Studies on the sub-unit structure of human properdin

Iramunocheraisrry, 1974, Vol. 11, pp. 361-368. Pergamon Press. Printed in Great Britain

STUDIES ON THE SUB-UNIT STRUCTURE OF HUMAN PROPERDIN*t J. ODURO MINTA and IRWIN H. LEPOW Department of Pathology, Division of Experimental Pathology, University of Toronto, Toronto, Canada and Department of Medicine, The University of Connecticut Health Center,.Farmington, Connecticut 06032, U.S.A. (First received 5 November 1973; in revised form 26 December 1973)

Abstract--Human properdin contains 9.8 % carbohydrate consisting of 3.8 % hexose, 0.7 % fucose, 1-5% hexosamine and 3'8% sialic acid. The partial specific volume for properdin calculated from amino acid composition and carbohydrate analysis was 0'7 cc/g. The tool. wt determined by high speed equilibrium ultra-centrifugation was 184,000 _+ 12,000. Results from SDS-polyacrylamide gel electrophoresis and SDS or guanidine agarose chromatography provide consistent evidence that properdin is comprised of four non-covalently linked subunits of similar mol. wt (46,000). The subunits combine in aqueous media to form a dimer (4-6 S, mol. wt = 88,000). On equal weight basis, the dimer has about 70-85 per cent of the antigenic properties and half the hemolytic activity of the native protein.

INTRODUCTION Properdin was described by Pillemer et aL (1954) as a protein of normal human serum which interacts with zymosan at 17°C in the presence of magnesium ions and certain non-dialyzable serum factors to form a complex that is capable of cleaving C3 :~ to C3a and C3b. The non-dialyzable factors were found to be a hydrazine-sensitive protein, designated Factor A (Pensky et al., 1959), and a heat-labile protein designated Factor B (Blum et al., 1959). Factor A is probably identical to C3 or to a large cleavage product of C3 resembling C3b and designated as activated hydrazinesensitive Factor, HSFa (M~ller-Eberhard and Gotze, 1972; Goodkofsky et al., 1973). Factor B has been identiffed with a serum proenzyme termed C3 proactivator, C3PA (Gotze and Miiller-Eberhard, 1971; Goodkofsky and Lepow, 1971) and with glycine-rich fl-glycoprotein, GBG (Alper et al., 1973). It has been shown by Gotze and Miiller-Eberhard (1971) that C3PA is cleaved and converted to an active enzyme, C3 activator (C3A) that is capable of cleaving C3 to C3a and C3b. Activation of C3PA was shown to be effected by a 3S serum enzyme designated C3 proactivator convertase (C3PAse) which * This investigation was supported by NIH Grant AI 08251 and the Canadian Medical Research Council, Ottawa, Canada. t Presented in part at the Fifth InternationalComplement Workshop, Coronado, California, 23 February, 1973. ~/Abbreviations used : The complement nomenclature follows the recommendations of the WHO (Immunochemistry (1970) 7, 137). C3 is the third component of serum complement; C3a and C3b are the fragments of C3 produced during the complement fixation reaction. C3PA, C3 Proactivator; C3A, C3 activator; C3PAse, C3 Proactivator convertase; HSFa, activated hydrazine-sensitivefactor.

required for its action, the presence of metal ions and HSFa (Miiller-Eberhard and Gotze, 1972). It has recently been suggested that properdin may act as an initiating agent in the alternate pathway of complement activation and may participate in the activation of C3PAse (Gotze and Miiller-Eberhard, 1973). However, the exact role and the mechanism of action of properdin in this reaction remains to be elucidated. We have, therefore, extended the work of Pensky et al. (1968) on the physico-chemical properties of human properdin with the goal of understanding its role and mechanism of action in the alternate pathway of complement activation. MATERIALS AND M E T H O D S

