The molecular weights of Arthropod hemocyanin subunits: Influence of tris buffer in SDS-PAGE estimations

Cornp. Biochem. Physiol. Vol. 79B, No. 1, pp. 41~,5, 1984

0305-0491/84 $3.00 +0.00 © 1984 Pergamon Press Ltd

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THE MOLECULAR WEIGHTS OF ARTHROPOD HEMOCYANIN SUBUNITS: INFLUENCE OF TRIS BUFFER IN SDS-PAGE ESTIMATIONS D. ROCHU and J. M. Firm Laboratoire d'Immunochimie des Prot+ines, Centre National de Transfusion Sanguine, 6, rue Alexandre Cabanel, 75015 Paris, France (Tel: 306-70-00) (Received 21 February 1984)

Abstract--l. Available molecular weights data for Arthropod hemocyanin subunits as measured by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate are analyzed. 2. Relationship between buffer composition and subunit mobility in SDS-PAGE is shown by studying Cancer pagurus hemocyanin. 3. Tris buffers are suspected to give erroneous molecular weight estimations for Arthropod hemocyanin subunits.

INTRODUCTION

Dissociation of Arthropod hemocyanin (Hc) leads to monomeric subunits with molecular weight close to 75,000. First molecular weight estimations were made by ultracentrifugation (Ellerton et al., 1970), and more accurate results were obtained by using polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS) (Loher and Mason, 1973; Waxman, 1975; Lambin et al., 1976a). SDS-PAGE is now a widely used technique for separation of polypeptides according to their molecular weights (MW). Its great resolving power contributes to the popularity of the method. We have measured the M W of Hc subunits in six species of decapod Crustacea (Rochu et al., 1978) using polyacrylamide gradient gel electrophoresis (PGGE) in the presence of SDS. This technique, giving results with a good accuracy, allowed us to estimate MW ranging from 72,500 to 79,500, in good agreement with the mean value described in Crustacea. But some authors have described a size heterogeneity in intraspecific subunit population, and Jeffrey and Treacy (1982) have discussed in this journal the reliability of MW determination of Hc subunits by SDS-PAGE. In view of such a divergence of opinion, we must see that standardization has not occurred in methodology, though it is generally recognized that binding of SDS to polypeptides is influenced by several parameters. In the present work, we analyze the influence of experimental parameters in SDS-PAGE, to explain the discordance observed in subunit size estimations. We examine M W data of Arthropod Hc subunits, with special reference to the technical conditions of analysis, and the role of buffer composition is described. MATERIAL AND METHODS Purification of hemocyanin Isolation and purity control of Cancer pagurus hemocyanin (CP Hc) were performed according to previously

41

described techniques (Rochu and Fine, 1978). Furthermore, enzymatic degradation of hemocyanin has been avoided by addition of an inhibitor of proteases prior to purification steps (Rochu and Fine, 1984). Electrophoresis Two methods of MW analysis by SDS-PGGE were employed. SDS-PGGE method A. The procedure described by Lambin et al. (1976b) was used in ultrathin layer gels. Hc samples were diluted to 1 mg/ml in a sodium phosphate buffer, 0.1M, pH 7.1, containing 1% SDS and 1% 2-mercaptoethanol. Samples were heated in a boiling water bath for 3 min. Electrophoreses were performed in ultrathin layer (0.5 ram) linear gradient gel (4-22%) with a phosphate buffer (sodium phosphate 0.1 M, pH 7.1, 0.1% SDS). SDS-PGGE method B. This technique was performed according to Grrg et al. (1981). Samples were diluted to 1 mg/ml in a 0.0375 M Tris-HC1 buffer, pH 8.8, containing 1% SDS and 1% 2-mercaptoethanol, and incubated at 100°C for 3 min. Electrophoreses were performed in ultrathin layer (0.5mm) linear gradient gel (4-22%) with a 0.375M Tris-glycine buffer, pH 8.3, 0.1% SDS. In both cases, horizontal electrophoresis ran at 600 V for 1.5 hr. Gels were cooled by a thermostatic circulator set to +4°C. After migration, gels were stained for 30 min at 20°C with 0.2% Coomassie Brilliant Blue R 250 dissolved in a methanol-acetic acid-water solution (5:1:4, v/v/v), and destained by several changes of the destaining solution (methanol-acetic acid-water 1: 1: 8, v/v/v). RESULTS

