Characterization of human complement components C6 and C7

,Molrcu/ur Immunoloyy Vol. 19. No. Il. pp. 1425-1431, Printed in Great Britain.

0161.5890,,82,1 ll425-07$03.00/O 0 1982 Pergamon Press Ltd.

1982.

CHARACTERIZATION OF HUMAN COMPLEMENT COMPONENTS C6 AND C7 RICHARD MRC Immunochemistry

G. DISCIPIO*

and JEAN GAGNON

Unit, Department of Biochemistry, University Oxford OX1 3QU, U.K.

(First receired

16 June 1981; accepted

in recised,form

of Oxford,

8 Murch

South

Parks

Road,

1982)

Abstract-Human complement components C6 and C7 have been purified and characterized. Component C6 is a single-chain plasma protein of mol. wt 104,800 containing 3.87; carbohydrate and it has alanine as the amino terminal residue. Component C7 is a single-chain plasma protein of mol. wt 92,400 containing 6.4’4 carbohydrate and it has serine as the amino terminal residue. Primary sequence analysis failed to provide evidence of homology between these two proteins.

INTRODUCTION

Complement components C6 and C7 are two of the five plasma proteins that assemble sequentially to form the membrane attack complex of the complement system. This membrane attack complex is a multiprotein complex consisting of equimolar amounts of CSb, C6, C7 and C8 and three moles of C9 Kolb & Miiller(Podack et al., 1976a; Eberhard, 1975). When assembled on cells, the membrane attack complex leads to leakage of the cell membranes followed by cytolysis. Some characterization of components C6 and C7 has been performed and these proteins were found to have similar features. Components C6 and C7 have been reported to be single-chain glycoproteins of approximate mol. wt 11 l,OOO-125,000, and 102,000-121,000, respectively. Circular dichroism studies have suggested that both proteins contain 20-30x ordered structure (Podack er al., 1976b, 1979). no primary sequence However, to date, analyses have been published. Rabbit C6 appears to have similar properties to human C6 (Rother et al., 1966, Nelson & Biro, 1968). A genetic relationship for components C6 and C7 has been suggested on the basis of a combined deficiency of C6 and C7 and on the basis of genetic polymorphism studies on these proteins (Lachmann & Hobart, 1978; Lachmann et al., 1978). The objective of this work has been to * Address correspondence to: Richard DiScipio, Scripps Clinic and Research Institute, Department of Molecular Immunology. 1066 North Torrey Pines Road, La Jolla, CA 92037, U.S.A.

characterize further human ponents C6 and C7.

MATERIALS

AND

complement

com-

METHODS

Sepharose CL4B, dextran sulfate and Sephacry1 S-300 were obtained from Pharmacia Fine Chemicals, Hounslow, U.K. Benzamidine HCl and polybrene were obtained from Aldrich Chemical Co. Ltd, Dorset, U.K. [‘4C]p-Chloromercuribenzoate was a product of CEA, GifSur-Yvette, France. [14C]Iodoacetamide was obtained from Amersham, Radiochemicals, Amersham, Bucks., U.K. Iodoacetamide was purchased from BDH, Poole, U.K. Trypsin (DPCC-treated) was a product of Sigma Chemical Corp. Ltd, Poole, U.K. Cellulose thin-layer sheets were obtained from MacheryNagel, Diiren, West Germany. Quadrol was Alto, California. All other sequencing chemicals were obtained from Rathburn Chemicals, Peebleshire, U.K. Benzamidine-Sepharose was prepared as described by DiScipio er al. (1977). A plasma deficient in component C6 was made by passing 50 ml of BaCl,-treated human plasma containing 5 mM EDTA over a rabbit anti-human C6 antibody Sepharose column (2.5 x 20 cm). A plasma deficient in component C7 was made similarly by using a rabbit antihuman C7 antibody Sepharose column (2.5 x 20 cm). Haemolytic assays for C6 and C7 were performed by using EAC14 cells with human plasma deficient in C6 and C7 respectively. The gelatin Verona1 buffers that were used for the assays were made as described by Nelson et al. (1966). Human complement components C6 and C7 1425

