditions used the larger molecules had the higher charges, and moved faster.
Production of antibodies The injection into rabbits of repeated doses of 10 mg ofB. undatum or N. antiqua haemocyanin proved to be a powerful antigenic stimulus. Antibodies to haemocyanin were detected in the serum by agar diffusion after the third injection. Injection of repeated doses of 1 mg. of the haemocyanin also led to the production of antibodies. The antibody titre (equivalence point) of the final bleeding in the quantitative precipitin test was in this case considerably less than when 10 mg. doses of haemocyanin had been injected (see below, and Fig. 5).
Cross-reactions between different haemocyanins A rabbit antiserum to B. undatum haemocyanin cross-reacted in agar with N. antiqua haemocyanin, and to a much lesser extent with Colus gracilis haemocyanin. Similarly the antiserum to N. antiqua haemocyanin cross-reacted strongly with B. undatum haemocyanin and weakly with C. gracilis haemocyanin (Fig. la). At pH 7.3 where there is no dissociation of the haemocyanins, single precipitin arcs were observed. The cross-reactions appeared to be those of partial identity. At a higher pH value (pH 8'3, Fig. lb) the same crossreactions occurred, but the precipitin lines were closer to the centre-well and were not so well defined. Ultracentrifugal analysis showed that at this pH the whole haemocyanin molecules were completely dissociated into one-tenth molecules. These have higher diffusion coefficients and therefore move closer to the centrewell before meeting antibody molecules (see Allison & Humphrey, 1960). It was also observed at this pH that some of the lines appeared to be double (Fig. lb). No cross-reaction of either B. undatum or N. antiqua haemocyanin antiserum was observed with the haemocyanins of Caliostoma zizyphimum, Littorina saxatilis, or Nassarius reticulatus (in some cases untreated haemolymph was used because of the small amounts of material available), but a weak cross-reaction was observed with the haemocyanin of Murex trunculus (Fig. ld). Similarly an antiserum to M. trunculus haemocyanin reacted very weakly with B. undatum and N. antiqua haemocyanins, showing that these haemocyanins have some antigenic determinants in common. Figure l(c) shows double precipitin lines more clearly and these were obtained by raising the pH until both whole and half molecules co-existed. Indeed both double and single precipitin arcs were obtained in the same experiment by controlling not only the pH but also the calcium ion concentration, since the latter represses dissociation of whole haemocyanin molecules. In Fig. lc the pH of the buffer in which the agar was made up was pH 8'0, and the buffer contained 0.005 M EDTA. The centre-well contained antiserum and the haemocyanins (except well C) were diluted in the same buffer and were allowed to stand at room temperature for 24 hr before being placed in the wells.
Immunochemical properties of haemocyanins
221
Fig. 1. Double diffusion in 0"75~oagar of haemocyanins against rabbit anti-haemocyanin. (a) The centrewell contained rabbit anti-N, antiqua serum, and the outer wells contained: A: N. antiqua haemocyanin, 10 mg/ml; B: B. undatum haemocyanin, 10 mg/ml; C: B. undatum haemocyanin, 5 mg/ml; D: N. antiqua haemocyanin, 5 mg/ml; F: B. undatum haemocyanin, 10 mg/ml; E: C. gracilis haemocyanin, i0 mg/ml. Buffer: Tris-EDTA-borate, pH 7.3. (b) As for Fig. l(a) except that the buffer had a pH of 8-3, and the haemocyanins were diluted in this buffer. (c) Tris-EDTA-borate buffer, pH 8"0, EDTA concentration, 0.005 M. Centre-well: neat rabbit anti-N, antiqua haemocyanin serum, The haemocyanins (except well C) were diluted into the above buffer and stood 24 hr before being placed in the wells. The haemocyanin in well C was diluted into buffer containing 0-05 M CaC12immediatelybefore being placed in the well. All haemocyanins 10 mg/ml. Well A: C. gracilis haemocyanin; wells B and C: N. antiqua haemocyanin; well D: B. undatum haemocyanin. (d) Normal Tris-EDTA borate buffer, pH 7.3 and haemocyanins (except wells A and D) diluted in the same. Centre-well: neat rabbit anti-N, antiqua haemocyanin serum. All haemocyanin 10 mg/ml. Haemocyanin in wells A and D diluted in Tris-EDTA borate buffer, pH 8-0. Wells A, B, and D: N. antiqua haemocyanin; wells C and F: M. trunculus haemocyanin; well E: C. gracilis haemocyanin. The haemocyanin in well C was diluted in buffer containing 0"05 M CaClz and the solution was placed in the well immediately after dilution. This haemocyanin therefore did not dissociate, while the others dissociated to yield a mixture of half and whole molecules, each of which produced their own precipitin line. Figure l(d) shows the same phenomenon in a slightly different way. Here the agar was made up in TEB buffer pH 7-3, and the haemocyanins, except those in wells A and D, were diluted in the same buffer. Single lines resulted at this pH since the haemocyanins did not dissociate. The haemocyanins in wells A and D were diluted in TEB buffer, pH 8.0. Dissociation into a mixture of whole and half molecules resulted, and reassociation did not take place in the EDTA-containing agar since re-association only takes place in the pres-
ence of calcium or magnesium ions under these conditions (Wood, 1973). These experiments illustrate the importance of controlling closely the conditions when antibody-antigen reactions are being performed with haemocyanins. Similar results were obtained with haemocyanin solutions in which one-half and one-tenth molecules co-existed, but here the precipitin lines due to the one-tenth molecules were much more diffuse. It is clear that some of the antigenic determinants of whole molecules and of sub-units are common, but there findings do not rule out the exposure of new determinants when dissociation takes place. lmmunoelectrophoresis
In immunoelectrophoresis a number of precipitin lines were observed and in some cases the patterns
222
E.J. WOOD
were very confusing. Often 3 lines were observed and these had different degrees of curvature and some of them appeared to extend from the starting well to beyond the furthest point to which protein travelled during electrophoresis. Figure 2 illustrates some of the patterns obtained, and Fig. 3 offers, in diagrammatic form, an explanation for the patterns in terms of the various factors involved, namely differences in mobility between whole molecules and sub-units, differences in diffusion coefficients, and differences in the rates of dissociation. Cellulose acetate membrane electrophoresis had shown that dissociation products moved more slowly than whole molecules. It therefore seems likely that the sharp, inner precipitin lines due to reaction with the fastest-moving species correspond to the precipitin
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Co •Fig. 3. Diagrammatic explanation for patterns observed in immunoelectrophoresis. The well initially contains haemocyanin, the agar is made up in a buffer of pH at which the haemocyanin would dissociate partially into one-half and one-tenth molecules,and the troughs contain antiserum. A: Electrophoresis performed after dissociation is complete, i.e. three molecular species present; B: electrophoresis performed immediately after dilution into the buffer, i.e. little dissociation during the electrophoresis, dissociation takes place during diffusion in agar; C: composite situation in which some dissociation takes place during electrophoresis and further dissociation of whole molecules takes place during diffusionafter electrophoresis has stopped.
Fig. 2. Electrophoresis and immunoelectrophoresis of haemocyanin in agar. Electrophoresis of B. undatum haemocyanin run immediately (a) and 1 week (b) after dilution into buffer, pH 8.1. The "tail" of dissociation products can be seen in b, c and d immunoelectrophoresis with similar agar gels: anti-B, undatum haemocyanin serum in trough. (e): more pronounced effect of dissociation during and after electrophoresis (see Fig. 3). Anti-B. undatum haemocyanin serum in top trough, anti-N, antiqua haemocyanin serum in bottom trough.