Ultra pure guanidine hydrochloride was obtained from Schwarz-Mann. Sodium dodecyl sulfate (SDS) was purchased from the British Drug House Ltd., and was reerystallized from ethanol. Sepharose 4B, Sephadex G-200, Blue Dextran 2000 and chymotrypsinogen A were products from Pharmacia. fl-galactosidase,phosphorylase a, pepsin and cytochrome c were obtained from Sigma Chemical Co. and were of the highest purity available. Five times crystallized ovalbumin was from Pentex Research Products Division. Human albumin was from Behringwerke and DNP-alanine from Mann Research Laboratories. 2-mercaptoethanol, acrylamide and N, N'-methylene-bisacrylamide was obtained from Eastman Organic Chemicals and N, N, N', N'-tetramethylenediamine was from Canalco Industrial Corporation. All other chemicals were of reagent grade and were used without further purification. Properdin Properdin was purified from human serum by the method of Pensky et al. (1968). The preparations used in this study were homogeneous as judged by immunoelectrophoresis, analytical ultracentrifugation and gel electrophoresis. Prop-

361

362

J. ODURO MINTA and IRWIN H. LEPOW

erdin concentration was measured by solid-phase radioimmuno-assay (Minta and Lepow, 1972; Minta et al., 1973) and properdin was assayed for functional activity by the hemolytic assays of Pillemer et al. (1956). Purified properdin was trace-labelled with 12sI iodine by the iodine monochloride method of Helmkamp et al. (1960) as described by Minta et al. (1973). Antiserum

Monospecific rabbit antiserum to human properdin was prepared as described by Pillemer et al. (1957). Carbohydrate analyses Carbohydrate analyses on solutions ofproperdin (1 mg/ml} were performed by standard chemical methods as already described (Minta et al., 1972). Hexose was determined by the Orcinol-sulfuric acid method (Winzler, 1955) using D-galactose as standard. Fucose was assayed by the cysteine-sulfuric acid reaction (Dische and Shettles, 1948) using L-fucose as standard. Sialic acid was determined by the thiobarbituric acid reaction of Warren (1959) with N-acetyl neuraminic acid as standard. Hexosamine was measured by the p-dimethylamino benzyaldehyde method (Boas. 1953)with l~-glucosamine as standard. Amino acid composition

Amino acid analysis was performed on a Beckman Automatic amino acid analyzer, Model 120C, according to Spackman et al. (1958). Samples (1 mg/ml) were dialyzed against distilled water and dried under vacuum. The residue was hydrolysed by treatment with 5.7 N HCI at 110°C in vacuo for 22 hr prior to analysis. Calculation o f the partial specific vol. (~) The partial specific volume of properdin was estimated from the amino acid composition by assuming a simple additivity of the specific volume of the amino acid residues (Cohn and Edsall, 1943) and was corrected for carbohydrate content. Amino terminal analysis Amino terminal analysis was performed according to the Dansyl-chloride (1-dimethylamino-naphthalene-5 sulfonyl chloride) method of Gray (1966). The protein 0 - 2 g) was dissolved in 0.02 M sodium phosphate buffer, pH 8.2, which was 5 M in urea, 25 per cent by volume dimethylformamide and 10 per cent by volume acetonitrile. The protein was reacted at room temperature for 30 min with a large excess of dansyl chloride in acetone (5 rag/rag protein). The dansylated protein was isolated by precipitation with 10Vo trichloroacetic acid, washed twice with acetone, dried in vacuo and bydrolysed for 5 hr at ll0°C in constant boiling HC1. The dried hydrolysate was extracted with 50 #1 ethyl acetate which had been saturated with water just before use and this solution was used for chromatography on polyamide plates (Ching Chen Trading Co., Taiwan), (Woods and Wang, 1967). Chromatograms were developed with 1.570 aqueous formic acid in the first dimension, followed after drying by benzene-acetic acid (9:1) in the second and ethyl acetate-methanol-acetic acid (20:1 : 1) in the third. The spots were identified by comparing their R s values with those of standard dansyl-amino acids. Ultracentrifugation studies Ultraeentrifugations were performed in a Beckman Spinco Model E afialytical ultracentrifuge equipped with both absorption and Rayleigh interference optics.