Analysis o f literature data concerning M W o f Hc subunits Most scientists working on the structure of Arthropod He have used SDS-PAGE to estimate the molecular weight of the polypeptide chains forming the monomeric subunits of these proteins. We have analyzed the literature data and observed that in the majority of cases, the authors select one of the commonly used procedures without attention to the influence of the experimental conditions on the results of their investigations. Strong discrepancies are observed in the subunit number distinguishable according to their molecular size analyzed by SDS-

42

D. ROCHU and J. M. FINE Table t. Arthropod species Limulus polyphemus Tachypleus tridentatus Heterometrus spp. Leirus quinquestriatus Pandinus pallidus Mastigoproctus brasilianus Trichodamon froesi Tarantula palmata Dugesiella californica Argiope bruennichii Cupiennius salei Ligia exotica Penaeus monodon Panulirus interruptus Panulirus regius Jasus edwardsii Cherax destructor Callinectes sapidus Carcinus maenas Macropipus puber Neptunus calidus Ovalipes catharus Cancer magister Cancer pagurus Maia squinado

Subunit number in SDS-PAGE 1 1 l 5 6 6 6 7 7

I 6 3

3 5

1 3

4 2

I I 1 3 6

1 1 WO, Weber and Osborn; L, Laemmli; LM,

Presence of Tris References Sullivanet al. (1976) Takagi and Nemoto (1980) Sugita and Sekigushi (1975) L + Klarman et al. (1979) L + Markl et al. (1979) L + Markl et al. (1979) L + Markl et al. (1979) L + Markl et al. (1979) L + Markl et al. (1979) WO Sugita and Sekigushi (1975) L + Markl et al. (1979) L + Terwilligerel al. (1979) LM + Ellerton and Anderson (1981) L + Van Eerd and Folkerts (1981) WO Rochu et al. (1978) LM + Robinson and Ellerton (1977) JT + Jeffreyand Treacy (1982) L + Hamlin and Fish (1977) WO Rochu et al. (1978) WO Rochu et al. (t978) WO Rochu et al. (1978) LM + Robinson and Ellerton (1977) L + Larson et aL (1981) WO Rochu et al. (1978) WO Rochu et al. (1978) Loher and Mason; JT, Jeffrey and Treacy.

P A G E . Table 1 shows the m a x i m u m n u m b e r of size isomers of Hc subunits f o u n d in three A r t h r o p o d classes ( M e r o s t o m a t a , A r a c h n i d a a n d Crustacea), relative to the presence or the absence of Tris in the buffer systems. It is n o t e w o r t h y t h a t size heterogenity o f the Hc subunits are exclusively observed w h e n S D S - P A G E are p e r f o r m e d with buffer systems c o n t a i n i n g Tris, i n d e p e n d e n t l y o f the phylogenic class a n d whoever the a u t h o r s m a y be. Since we have never observed any heterogeneity of s u b u n i t size, we have c o m p a r e d the results of analysis p e r f o r m e d according to the m o r e accurate technique ( S D S - P G G E in u l t r a t h i n layer), with two different buffer systems, the first one c o n t a i n i n g p h o s p h a t e ions, the o t h e r c o n t a i n i n g Tris ions.

Buffer system WO WO WO

-

-

-

-

-

a

b -4%

MW of Cancer pagurus He subunits by SDS-PGGE in u l t r a t h i n l a y e r

To study the influence o f Tris on the o b s e r v a t i o n o f size heterogeneity in Hc subunits o f A r t h r o p o d s , we have p e r f o r m e d M W estimations in S D S - P G G E according to L a m b i n e t al. (1976b), in u l t r a t h i n layer gels. Hc subunits of C a n c e r p a g u r u s o b t a i n e d by dissociation with SDS have been analyzed with the p h o s p h a t e buffer system of W e b e r a n d O s b o r n (1969), a n d with the Tris buffer r e c o m m e n d e d by G 6 r g e t al. (1981). As s h o w n in Fig. l, S D S - P G G E in p h o s p h a t e buffer exhibits a single b a n d c o r r e s p o n d i n g to the Hc s u b u n i t s o f C a n c e r p a g u r u s , whereas S D S - P G G E in Tris buffer reveals two constituents with distinct mobilities. The p a t t e r n o b t a i n e d after S D S - P G G E in Tris buffer m u s t be c o m p a r e d to the one observed by electrophoresis of CP Hc in a n inert m e d i u m such as cellulose acetate, p e r f o r m e d with a dissociating buffer, which exhibits two fractions c o r r e s p o n d i n g to the m o n o m e r i c subunits separated according to their difference o f net charge ( R o c h u a n d Fine, 1984).