reaction with whole molecules (Fig. 2), and the outer, more diffuse lines to the reaction with one-tenth molecules. The fact that the inner lines were sharp and close to the antigen zone confirms that the inner lines are due to associated haemocyanin i.e. having a low diffusion coefficient, and a high mobility. The time required for dissociation to come to completion is also important. Thus if haemocyanin is freshly diluted into buffer and electrophoresis takes place immediately, a complicated situation may arise. During electrophoresis (2 hr max.) the whole molecules continue to dissociate and since the dissociation products move slower than the wholes, they form a "tail" (Fig. 2b). When electrophoresis stops all the species continue to diffuse in a radial direction (12-24 hr), but the wholes and possibly the halves also, continue to dissociate. The dissociation products now move faster (radially) than the wholes. Eventually the different molecules meet the antiserum (which reacts with wholes, halves and tenths) and form immobile precipitin bands. If on the other hand dissociation has been allowed to come to completion before electrophoresis, a completely different pattern would be expected. In this case up to 3 lines should be seen and where these approach one another they may or may not show reactions of identity depending on whether the antigenic determinants of wholes are completely similar to those of tenths or not. An even more complex situation may result if the antibodies to the haemocyanin are present as IgG and IgM, which have different diffusion coefficients and are perhaps directed to different determinants.
Immunochemical properties of haemocyanins
223
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Fig. 4. Quantitative precipitin test with Buccinum undatum haemocyanin. Serum diluted 1:4 in buffer of the appropriate pH and mixed with an equal volume (0-5 ml) of buffer containing the stated amount of haemocyanin. Finally precipitate taken up in 0.5M NaOH and absorbance at 280 nm recorded. (O) associated haemocyanin molecules (reaction at pH 7.0 wholes); (e) dissociated haemocyanin molecules (reaction at pH 9.0 tenths). The arrows show the first tube with antigen excess. Inset: Precipitation of B. u,datum haemocyanin by its rabbit antiserum. - - ( 3 - - and - - ~ - - refer to the antibody reaction with associated and dissociated haemocyanin respectively (left abscissa), and - - A - - and - - & - - refer to the antibody-antigen ratios with associated and dissociated haemocyanins respectively (right abscissa).
The quantitative precipitin test The results of the quantitative precipitin reaction between B. undatum haemocyanin and its rabbit antiserum (raised by injection 6 x 10 mg. of the haemocyanin) are shown in Fig. 4. Different equivalence points were obtained with the associated (whole molecules) haemocyanin as compared with the dissociated (one-tenth molecules) haemocyanin. (Similar results were obtained for N. antiqua haemocyanin and its antiserum). This observation may be explained by the great difference in size between antigen and antibody molecules. Assuming a mol. wt of about 150,000 for the antibody molecule, a molecular ratio of antibody molecules to antigen molecules of 1:1 would corre-
spond to a weight ratio of about 1:56 for associated haemocyanin, and for dissociated haemocyanin about 1:5.6. Thus depending on the actual combining ratio, very small weights of antibody can precipitate large weights of whole haemocyanin. In fact for whole haemocyanin molecules the equivalence ratio (on a weight basis) was about 0.25 while the figure for tenth molecules was about 2.2 (Fig. 4). Figure 5 shows the precipitin curve for dissociated haemocyanins in more detail, and also shows quantitatively the cross-reaction between B. undatum and N. antiqua haemocyanins. For example with antiserum to B. undatum haemocyanin, maximal precipitation took place at about 1.7 mg haemocyanin/tube, whereas that
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Fig. 5. Quantitative precipitin reaction with haemocyanins. In each case the antiserum was to B. undatum haemocyanin, and the conditions, except for the buffer pH, were the same as those in Fig. 4. O: associated B. undatum haemocyanin; A: dissociated B. undatum haemocyanin; O: associated N. antiqua haemocyanin; &: dissociated N. antiqua haemocyanin; El: associated B. undatum haemocyanin with the antiserum obtained by immunization with doses of I mg/ml haemocyanin instead of 10 mg/ml.