The sedimentation velocity of properdin in phosphate buffered saline, PBS, (y 0.15 pH 7.4) was determined according to the procedures of Schachman (1957) at 60,000 rev/min and 17_+0.1 °C. The sedimentation coefficients were corrected for the effects of temperature, viscosity and density of the solvent systems. No correction was made for the dependence of sedimentation coefficient on protein concentration. Molecular weight was determined in the same buffer by the high speed equilibrium method of Yphantis (1964). The rotor was maintained at 17±1°C and was operated at speeds between 15,000 and 22,000 rev/min and protein concentrations of 0.05-0-170 were used. The high speed equilibrium data were measured from O.D. tracings or from photographs of Rayleigh interference fringes using Gaertner microcomparator and were processed using the computer program of Roark and Yphantis (1968) to obtain weight-average tool. wt as a function of protein concentration. Gel electrophoresis Analytical polyacrylamide gel electrophoresis was performed at room temperature on 770 acrylamide gels in Tris-HCl-glycine buffer, pH 8'6, according to Davis 0964). Protein bands were detected by staining the gels in 0-170 solution of Amido Black in 770 acetic acid for 7-18 hr followed by electrolytic removal of excess unbound dye. Polyacrylamide disc gel electrophoresis in the presence of 0'1~o sodium dodecyl sulfate was carried out at room temperature on 1070 acrylamide gels according to the method of Weber and Osborne (1969). The proteins were dissolved at about 1 mg/ml in 0.01 M sodium phosphate buffer, pH 7.2 containing 1~ SDS and 1~o mercaptoethanol and were incubated at 37°C for 1 hr or at 100°C for 5 rain before electrophoresis. Electrophoretic migration was determined on gels containing both the test protein and cytochrome c or bromophenol blue as a visible marker and was expressed relative to the marker. Electrophoresis was for 5 hr at a current of 8 mA/gel. When 125I trace labelled proteins were employed, the gels were sliced into 2 mm thickness and counted for radioactivity. Otherwise the gels were stained in 0"2570Coomassie Brilliant Blue for 18 hr at room temperature and then destained electrophoretically in acetic acid-methanol-water mixture (1-5:1:17.5). The tool. wt of properdin in sodium dodecyl sulfate was determined from a calibration curve of log mol. wt vs electrophoretic mobilities of standard proteins. Gel filtration Gel filtration was performed on Sephadex G-200 columns in phosphate buffered saline (PBS) at 4°C. Gel filtration of denatured proteins on 4% agarose columns (Sepharose 4B) in 6 M guanidine hydrochloride or in 1% sodium dodecyl sulfate in the presence or absence of 10-2 M dithiothreitol were performed at room temperature as described by Davison (1968) and Fish et al. (1969), Fractions (1-ml aliquots) were collected directly into 12 x 75 mm disposable glass tubes and counted for radioactivity in an automatic gamma counter (Model 4230, Nuclear Chicago Corp., Des Plaines, Illinois). The u.v. absorbances (A2s0 #m) of the fractions were measured to determine protein concentration. Reference proteins of known tool. wt were used to calibrate the Sephadex G-200 and the Sepharose 4B columns (Andrews, 1965; Davison, 1968; and Fish et al., 1969). The void volume of the column (V0) was determined

Sub-unit Structure of Properdin by measuring the elution volume of Blue Dextran 2000 at 625 /~m. The total volume of the column (V~) was determined by measuring the elution volume of DNPalanine at 330 #m. The distribution coefficient, Kd, is defined as K,i = ( l i e - Vo)/(Vi- 1Io) where Ve is the elution volume of the protein. From calibration curves of log mol. wt of reference proteins against distribution coefficients, the mol. wt for properdin was estimated. RESULTS Carbohydrate composition Table 1 shows the results of carbohydrate analyses of properdin. The total carbohydrate content was 9.8 per cent suggesting that properdin is a glycoprotein. Amino acid composition Table 2 shows the average of the amino acid compositions on two different preparations of properdin. The amino acid compositions were in general very similar and in no case was the difference between the corresponding residues greater than 1 mole per cent. The amino acid composition indicated a high molar content of glutamic acid, glycine and proline and a low molar

363

concentration of tyrosine, phenylalanine, isoleucine and methionine. The ratio of acidic (Glu, Asp) to basic (Arg, Lys)amino acids was about 1.2. Tryptophan content was not determined and the molar concentration of half cystines listed in Table 2 is quite approximate since the protein was not subjected to performic acid oxidation prior to hydrolysis. However, the data obtained from gel filtration and gel electrophoresis studies in dissociating solvents preclude the possibility that interchain disulfide bonds are present in properdin. The amino acid composition was used to calculate the partial specific volume (V) according to the method of Cohn and Edsall (1943). From the data on Table 2, the partial specific volume of properdin was determined to be 0-7 cc/g after correction for carbohydrate content. In three separate experiments, we were unable to detect any free amino terminal amino acid residue in properdin using the dansyl chloride method (Gray, 1966).