1426

RICHARD

G. DISCIPIO

were purified as byproducts of the procedure (DiScipio, 1981) for the purification of components C3 and CS. The earlier steps included BaCI, treatment of one 1. of outdated human plasma, 5-12% polyethylene glycol 4000 precipitation, plasminogen depletion on lysinecolumn DEAE-Sephadex Sepharose and chromatography. The DEAE-Sephadex column (3.5 x 32cm) was equilibrated with buffer A: 25 mM pH 7.0, 0.075 M NaCl, imidazoleeH,P04, 20 mM e-aminocaproic acid, 10 mM EDTA, 2 mM benzamidine, sodium azide 0.01% (w/v), glycerol 20”/, (v/v). After sample application, the column was washed with 400ml of buffer A, and then a linear gradient consisting of 450 ml each of O-0.35 M NaCl in buffer A was used to develop the column. The C7 activity passed through in the latter half of the breakthrough. The C6 activity eluted in a broad zone between 0 and 0.15 M NaCl in buffer A. The first half of the C6 activity was pooled. The C7 pool was diluted one-to-one with distilled water and this pool was applied to a CM-Sephadex column (3.5 x 32cm) equilibrated in buffer A that was diluted one-to-one with distilled water. A linear gradient consisting of 400 ml each of O-O.4 M NaCl in the same buffer was applied. The C7 activity eluted between 0.1 and 0.2 M NaCI. This activity was separated effectively from the factor B activity by this column. The C7 pool was dialysed against buffer B: 15 mM imidazole-HCl, pH 7.4, 0.03 M NaCl, 2 mM benzamidine, 0.01% (w/v) sodium azide, and applied to a benzamidineesepharose column (1.6 x 20 cm) equilibrated in buffer B. After sample application a linear gradient consisting of 150 ml each of O-O.5 M NaCl in buffer B was employed to develop the column. The C7 activity eluted between 0.1 and 0.15 M NaCl in buffer B. The C7 was essentially pure at this stage by the criterion of SDSpolyacrylamide gel electrophoresis. The yield (10 mg from 1 1. of plasma) reflected about a 15% recovery. The C6 fractions from the DEAE-Sephadex column were applied to a dextran sulfate Sepharose column (1.6 x 20 cm) that was equilibrated in buffer A. A linear gradient consisting of 200 ml each of o-0.6 M (NH&SO4 in buffer A was used to develop the column. The C6 activity eluted between 0.5 and 0.6 M (NH&SO,. The C6 activity was pooled and passed through a rabbit anti-human IgG Sepharose column (2.6 x 15 cm) that was equi-

and JEAN

GAGNON

librated with 0.5 M (NH&SO4 in buffer A. The C6 activity was pooled and this protein was judged pure by the criterion of SDSpolyacrylamide gel electrophoresis. The yield was about 8 mg reflecting a recovery of about 11%. The C5b6 complex was formed by incubating component C5 (1 mg/ml final concentration) with component C6 (0.6 mg/ml final concentration) with a l/50 molar ratio of the cobra venom factor C5 convertase (CVFBb) for 10 hr at 37°C in 10 mM imidazoleeHCl buffer, pH 7.3, 0.075 M NaCl. The C5b6 complex was assayed for haemolytic activity by the following procedure. Aliquots containing between 1 and 50 ng of C5b6 were preincubated with 10’ EAC143 cells for 5 min at 37°C in VBSEDTA: 5 ng of C7, 5 ng of C8, and 15 ng of C9 were then added to each tube. After 30 min at 37-C the amount of lysis was determined by absorption at 413 nm. SDS-polyacrylamide gel electrophoresis was performed by the method of Weber & Osborn (1969) as modified by Kisiel et ul. (1976). The mol. wts of components C6 and C7 were estimated by interpolation from calibrated plots of the In (mol. wt) vs distance of migration using the following standards: factor H (mol. wt 160,000); phosphorylase a (mol. wt 97,000); bovine serum albumin (mol. wt 68,000); ovalbumin (mol. wt 45,000); bovine carbonic anhydrase (mol. wt 29,000). Amino acid analysis was carried out by the method of Moore & Stein (1963) and Spackman et ul. (1958). Hexosamine was determined after the proteins were hydrolysed with 2 M HCl for 16 hr at 110°C. Half-cystine was determined as cysteic acid by the method of Hirs (1967). The amino acid and hexosamine samples were analyzed on a Durrum D500 amino acid analyzer. Tryptophan content was quantitated by the method of Edelhoch (1967). Sialic acid was determined by the method of Warren (1959), and neutral sugar was analysed by the method of Dubois (1954). The partial specific volumes were calculated from the amino acid (Cohn & Edsall, 1943) and carbohydrate content (Gibbons, 1966). Attempts to determine the content of free sulphydryl groups in components C6 and C7 were performed by measuring the incorporation of [ “C]iodoacetamide or [’ 4C]p-chloromercuribenzoate into these proteins. The iodoacetamide reaction was performed essentially