224
E.J. WOOD
for N. antiqua haemocyanin against the same antiserum was about 0.2 mg haemocyanin/tube. The corresponding figures for dissociated haemocyanins were about 0-1 and 0.04 mg haemocyanin/tube. It may be observed here that the precipitin curves for the dissociated haemocyanins appear to be somewhat irregular. A possible explanation for ,this is that at pH 9'2 it is highly likely that the material is not in fact homogeneous one-tenth molecules, but that some one-twentieth molecules may also be present. In support of this is may be mentioned that in the analytical ultracentrifuge, the material at pH 9.2 does not sediment as a symmetrical peak (see Wood & Peacocke, 1973). Also shown in Fig. 5 is the curve obtained with the rabbit antiserum raised by the injection of 6 doses of 1 mg haemocyanin instead of 6 doses of 10mg. Maximum precipitation for associated haemocyanin then occurred at about 0.12mg haemocyanin/tube, compared with about 1.7 mg haemocyanin per tube. DISCUSSION It has been shown repeatedly that haemocyanins are powerful antigens This may be related to their relatively high content of carbohydrate or to the nature of the carbohydrate (see below), but it is also true to say that haemocyanins are distinctly "foreign" proteins as far as mammals are concerned (Malley et al., 1965; Dixon et al., 1966; Wood et al., 1968). Haemocyanins have therefore been used as challenge antigens in the investigation of the immunological response (Mannick et al., 1962; Dixon et al., 1966). Mannick et al. (1962) found that keyhole limpet (Megathura crenulata) haemocyanin given to dogs either intravenously, subcutaneously or intradermally, always resulted in the production of precipitating antibodies, and they considered haemocyanin superior to any other common antigen. Haemocyanins have an added advantage that mammals will almost certainly never have encountered them before, and therefore the primary immunological response can be observed; the same is difficult to guarantee to be true for bacterial antigens. It may be noted that Dixon et al. (1966) showed that 13 li_labelled haemocyanin injected into rabbits disappeared very rapidly from the circulation. Thirty min after injection of 2mg, 24~o of the 1311 was present in precipitable form in the total serum volume and after 6 hr, only 1"5~o. In contrast, Aisen et al. (1964) had shown that the half-life of coeruloplasmin in rabbits was 56 hr and in rats 27 hr. This was of course homologous coeruloplasmin. However Ashwell's group (van den Hamer et al., 1970) have more recently shown that if some of the sialic acid residues are removed from coeruloplasmin, the modified material has a half-life of minutes. It therefore seems that the terminal sialic acid residues of glycoproteins somehow protect them against catabolism and it is interesting to recall that purified haemocyanins contain virtually no sialic acid (Dijk et al., 1970; Albergoni et al., 1972). It seems likely that haemocyanins are rapidly catabolised in mammals but that a small fragment or fragments are retained which serve as the immunological stimulus (see Campbell & Garvey, 1961). In the present work it has been confirmed that mollusc haemocyanins are l~ighly antigenic in rabbits, even in doses of 6 x 1 mg. Dixon et al. (1966) mention that with keyhole limpet haemocyanin in rabbits maximal
primary responses were elicited by 2 mg. haemocyanin. From both the immunological and the biological viewpoints a thorough understanding of the mechanism of the reaction between antibody and haemocyanin molecule is essential in evaluating results from the study of immune responses and biological cross-reactions. Denuc6 & Cushing (1963) studied many cross-reactions between crustacean haemocyanins but had difficulty in distinguishing between a haemocyanin that contained several antigens and a single haemocyanin that dissociated and produced several precipitin lines. The present work has confirmed the work of Tournabene & Bartel (1962), and of Weigle (1964), both of whom used M. crenulata haemocyanin. A pure antigen may indeed give rise to more than one precipitin line if it can dissociate partially and if the products of dissociation are active immunologically. This is