APPROACH TO EQUILIBRIUM ULTRACENTRIFUGATION OF ZYMOSAN-PURIFIED HUMAN PROPERDIN

Table 1. Carbohydrate content of zymosan-purified human properdin Hexose Hexosamine Sialic acid Fucose

4.

3.8 1.5 ~o 3.8 ~o 0.7~

3.

9-8% 2.

Table 2. Amino acid composition ofzymosanpurified human properdin Amino acid Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half cystine Valine Methionine lsoleucine Leucine Tyrosine Phenylalanine

v

Moles ~o" 4.70 2-73 8.85 4-74 5.70 7.26 15.30 11-60 12.74 5-58 4-96 4.63 1.43 1.32 5.07 1.45 1.90

Average of two preparations of properdin. Partial specific volume (V)calculated from amino acid and carbohydrate analyses = 0-70 cc/g.

0-

-I

49.~

56.0

50.5

51,0

51.5

52.0

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Fig. 1. Representative plot of high speed sedimentation equilibrium ultracentrifugation run used in the calculation of the mol. wt of properdin. The data was obtained from interference photographs of the equilibrium distribution in the cell at 48 hr (speed = 12,000 rev/min, T= 16-9°C, protein concentration in PBS = 1 mg/ml) expressed as log of concentration (In c) vs the square of distance (r) from the axis of rotation. The units of concentration are mm of fringe displacement. The tool. wt calculated from the slope of the plot was 186,000.

364

J. ODURO MINTA and IRWIN H. LEPOW

U ltracentrifugation studies on properdin Sedimentation velocity studies on properdin at protein concnt of 0"5 and l mg/ml both gave an S~ow value of 5.2 which was in agreement with the findings of Pensky et al. (1968). The protein sedimented as a single symmetrical boundary and there was no evidence of aggregate formation. The mol. wt of properdin in phosphate buffered saline was estimated by sedimentation equilibrium experiments using the high speed technique (Yphantis, 1964). A typical sedimentation equilibrium data displayed as a plot of log concentration vs the square of the distance from the axis of rotation (Fig. 1) was linear indicating homogeneity and the absence of any protein association or dissociation. From 11 determinations on three different preparations, the average value of the mol. wt of properdin was calculated to be 184,000 -1- 12,000.

POLYACRYLAMIDE DISC ELECTROPHORESIS OF ZYMOSAN-PURIFIED

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Polyacrylamide gel electrophoresis in Tris-~31ycinebuffer, pH 8"6. Figure 2 shows the electrophoretic mobility of properdin on 7% acrylamide gel in Tris-glycine buffer, pH 8.6. The sample was applied between the two halves of

Fig. 3. Polyacrylamide gel electrophoresis of properdm in the presence of sodium dodecyl sulfate. Electrophor~is was carried out on 10% acrylamide gels at 8 mA per gel for 5 hr at room temperature. Electrophoretic migration of properdin was determined on gels containing both properdin (containing a trace of ~2sI properdin) and cytochrome and was expressed relative to cytochrome c. The gels were stained in 0.25% Coomassie Brilliant Blue in acetic acidmethanol-water mixture ( 1.5:1 : 17.5)overnight and destained electrophoretically in 7% acetic acid or sliced into 2 mm thicknesses and counted for radioactivity. the gel and it is seen that properdin has a very slow cathodal mobility.