Characterization of Human

Complement

as described by Crestfield et ul. (1963). Components C6 and C7 (600 pmoles each) were incubated with 120 nmoles of [ “C]iodoacetamide (50 nci~nmole) in 200 ,~l of 0.1 M Tris-HCl buffer, pH 8.6, 10mM EDTA in the presence and absence of 6 M guanidine-HCI. The reaction with p-chloromercuribenzoate was performed by incubating 600 pmoles each of C6 and C7 with 75 nmoles of [i4C]p-chloromercuribenzoate (15 nCi/nmole) in 200 ~1 of 20mM Mes-Tris buffer, pH 5.0, in the presence and absence of 6 A4 guanidine-HCl. The incorporation of the radiolabel into these proteins was measured by liquid scintillation counting after the samples were dialysed. Sedimentation equilibrium ultracentrifugation was performed by the method of Yphantis (1964) using a Beckman Model E analytic ultracentrifuge that was equipped with a photoelectric scanner and an electronic speed control For both C6 and C7 the motor speed was 16,00Orev/min, and three protein concentrations were used (0.3, 0.2 and 0.15 mg/ml). Sedimentation velocity runs were performed at 52,000 rev/min to determine the sedimentation coefficients of these proteins. The diffusion coefbcients were determined from gel filtration (Ackers, 1975) of C6 and C7 on a calibrated Sephacryl S300 column (2.6 x 95 cm). The calibration standards were: IgM, Dzo,w = 1.7 x 10e7 cm2/sec; apoferritin, IgG, Dzo,, = D20,w = 3.6 x lo-‘cm’/sec; 4.0 x 1O-7 cm”/sec; catalase, D20,w = 4.2 x 10e7 cm2,%ec; bovine serum albumin, D20,w = 5.9 x IO-’ cm’/sec; ovalbumin, DJo,,, = 7.8 x 1O- ’ cm’/sec. The frictional ratios were calculated as described by Ackers (1975). The extinction coefficients were determined by employing a Beckman Model E analytical ultracentrifuge to determine protein concentration from synthetic boundary experiments (Babul & Stellwagen. 1969). The OD at 280 nm was corrected for Rayleigh light scattering as described by Shapiro & Waugh (1966). Amino-terminal amino acid sequences were determined by automated Edman degradation in a Beckman 890~ sequencer by the methodology described in Johnson et al. (1980). About 30 nmoles each of C6 and C7 were analyzed. RESULTS

Identijicution Component

of components

C6 and C7

C6 was identified

on the basis of

Components

C6 and C7

1427

its ability to restore the haemolytic capacity to C6-deficient rabbit serum and C6-depleted human plasma. Component C6 that was purified by the method described in this paper contained 1.0 x lo6 CH,, units of haemolytic activity per milligram of protein. This purified protein was devoid of any detectable haemolytic activity of C3, C5, C8, C9 or factor B. Component C6 could complex with nascent C5b to form the C5b6 complex, which exhibited a sedimentation coefficient of 11.2S, confirming an earlier report (Podack et al., 1978). Component C7 was identified on the basis of its ability to restore the haemolytic capacity to C7-depleted human plasma. Component C7 that was isolated by the method described in this paper contained 1.2 x lo6 CH,, units of haemolytic activity per milligram of protein, and this purified protein was devoid of any detectable haemolytic activity of C3, C5, C6, C8, C9 or factor B. Component C7 could complex with C5b6 resulting in the loss of both C7 and C5b6 haemolytic activity as has been previously observed (Thompson & Lachmann, 1970). The (C5b67), complex had an observed sedimentation coefficient of greater than 2OS, confirming earlier findings (Podack et ctl., 1980). SDS-pol~N1~r~lumide gel electrophoresis SDS-polyacrylamide gel electrophoresis patterns of components C6 and C7 are shown in Fig. I. It is observed that both C6 and C7 are single-chain proteins. The apparent mol. wt by this criterion of reduced C6 is 120,000, and that of reduced C7 is 105,000.