especially true where the dissociation is from a very large molecule (having a low diffusion coefficient) to a small molecule (having, comparatively, a high diffusion coefficient). The position occupied by a precipitin line in agar has been shown to be determined by the diffusion coefficient both of the antibody and of the antigen (Allison & Humphrey, 1960). Furthermore the equivalence point can vary, as in the present case, depending upon the pH at which the precipitin reactions are performed. This phenomenon is explainable in terms of our knowledge, from ultracentrifugal and electrophoretic analysis, of the behaviour of haemocyanin at different pH values. The immunoelectrophoretic patterns obtained in the present work were very similar to those obtained by Masseyeff et al. (1963) with Cymbium neptuni haemocyanin and by Denuc6 & Cushing (1963) with a variety of haemocyanins. Three or more lines were observed in immunoelectrophoresis at pH 8.6. The patterns consisted of short inner lines and much longer more diffuse outer lines extending almost from the origin well. It seems likely that the short inner lines correspond to the precipitin reaction with whole molecules and the outer diffuse lines to that with one-tenth molecules (Fig. 3). Bartel & Campbell (1959) and Weigle (1964) were able to show that in the process of dissociation of whole molecules, some new antigenic determinants were revealed. Weigle also emphasised the lack of precision inherent in a precipitin reaction where antibody and antigen molecules differed markedly in size. A small error in measurement of the total precipitiate results in a large error in the calculated value for antibody. The finding of a cross-reaction between haemocyanins from B. undatum and N. antiqua, and to a lesser extent with that from C. gracilis, is not surprising in view of the close similarities of the animals. Haemocyanins from other whelks that were obtainable did not cross-react, with the exception of that from Murex trunculus. It may be noted that the haemocyanins of M. trunculus and M. brandaris partially cross-reacted when tested with anti-M, trunculus serum (Wood et al., 1968). The results from the quantitative precipitin assay are in agreement with those of Weigle (1964) where at equivalence the antibody-antigen weight ratio was about 0"22 for whole molecules and 2.0-2-5 for dissociated molecules. These latter are probably one-tenth molecules (Wood, 1973) and it may be significant that
Immunochemical properties of haemocyanins
225
BARTELA. H. & CAMPBELLD. H. (1959) Some immunochemical differences between associated and dissociated haemocyanin. Archs Biochem. Biophys. 82~ 232-234. BOYD W. C. (1937) Cross-reactivity of various haemocyanins with special reference to the blood protein of the black widow spider. Biol. Bull. 73, 181-183. CAMPBELLD. H. & GARVEYJ. S. (1961) The fate of foreign antigen and speculations as to its role in immune mechanisms. Lab. Invest. 10, 1126-1150. DENUCr~J. M. & CUSHINGJ. E. (1963) Comparative serology of crustaceans and molluscs of the coast of South California. Protides of the Biological Fluids ll, 146-149. D1JK J., BROUWERM., COERT A. & GRUBER M. (1970) Structure and function of haemocyanins. VII. Biohim. biophys. Acta 221,467-479. DIXON F. J., JACOT-GUILLARMODH. & MCCONAHEYP. J. (1966) Antibody responses of rabbits and rats to haemocyanin. J. Immunology 97, 350-355. HEIRWEGHK. & LONT1ER. (1960) Decrease of the copper bands of Helix pomatia haemocyanin. Nature, Lond. 185, 854-855. MALLEYA., SAHAA. & HALL1OAYW. J. (1965) Immunochemical studies ofhaemocyanin from the giant keyhole limpet (Megathura crenulata) and the horseshoe crab (Limulus polyhemus). J. Immunology 95, 141-147. MANNICK J. A., LEE H. M. & EDGAHLR. H. (1962) Effect of 6-mercaptopurine on the immunological reactivity of dogs to haemocyanin and kidney homotransplants. Ann. N.Y. Acad. Sci., U.S. 99, 762-767. MASSEYEFFR., GOMBERTJ., TANGUYU. & NEUZILE. (1963) Contribution a l'6tude biochimique de l'h6mocyanine de Cymbium neptuni II l~tude 61ectrophor6tique et immuno61ectrophor6tique. Bull. Soc. Chim. Biol. 45, 1133-1144. SEIZENR. J. & VAN DRIELR. (1973) Structure and properties of haemocyanins. VIII--Microheterogeneity of ~haemocyanin of Helix pomatia. Biochim. biophys. Acta 295, 131-139. TOURNABENET. & BARTELA. H. (1962) Antigen dissociation as a factor in the immunodiffusion analysis of haemocyanin. Tex. Rep. Biol. Med. 20, 683-685. VAN DEN HAMERC. J. A., MORELLA. G., SCHEINBERGI. H., HXCKMANJ. & ASHWELLG. (1970) Physical and chemical studies on ceruloplasmin. IX. J. Biol. Chem. 245, 439% 4402. WEETALLH. H. & WELIKYN. (1965) Immunoabsorbent for Acknowledgements--I am indebted to Mrs. J. Waterhouse the isolation of purine-specific antibodies. Science, N. Y for collaboration in the early part of this study, and to Miss 148, 1235-1237. Lindsey Mosby for skilled technical assistance. My thanks are due to the staff of the Wellcome Marine Station, Robin WEIGLE W. O. (1964) Immunochemical properties of haemocyanins. Immunochemistry 1,295-302. Hood's Bay, U.K., for supplying the whelks and to Dr. A. Graham, University of Reading, Reading, U.K., for help WOODE. J. (1973) Gastropod haemocyanins: dissociation of haemocyanins from Buccinum undatum, Neptunea antiqua, with their identification. and Colus gracilis in the region pH 7.5-9.2. Biochim. biophys. Acta 328, 101 106. Abbreviations used: EDTA--ethylenediaminetetracetic WOOD E. J., BANNISTERW. H., OLIVER C. J., LONTIE R. & WITrERS R. (1971) Diffusion coefficients, sedimentation acid; TEB buffer--Tri~EDTA-borate buffer. coefficients, and molecular weights of some gastropod haemocyanins. Comp. Biochem. Physiol. 40B, 19-24. WOODE. J. & PEACOCr,E A. R. (1973). Murex trunculus haeREFERENCES mocyanin. Physical properties and pH-induced dissociaAISEN P., MORELLA. G., ALPERTS., ~¢. STERNLIEBI. (1964) tion Eur. J. Biochem. 35, 410-420. Bilary excretion of coeruloplasmin copper. Nature, Lond. WOODE. J., SALISBURYC. M., FORMOSAN. & BANNISTERW. 203, 873-874. H. (1968) An electrophoretic and immunologic study of ALBERGONIV., CASSINIA. • SALVATOB. (1972) The carboMurex trunculus haemocyanin. Comp. Biochem. Physiol. hydrate portion of haemocyanin from Octopus vulgaris. 26, 345-351. Comp. Biochem. Physiol. 41B, 445-431. ALLISONA. C. & HUMPHREYJ. H. (1960) A theoretical and experimental analysis of double diffusion precipitin reactions in gels, and its application to characterization of Key Word lndex--Haemocyanin; Buccinum undatum; antigens. Immunology 3, 95-106. Immunochemistry; Neptunea antiqua. the weight ratios differ by a factor of about 10. Working with M. crenulata haemocyanin that had been modified by coupling to it 6-trichloromethylpurine, Weetall & Weliky 0965) found a weight ratio of antibody to antigen of 0-34. For disSOciated keyhole limpet haemocyanin, Dixon et al. (1966) found an antibodyantigen nitrogen ratio of 2'5-3'0. In the quantitative precipitin assay it was observed that in the region of antibody excess, the amount of antibody precipitated by a given amount of dissociated haemocyanin was greater than by a similar amount of associated haemocyanin. In order to precipitate the maximum amount of antibody protein much larger amounts of associated haemocyanin were needed than dissociated haemocyanin. The antibody-antigen ratio at equivalence was about l0 times higher with dissociated haemocyanin than with associated. Both curves showed classical behaviour in that in the antigen excess region addition of more antigen resulted in inhibition of precipitation. In contrast, Weigle (1964) found it difficult to establish the equivalence point with the system antiserum-associated haemocyanin. Malley et al. (1965) found that the amount of rabbit antibody produced to Limulus polyhemus was 2"7-4-0 mg. antibody protein/ml, serum. A value of about 2-4 mg./ml may be calculated from the present work where B. undatum haemocyanin was used as antigen in doses of 6 x 10mg. The present work has yielded no information about the proposed microheterogeneity of haemocyanin (see Siezen & van Driel, 1973), These workers showed by a number of techniques that an apparently homogeneous preparation of H. pomatia ~-haemocyanin consisted of a family of closely similar but not identical molecules. It may be that the combination of immunological techniques together with such methods as isoelectric focussing may in the future be able to throw some light on this problem.