Polyacrylamide gel electrophoresis in the presence of SDS Figures 3a and 3b show the electrophoretic migration of properdin containing a trace amount of radiolabeled properdin on polyacrylamide gels in the presence of SDS. Electrophoretic migration was determined on gels containing both protein and cytochrome c as a visible marker (Fig. 3b). Properdin migrated as a single protein band and was completely overlapped by the radiolabeled properdin peak. The mol. wt of dissociated properdin was estimated by comparing the relative migration distances of marker proteins of known mol. wt with those of properdin. A typieal calibration curve is shown in Fig. 4 as a plot of the log of the tool. wt vs the relative mobility for each of the reference proteins. A mean mol. wt of 46,000 was estimated for dissociated properdin and this value did not change when 2-mercaptoethanol (0.1 M) was excluded from the experiment. These findings suggest that properdin is composed of four non-covalently linked subunits of similar mol. wt. Fig. 2. Polyacrylamide gel electrophoresis of properdin on 7% acrylamide gels in Tris-glycine buffer, pH 8-6. Electrophoresis was carried out at 5rnA/gel for 90 min at room temperature.

Gel filtration in the presence of guanidine hydrochloride and sodium dodecyl sulfate The elution profile obtained when 12sI properdin was subjected to gel filtration on Sephadex G-200 in

Sub-unit Structure of Properdin Studies on the association of the subunits

POLYACRYLAMIDE GEL ELECTROPHORESIS OF Z Y - H U M A N PROPERDIN IN 11[ SDS

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Fig. 4. Polyacrylamide gel electrophoresis of properdin in the presence of sodium dode~yl sulfate and proteins of known mol. wt. The data are graphed as a semi-logarithmic plot of the mol. wt of the proteins vs their relative mobilities. The relative mobilities were determined by reference to the electrophoretic mobility of cytochrome ¢. The relative mobility of properdin is indicated by arrows and corresponds to a mol. wt of 46,000.

PBS is shown in Fig. 5a. t2sI properdin was ¢luted in a single symmetrical peak before bovine ~G. However, on gel filtration on a Sepharose 4B column in 6 M guanidine hydrochloride (Fig. 5b) properdin was eluted as a single peak in the region of ovalbumin. Estimation of the mol. wt of the dissociated properdin by measuring the distribution coefficients of several standard proteins of known reel. wt and plotting these values against the log of their mol. wt (Fig. 5b, insert), (Davison, 1968) gave a mean subunit reel. wt of 45,500±3,000. This value was not altered by the addition of dithiothreitol (10 -2 M) to the ¢lutrient confirming our findings from SDS gel elcctrophoresis that properdin is a tetrainer of four non-covalently linked subunits of comparable or identical reel. wt. 12sI properdin was also eluted from SDS (1%) gel chromatography on 4% agarose columns in the presence or absence of 10 -2 M dithiothreitol as a single radioactivity peak with elution volume (distribution coefficients) similar to that of ovalbumin. The column calibrated by relating the distribution coefficients of standard proteins to the log of their mol. wt gave a subunit mol. wt of 46,000 for properdin. This value was in good agreement with those obtained from agarose chromatography in 6 M guanidine hydrochloride (45,500_+ 3,000) and SDS gel electrophoresis (46,000).