The amino acid and carbohydrate compositions of components C6 and C7 are shown in Table 1. There were no unusual features in the compositions of these proteins. After mol. wt adjustments of the amino acid determinations of Podack et crl. (1979), several differences are noted in comparison with the data presented in Table 1. The adjusted values from Podack et al. (1979) for the content in C6 or half-cystine (48.9) and the content in C7 of half-cystine (40.0), tryptophan (17.1) and arginine (37.7) are higher than the corresponding values in Table 1. The reason for these differences is not known. There were no detectable free sulphydryl groups in either C6 or C7. Both C6 and C7 are glycoproteins with C6 having 3.87; carbohydrate and C7 having 6.4:< carbohydrate.

1428

RICHARD

G. DISCIPIO

and JEAN

GAGNON

Fig. 1. SDS-polyacrylamide gel electrophoresis of C6 and C7 (UP;, polyacrylamide gels and 25 pg of protein applied to each gel). The identity of the gels is as follows: component C6 (unreduced); component C6 (reduced); component C7 (unreduced); component C7 (reduced).

The physical parameters of components C6 and C7 are shown in Table 2. Several methods were used to estimate the mol. wts of components C6 and C7. The most reliable method, sedimentation equilibrium ultracentrifugation, provided a mol. wt for C6 of 104,800 and for C7 of 92,400. These values are somewhat lower than the values determined by SDS-poiyacrylamide gel electrophoresis, but because C6 and C7 are glycoproteins the mol. wt estimates from gel electrophoresis analyses are expected

to be higher than the correct values, as giycoproteins bind less SDS than apoproteins (Segrest & Jackson, 1972). Assuming a normal degree of hydration, the frictional ratios of 1.47 for C6 and I .36 for C7 suggest that both structures possess some degree of asymmetry with the C7 being the more globular of the two proteins. The diffusion coefficients reported here are somewhat higher than the values of Podack et ul. (1976b) for C6 (DZO,w= 4.09 x 10W7cm”/sec) and C7 (D20,w = 4.23 x 1O- ’ cm’jsec).

Characterization

of Human Complement Components C6 and C7

1429

Table 1. The amino acid and carbohydrate content of components C6 and C7 C6

c7

(residues/mole)

(residues/mole)

Asx Thr Ser Glx Pro Gly

92.1 54.2 74.5 120.9 54.9 75.9

60.3 53.5 76.4 88.2 66.6 68.1

Ala ‘iCYS Val

53.7 27.6 48.6

54.0 23.9 59.7

Met Ile Leu Tyr Phe His LYs

6.8 37.8 75.7 26.1 30.0 17.6 64.3

6.0 16.5 66.7 26.4 29.2 14.2 42.7

Arg

37.8 10.2 908.7 100,200

Tm

residues apoprotein mol,wt apoprotein hexose N-Acetyl-glucosamine Sialic Acid residues carbohydrate mol.wt.carbohydrate

(residues/mole) 11.1 4.1 4.1 19.3 3,999

percent carbohydrate mol_wt alvcowrotein

3.0% 104.200

The extinction coefficients reported in Table 2 for C6 and C7 differ by about two-fold from the values published by Podack et al. (1979). They calculated that A@%“,,, Fm was 17.1 for C6 and 19.2 for C7.