22% Fig. 1. Electrophoresis of Cancer pagurus hemocyanin in ultrathin layer linear gradient (4-22~o) polyacrylamide gel, in the presence of SDS. (a) Phosphate buffer, pH 7.1; (b) Tris-glycine buffer, pH 8.3.

The molecular weights of arthropod hemocyanin subunits

Stacking gel buffer Separation gel buffer Electrode buffer

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Table 2. Buffer in SDS-PAGEanalysisof hemocyaninsubunits Continuous buffer systems Multiphasicbuffer systems Weber and Osborn Jeffrey and Treacy Loher and Mason Laemmli 0.125 M Tris-HCl None None None pH 8.8 0.1% SDS 0.1 M sodium phosphate 0.05 M Tris 0.035 M Tris-sulfate 0.375 M Tris-HCl pH 7.1 pH 7.8 0.0001 M EDTA pH 8.8 pH 8.8 0.1% SDS 0.1~ SDS 0.2% SDS 0.1% SDS 0.1 M sodium phosphate 0.05 M Tris 0.03 M Tris-acetate 0.025 M Tris pH 7.1 pH 7.8 0.001 M EDTA 0.192 M glycine 0.1% SDS 0.1~ SDS pH 8.3 0.1% SDS pH 8.3 0.02% SDS

DISCUSSION Influence o f Tris in S D S - P A G E Reliability o f molecular weight determination methods Analysis of literature data, and our own results Ultracentrifugation. Ultracentrifugation has been show that estimations of M W performed by SDSused for estimation of the size of particles since PAGE with buffers containing Tris, lead to an anomSvedberg (1925) published his "theory of molecular alous number of subunits differing by their molecular weight measurement, or sedimentation equilibrium", size. and the technique has given many good results. M W estimations of Hc subunits by SDS-PAGE are Nevertheless, we must know that this method performed with one of the four buffer systems depends on an accurate knowledge of the partial described in Table 2: two continuous buffer systems, specific volume 6 of the protein analyzed, usually according to Weber and Osborn (1969) with phosranging from 0.70 to 0.75, and that a 1% error in phate ions, or according to Jeffrey and Treacy (1982) leads to an error of about 3% in MW. The partial with Tris ions, and two multiphasic buffer systems, specific volume being essentially accessible by the according to Loher and Mason (1973) with analysis of the amino acid composition of the mole- Tris-sulfate and Tris-acetate ions, or according to cule, the limits of the accuracy of MW determinations Laemmli (1970) with Tris and Tris-glycine ions. by sedimentation equilibrium are clearly defined. The various SDS-PAGE systems described above This method is always useful but its lack in precision have allowed many authors to estimate the MW of explains why it is now replaced by methods found the monomeric subunits of Arthropod He. But, in a upon analysis of electrophoretic mobilities in sieving recent work, Jeffrey and Treacy (1982) calculated an media (polyacrylamide gel), after addition of an uncertainty between +30% and + 10% in the proanionic detergent (SDS) giving the same electric cedure using the relation log M W vs Rf. As for us, charge to the various proteins. we consider SDS-PAGE as an accurate method for SDS-PAGE. Many SDS-PAGE procedures were MW determination. described for the determination of MW polypeptides. The analysis of the results published in the literaGels with constant polyacrylamide concentration, or ture set several problems. Indeed, in the same animal gradient gels, cast either into tubes or in slabs, can be species, divergent Hc subunit MWs are observed, used with continuous or multiphasic buffer systems. according to the experimental conditions used by the Two main mathematical relations allow the deter- authors. mination of protein MW. The first one, according to For example, Larson et al. (1981) have described in Weber and Osborn (1969), concerns PAGE at con- Cancer magister He, two or six subunits with different stant concentration, where log M W is related to the MW by using either the Loher and Mason or the electrophoretic mobility of the protein. This method Laemmli buffer systems, the latter one allowing them gives an accuracy of about 10% and permits an to distinguish subunits with a difference of size of estimated MW from 12,000 to 200,000. More 2.5%. Such a discrepancy in the confidence conceded recently, a second relationship has been described b~_- to the method of M W estimation by SDS-PAGE Lambin et al. (1976b) in SDS-PGGE. In a liriear must be analyzed. In our opinion, particular attengradient of polyacrylamide concentration, log MW is tion must be focused on the presence of Tris in buffers related to log of the polyacrylamide concentration T used for electrophoresis. On this subject Gielens et al. reached by SDS-protein complexes. This method has (1981) have done a valuable observation that they did an accuracy of 5% (Lambin, 1978), and allows esti- not fully exploit. Indeed, these authors have observed mations of M W between 13,000 and 950,000, in a that Hc and some other proteins bind only 0.7 g single gel slab. More recently, G6rg et al. (1981) SDS/g protein in Tris-HC1 and Tris-glycine buffers, introduced ultrathin layer polyacrylamide gradient when they bind 1.4g SDS/g protein in phosphate gels which allows increased sensitivity and separation buffers. in SDS-PGGE. It is now evident that best estimations We think that the key of the problem is located at of M W by SDS-PAGE are obtained in gradient slab the level of the binding of SDS by polypeptides in the gels by relating log M W vs log T according to presence of the ions introduced to secure the Lambin et al. (1976b). In gradient gels, proteins buffering effect during electrophoresis. The use of migrate through progressively smaller pores, are Tris leads to an unsaturation of proteins by the stacked in sharp zones, and their diffusion is virtually detergent and does not allow the removal of their eliminated. In slab gels, a comparative analysis of electric charges. Under these conditions, SDS-PAGE standards and sample mobilities is allowed. separates polypeptides according to size and charge