When the dissociated properdin ¢luted from guanidine agaros¢ columns was extensively dialysed against phosphate buffered saline in the cold and rechromatographed on a Sephadex G-200 column, th~ protein was eluted in a single peak with an estimated molecular weight of 85,000 (Fig. 6). In some experiments, about 5-10 per cent of the material applied to the column eluted with the void vol. of the column as a large mol. wt aggregated material. I~termination of the reel. wt of the main component by the high speed equilibrium procedure (Y.phantis, 1964) gave a mean mol. wt of 88,000+_10,000. The sedimentation coefficient of this material determined in the analytical ultracentrifuge was 4.6 S. When this protein was subjected to SDS acrylamid¢ gel electrophoresis or treated with 6 M guanidine hydrochloride and rechromatographed on guanidineagarose columns, a subunit mol. wt of 46,000 was obtained. This suggests that in aqueous media the monomer subunits associate with one another with the formation of dimers. The dimers on treatment with denaturation solvents dissociate to the monomer subunits. On equal weight basis, the radiolabelled dimer was only 20-30 per cent less efficient compared to 125I properdin in binding to rabbit anti-human properdin adsorbed onto polystyrene tubes (Fig. 7)~ (Minta et al., 1973) suggesting the retention of most of the antigenic determinants of the parent molecule in the dimer species. On equal weight basis, the dimer protein had about 50 per cent of the functional activity of properdin in the zymosan assay (Pillemer et al., 1954). DISCUSSION Data has been obtained tb suggest that properdin is a glycoprotein containing about 9-8% carbohydrate. The amino acid analysis revealed a high content of glycine and prolin¢ which are quite reminiscent of Clq (Yonemasu et al., 1971). The partial specific volume of properdin calculated from amino acid composition and corrected for carbohydrate content gave a value of 0.7 cc/g. This must be considered as an approximate value since tryptophan content has not been taken into account in the calculation and the half cystine content is quite approximate. Using this value in the calculation of the reel. wt of properdin from a high speed equilibrium ultracentrifugation data, a mean value of 184,000 ± 12,000 was obtained. This value is in agreement with the value of 223,000 obtained by Pensky et al. (1968) when a partial specific volume of 0.75 cc/g assumed by these workers was used in our calculation. The sedimentation coefficient of properdin determined by analytical ultracentrifugation was 5-2 S and is in good agreement with the results of Pensky et al. (1968). The low sedimentation rate and the high reel. wt values indicate that properdin is an asymmetric molecule.

366

J. ODURO MINTA and IRWIN H. LEPOW SEPHADEX G'200-

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Fig. 5a. Chromatography of 1251 properdin and proteins of known mol. wt on Sephadex G-200 in PBS (78 x 2 cm). The column was run at 1 ml/min and 2.3 ml fractions were collected into (12 x 75 mm) disposable glass tubes. 12sI properdin was located by counting the fractions in an automatic ~,-counter. Reference markers were located by their absorbancy in a Zeiss PMQ11 spectrophotometer and have been indicated on the graph by arrows corresponding to their elution peaks. Fig. 5b. Chromatography of properdin (containing a trace of 12sI properdin) and proteins of known mol. wt on Sepharose 4B column in the presence of 6 M guanidine hydrochloride. The column measured 78 x 2 cm and the sample volume applied to the column was 1 ml. Blue dextran, DNP alanine, protein and radio-activity were located in the fractions as already described. Fig. 5b. (insert). The data of Fig. 5b are graphed as a semi-logarithmic plot of the mol.wt of protein vs distribution coefficient (Ke). The K~ values were determined as described in the text. From the graph a subunit mol.wt of 45,000 was obtained for properdin as indicated by an arrow. The data presented above provide consistent evidence that human properdin is a stable tetramer of four non-covalently linked subunits of similar mol. wt. The protein is dissociated in the absence of reducing agents under conditions where properdin is probably fully denatured and using the average mol. wt of 184,000 for properdin, the data are in agreement with four of the 46,000 mol. wt subunit/properdin molecule. In two independent systems which are potentially capable of detecting differences in subunit structure, namely polyacrylamide gel electrophoresis in SDS and agarose chromatography in guanidine hydrochloride or SDS, no evidence of non-identical subunits was observed. This finding is significant since these systems depend on different manifestations of different polypeptide chain length. It is believed that SDS anions bind to proteins and swamp all the charge effects so that the

proteins migrate in gels as rod-like polypeptide chains almost entirely according to their molecular size (Reynolds and Tanford, 1970) whereas concentrated guanidine causes most (reduced) proteins to lose their non-covalent structure and are dissociated to their constituent polypeptide chains as random coils. However, these results do not preclude the possibility of similar but distinct subunits in properdin. Our failure to detect an amino terminal amino acid residue for properdin may be due to the presence of a blocked amino acid residue at the amino terminal end of the molecule. The finding that the subunits tend to associate in aqueous media at p H 7.4 to form only dimers (mol. wt = 88,000) but not tetramers and that on equal wt basis, the dimer possesses only 50 per cent of the functional activity of the parent molecule would suggest that some differences in chemical corn-

Sub-unit Structure of Properdin 200,000.

~

i

GLOBULIN

100,000.