30.6 11.7 794.7 86,200 (residues/molf$ 14.4 9.7 5.1 29.2 5,909 6.4% 92.100

~~~~ary sequence analyses

The primary sequence analysis of the aminoterminals of components C6 and C7 are presented in Fig. 2. It is observed that there is no homology between the amino-terminals of

Table 2. Physical parameters of components C6 and C7

Sedimentation coefficient,a 2. w

‘

C6 6.09

c7 5.6s

diffusion coefficient, D2o w

4.7 x lo"? cm'fsec

5.3 x lo-7czn2fsec

partial specific volume, 5

0.72Gml/gm

0.721 ml/gm

mol.wt, from s20 w and D20 w

111,100 daltons

92,200 daltons

mol,wt,

120,000 daltons

105,000 daltons

t

,

from SDS-polyacrylamide gels

mol,wt, sedimentation equilibrium extinction coefficient, A 1% 280Nn lcm

,

frjctional

ratio, f/f0

isoelectric point* ,'I *Datafrom Podacketal.(1979).

104,800;t5,700 daltons

92,400+ 3,300 daltons

9.3

9.9

1.47

1.36

6.15-6.55

6.X-6.70

RICHARD

1430

I

G. DISCIPIO

and JEAN

5

GAGNON

JO

Vat/Met

Am

Cys

Gln

Trp

Asp

Phe

Tyr

Ala

Pro

?

2:r

?

ASP

His

Tyr

Ak

GLu

(Leuf

Gln

?

Tyr

Pro

Asn

Gty

Asn

Gly

C7)

Ser

Ser

Pro

C6)

Ato

Phe

C71

16 Glu

Cys

031

Lys

Fig. 2. The amino-terminal sequences of components C6 and C7. Components C6 and C7 were subjected to primary sequence analysis as described in Materials and Methods. ? refers to amino acid residues that have not been unambiguously identified; residues in brackets are tentative assignments.

these two structures. Furthermore, these sequences are not homologous to any protein known to us. DISCUSSION

Human complement components C6 and C? are single-chain proteins of mol. wt 104,800 and 92,400, respectively. Both proteins are glycoproteins with C6 having 3.8% carbohydrate and C7 having 6.4:1, carbohydrate. Both proteins possess some degree of asymmetry with component C6 being the more ellipsoid of the two proteins. The extinction coefficient is an important parameter for the evaluation of protein concentration and sto~ch~ometries of molecular interaction. The values reported in Table 2 for C6 (A;& nm,lcm = 9.3) and C7 (A$$,.,,, cm = 9.9) are each about half the values published by Podack et al. (1979). These investigators used amino acid analysis to quantitate protein concentration. This method can result in an underestimate of protein concentration, resulting in an overvalue in the determined extinction coefficient. Podack et al. (1979) have suggested homology between C6 and C7 based on similarities in the amino acid composition, size, chain structure, and circular dichroism characteristics; and on the results of genetic polymorphism studies that suggested linkage of the genes of C6 and C7 (Lachmann & Hobart, 1978; Lachmann et al., 1978). However, when considering the properties of mol. wt, frictional ratio, isoelectric point, carbohydrate content, and diffusion coefficient, component C7 shows more similarity to factor B (Curman et al., 1977) than it does to component C6. There exists no evidence of homology between C6 and C7 based on amino-terminal sequence anaiyses (Fig. 2). It may be true that C6 and C7 share regions of internal sequence homology. The evaluation of this possibility awaits further work. Procedures for the purification of human

complement components C6 and C7 are described in Materials and Methods. These proteins are purified from the same preparation that is used in the isolation of C5, C3, factor B, factor H, properdin, plasminogen and the vitamin K-dependent proteins (DiScipio et al., 1977; DiScipio, 1981). This allows for optimal use of human plasma for the isolation of several plasma proteins. The complete structural analysis of C6 and C7 would be of great interest not only for possible homology relationships, but also because this information would provide a structural basis for understanding the molecular dynamics of the membrane complex, where five soluble proteins (C5b, C6, C7, C8 and C9) associate to form an integral membrane multiprotein complex. At the time that this manuscript was being revised for publication, a paper appeared by Kolb et u/. (1982) dealing with the characterization of complement component C6. In that paper the authors suggested that component C6 was a serine active site protease. However, the C6 that we purified was not inactivated by 2 mM diisopropyl-fiuoro-phosphate or 0.2 mM phenyImethylsulphony1 fluoride. Our C6 exhibited no amidase activity toward the following synthetic substrates: H-D-valyl-L-leucyl+lysine-p-nitroanilide dihydrochloride; H-D-prolylL-phenylalanyl-L-arginine-p-n~troanilide dihydrochloride; ~-benzoyl-L-phenylalanyl-L-valylL-arginine-p-nitroanilide hydrochloride. For the characterization of the C6 protein Kolb et elf. (1982) obtained some results that varied from our findings. (a) Kolb et al. (1982) observed 4.7-6.3 free sulphydryl groups in C6; however, we did not observe any free sulphydryl groups in this protein. (b) The carbohydrate content of 11.39; published by Kolb ef ul. (1982) differs significantly from our findings of 3.8%. (c) For the amino acid composition Kolb et al. (1982) observed 6.25 residues of halfcystine/lOO residues of apoprotein and 2.52 residues of tryptophan/iOO residues. This con-