44

D. ROCHU and J. M. FINE

and must not be used for M W estimations. On the contrary, phosphate buffers permit the saturation of polypeptides by SDS and can be used in S D S - P A G E experiments performed with intent to estimate MW. Binding o f S D S to hemocyanin subunits

Binding studies of a variety of different proteins indicate that above an SDS m o n o m e r concentration of 8 - 1 0 - 4 M (0.02~o) 1.4g of SDS are bound per gram of protein (Pitt-Rivers and AmbesiImpiombato, 1968; Reynolds and Tanford, 1970a,b). This ratio corresponds approximately to one SDS molecule bound to two amino acid residues of the polypeptides chain. This " S D S - c o a t " masks the net charge of the protein and an approximately constant negative charge per unit mass is obtained, due to the SO 3 heads of the detergent. Moreover, binding of SDS to proteins depends upon the equilibrium concentration of the m o n o m e r and not upon total SDS which includes micelles. A r t h r o p o d Hc subunits are characterized by a special amino acid composition. Indeed, they all contain 25% of dicarboxylic residues (Asp and Glu) which are negatively charged at the p H values used in all S D S - P A G E techniques. A possible attraction of Tris ions (with both positively charged and hydrophilic poles) by a quarter of the residues could prevent a normal fixation of the detergent on the polypeptide chain. Moreover, Tris ions could decrease the number of SDS monomers in solution by promoting formation of micelles. On the contrary when incubation of proteins are performed in the presence of phosphate ions (negatively charged), these could not specially interfere in the binding process of SDS to polypeptide chains. This hypothesis will be extended by precise chemical studies on the capacity of proteins to bind SDS, which will take into account the environmental conditions existing in experimental media. In conclusion, the nature of ions used in buffer systems can influence the results of M W estimation by S D S - P A G E , concerning the subunits of A r t h r o p o d Hc. Tris containing buffers lead us to observe a false size heterogeneity of Hc subunits, due to unsaturation of polypeptides by the detergent.

REFERENCES

Ellerton H. D. and Anderson D. M. (1981) Subunit structure of the hemocyanin from the prawn Penaeus monodon. In Invertebrate Oxygen Binding Proteins. Structure, Active Site and Function (Edited by Lamy J. and Lamy J.), pp. 159-169. Marcel Dekker, New York. Ellerton H. D., Carpenter D. E. and Van Holde K. E. (1970) Physical studies of hemocyanin. V. Characterization and subunit structure of the hemocyanin of Cancer magister. Biochemistry 9, 2225-2232. Gielens C., De Jonghe Ch., Preaux G. and Lontie R. (1981) The binding of sodium dodecyl by molluscan and arthropodan hemocyanins in Tris buffer. In Invertebrate Oxygen Binding Proteins. Structure, Active Site and Function (Edited by Lamy J. and Lamy J.), pp. 41-47. Marcel Dekker, New York. Gorg A., Postel W., Westermeier R., Gianazza E. and Righetti P. G. (1981) SDS-gel gradient electrophoresis, isoelectric focusing and high resolution two-dimensional electrophoresis in horizontal, ultrathin-layer poly-