MW=85,000 " = ' - ~ U M I N 0

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6

20,000-

10,000 0,2

0'.3

o'.4

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Ka Fig. 6. Gel filtration on Sephadex G-200in PBS (78.5 x 2 cm) of proteins of known tool. wt and of properdin eluted from Sepharose 4B-guanidine hydrochloride column and extensively dialysed against PBS. The data are expressed as a semilogarithmic plot of the mol. wt of the protein vs the distribution (Ka), The Ka for properdin corresponded to a tool. wt of 85,000. position might exist between the subunits. Such differences, if they indeed exist would have to be small since the dimer retains most of the antigenic properties of the parent protein as measured by their ability to bind to antiproperdin adsorbed on polystyrene tubes. It is possible that free sulfhydryl groups might be present in the intact molecule that could be involved 80-

60

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20

367

in the formation of the dimer species when the dissociated molecule is dialysed against phosphate buffered saline. However, our finding that the dimer species readily dissociates to the monomer species in denaturing solvents in the absence of reducing agents makes this possibility unlikely. The possibility that properdin exists in serum as a dimer which undergoes tetramer formation upon interaction of serum with zymosan was excluded by showing that when serum was fractionated by gel filtration on Sephadex G-200 column and the fraction analyzed for their ability to compete with 1251 properdin for binding to antiproperdin coated polystyrene tubes, the properdin activity overlapped the elution profile of zymosan purified properdin. It has been reported by Kungs and Knight (1971) that negative charge effects possibly resulting from carbohydrate residues are not eliminated by SDS polyacrylamide gel electrophoresis and may cause errors in estimation of mol. wt. Our findings that the subunit mol. wt for properdin obtained from agarose--guanidine chromatography was in agreement with the value determined by SDS polyacrylamide gel electrophoresis would suggest that the high carbohydrate content in properdin does not greatly influence its electrophoretic mobility in SDS gels. Quite recently, Gotze and Miiller-Eberhard (1973) have reported that incubation of properdin with fresh human serum resulted in the conversion of C3PA to C3A and the cleavage of C3 to C3a and C3b as judged by immunoelectrophoresis. Cleavage of C3 did not occur when properdin was added to C3PA depleted serum. They have suggested that properdin interacts in serum to generate a C3PAse activating capacity. We have observed that the dimer species also interacts in serum to effect the activation of C3PA and cleavage of C3. However, on equal weight basis, it was less efficient compared to undissociated properdin. The importance of the carbohydrate moiety of properdin in the activation of C3PA is yet to be determined. Milder conditions for dialysis of the dissociated properdin subunits are currently being sought with the goal of generating the tetramer species. Acknowledgements--We wish to express our gratitude to Dr. E. Wampler for his advice in the ultracentrifugation experiments and to Dr. J. Ozols for his assistance in the amino acid analysis. The technical assitance of Miss Phyllis Jezyk is gratefully acknowledged. REFERENCES

1 2 4 8 16 32 64 128 256 RECIPROCALOF DILUTIONOF ANTISERUM (xl0"3} Fig. 7. Binding of 12"1 properdin ( o - - - e ) and 1251dimer properdin (O- O), (40 ng/ml) to polystyrene tubes coated (2 hr, 23°C) with increasing dilutions of rabbit anti-human properdin antiserum. The antibody coated tubes were incubated with 40 ng of the radiolabelled protein 0"5~o BSA in PBS for 18 hr at 37°C as described by Minta et al. (1973}.

Alper C. A., Goodkofsky I. and Lepow I. H. (1973) J. exp. Med. 137, 424. Andrews P. (1965) Biochem. J. 96, 595. Blum L., Pillemer L. and Lepow I. H. (1959)Z. Immun. forsch. 118, 349. Boas M. F. (1953) J, biol. Chem. 204, 553. Cohn E. J. and Edsall J. T. (1943) Proteins, Amino Acids and Peptides (Edited by Cohn E. J. and Edsall J. T.), p. 370. Reinhold, New York. Dische Z. and Shettles L. B. (1948) J. biol. Chem. 175, 595.

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J. ODURO MINTA and IRWIN H. LEPOW

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