Characterization

of Human

Complement

trasts with our results of 3 residues of halfcystinejlO0 residues and 1.1 residues of tryptophan/lOO residues. At this time it is not clear why such major differences in the characterization of this protein should be observed by different investigators. It is hoped that future work will clarify this situation.

Acknowledgements-The authors wish to express their gratitude to Jane Heritage for excellent technical assistance with the analytical ultracentrifuge. Thanks also go to Mr Tony Ciascoyne for performing the automated amino acid analyses. We are also grateful to Mr Tony Willis for technical assistance with the automated protein sequencer. We would like to express our thanks to Prof. Peter J. Lachmann for a gift of rabbit C6 deficient plasma. Our gratitude also goes to MS Beryl MotIat for excellent technical assistance.

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Components

C6 and C7

1431

Kisiel W., Ericsson L. H. & Davie E. W. (1976) Proteolytic activation of protein C from bovine plasma. Biochemisrry 15, 489334900. Kolb W. P., Kolb L. M. & Savary J. R. (1982) Biochemical characterization of the sixth component (C6) of human complement. Biochemistry 21, 294301. Kolb W. P. & ‘duller-Eberhard H. J. (1975) The membrane attack nlechanism of complement: isolation and subunit composition of the C5b9 complex. J. exp. Med. 141, 724-735. Lacbmann P. J. & Hobart M. J. (1978) C66C7: a further complement supergene. J. Immun. 120, 1781&1782. Lachmann P. J., Hobart M. J. & Woo P. (1978) Combined genetic deficiency of C6 and C7 in man. Clin. esp. Immun. 33, 193-201. Moore S. & Stein W. H. (1963) Chromatographic determination of amino acids by use of automatic recording equipment. ~~f,Z~. Enqm. 6, 819-831. Nelson R. A. & Biro C. E. (1968) Complement components of a haemolytically deficient strain of rabbits. Immunology 14, 5255540. Nelson R. A., Jensen J., Gigli I. & Tamura N. (1966) Methods for the separation. purification and measurement of nine components of hemolytic complement in guinea pig serum. Immunochemistry 3, 11 t-135. Podack E. R., Biesecker G. & MtilIer-Eberhard H. 3. (19760) Membrane attack complex of complement: generation of high affinity phospholipid binding sites by fusion of five hydropilic plasma proteins. Proc. nufn. Acud. Sci. U.S.A. 76, 897-901. Podack E. R., Esser A. F., Biesecker G. & MilllerEberhard H. J. (1980) Membrane attack complex of complement. J. exp. Med. 151, 301-313. Podack E. R., Kolb W. P., Esser A. F. & Miiller-Eberhard H. J. (1979) Structural similarities between C6 and C7 of human complement. /. Immun. 123, 1071-1077. Podack E. A., Kolb W. P. & Mtiller-Eberhard H. J. (19766) Purification of the sixth and seventh components of human colnplement without loss of haemolytic activ*ity. .f. lmmuii. 116, 263-269. Podack E. R., Kolb W. P. & Miiller-Eberhard H. J. (1978) The complex: formation, isolation and inhibition of its activity by lipoprotein and the S protein of human serum. d. Immun. 120, 1841-1848. Rother K., Rother U., Miiller-Eberhard H. J. & Nilsson U. R. (1966) Deficiency of the sixth component of complement in rabbits with an inherited complement defect. J. e.xp. Med. 124, 7733785. Segrest J. P. & Jackson R. L. (1972) Molecular weight determination of glycoproteins by polya~rylamide gel electrophoresis in sodium dodecyf sulfate. Meth. Enzym. 28, 54-63. Shapiro S. S. & Waugh D. F. (1966) The puriiication of human prothrombin. Thromh. Diath. haemorrh. 16, 469-490. Spackman D. H., Stein W. H. & Moore S. (1958) Automatic recording apparatus for use in the chromatography of amino acids. Analyt. Chem. 30, 1190. Thompson R. A. & Lachmann P. J. (1970) Reactive Iysis: the complement-mediated lysis of unsensitized cells. f. exp. Med. 131, 629-641. Warren L. (1959) The thiobarbituric acid assay of sialic acids. J. bid. Chem. 234, 1971-1975. Weber K. & Osborn M. (1969) The reliability of molecular weight determinations by sodium dodecyl sulfate polyacrylamide gel electrophoresis. 1. bid. Chem. 244, 4406-44 12. Yphantis D. A. (1964) Equilibrium ultracentrifugation of dilute solutions. ~i~c~~emisfry 3, 297-317.