acrylamide gels. In Electrophoresis 1981 (Edited by Allen R. C. and Arnaud P.), pp. 259-270. de Gruyter, Berlin. Hamlin L. M. and Fish W. W. (1977) The subunit characterization of Callinectes sapidus hemocyanin. Biochim. biophys. Acta 491, 46-52. Jeffrey P. D. and Treacy G. B. (1982) The molecular weights of Arthropod hemocyanin subunits. A brief survey with special reference to Cherax destructor hemocyanin. Comp. Biochem. Physiol. 73B, 983-990. Klarman A., Gottlieb J. and Daniel E. (1979) Quaternary structure and arrangement of subunits in hemocyanin from the scorpion Leirus quinquestriatus. Biochemistry 18, 2239-2244. Laemmli U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, Lond. 227, 680-685. Lambin P. (1978) Reliability of molecular weight determination by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate. Anal~,t. Biochem. 85, 114-125. Lambin P., Rochu D., Ghidalia W. and Fine J. M. (1976a) Etude biochimique des subunit6s de l'h+mocyanine chez quatre esp6ces de Crustac~s D~capodes. Cahiers de Biologie Marine, 17, 21-29. Lambin P., Rochu D. and Fine J. M. (1976b) A new method for determination of molecular weights of proteins by electrophoresis across a sodium dodecyl sulfate (SDS) polyacrylamide gradient gel. Ana(vt. Biochem. 74, 567 575. Larson B. A., Terwilliger N. B. and Terwilliger R. C. (1981) Subunit heterogeneity of Cancer magister hemocyanin. Biochim. biophys. Acta 667, 294-302. Loher J. S. and Mason H. S. (1973) Dimorphism of Cancer magister hemocyanin subunit. Biochem. biophys. Res. Commun. 51, 741-745. Markl J., Markl A., Schartau W. and Linzen B. (1979) Subunit heterogeneity in Arthropod hemocyanins: I. Chelicerata. J. comp. Physiol. 130, 283-292. Pitt-Rivers R. and Ambesi-lmpiombato F. S. (1968) The binding of sodium dodecyl sulfate to various proteins. Biochem. J. 109, 825 830. Reynolds J. A. and Tanford C. (1970a) Binding of dodecyl sulfate to proteins at high binding ratios. Possible implications for the state of proteins in biological membranes. Proc. natn. Acad. Sci. U.S.A. 66, 1002 1007. Reynolds J. A. and Tanford C. (1970b) The gross conformation of protein-sodium dodecyl sulfate complexes. J. biol. Chem. 245, 5161-5165. Robinson H. A. and Ellerton H. D. (1977) Heterogeneous subunits of the hemocyanin from Jasus edwardsii and Ovalipes catharus. In Structure and Function of Hemocyanin (Edited by Bannister J. V. ), pp. 55-70. SpringerVerlag, Berlin. Rochu D. and Fine J. M. (1978) Antigenic structure of the hemocyanin in six species of decapod Crustacea. Comp. Biochem. Physiol. 59A, 145-150. Rochu D. and Fine J. M. (1984) Characterization of the subunits of Cancer pagurus hemocyanin. Comp. Biochem. Physiol. 77B, 333-336. Rochu D., Lambin P., Ghidalia W. and Fine J. M. (1978) Hemocyanin subunits and their polymeric forms in some decapod Crustacea. Comp. Biochem. Physiol. 59B, 117-122. Sugita H. and Sekigushi K. (1975) Heterogeneity of the minimum functional unit of hemocyanins from the spider (Argiope bruennichii), the scorpion (Heterometrus spp.) and the horseshoe crab (Tachypleus tridentatus). J. Biochem, Tokyo 78, 713 718. Sullivan B., Bonaventura J., Bonaventura C. and Godette G. (1976) Hemocyanin of the horseshoe crab, Limulus polyphemus. J. biol. Chem. 251, 7644-7648. Svedberg T. (1925) Kolloid Z. (Zsigmondy-Festschr.) 36, 53. Takagi T. and Nemoto T. (1980) Tachypleus tridentatus

The molecular weights of arthropod hemocyanin subunits Hemocyanin. Separation and characterization of monomer subunits and studies of sulfhydryl groups. J. Biochem. 87, 1785-1793. Terwilliger N. B., Terwilliger R. C., Applestein M., Bonaventura C. and Bonaventura J. (1979) Subunit structure and oxygen binding by hemocyanin of the Isopod Ligia exotica. Biochemistry 18, 102-108. Van Eerd J. P. and Folkerts A. (1981) Isolation and characterization of five subunits of the hemocyanin from

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the spiny lobster Panulirus interruptus. In Invertebrate Oxygen-Binding Proteins. Structure, Active Site and Function (Edited by Lamy J. and Lamy J.), pp. 139-149. Marcel Dekker, New York. Waxman L. (1975) The structure of Arthropod and Mollusc hemocyanins. J. bioL Chem. 250, 3796-3806. Weber K. and Osborn M. (1969) The reliability of molecular weight determination by dodecyl sulfate polyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406-4412.