Isolation of human complement factors C3, C5 and H

Journal oflmmunologicalMethods,

81 (1985)147-160

147

Elsevier JIM 03546

Isolation of Human Complement Factors C3, C5 and H Ake Lundwall * and G6sta Eggertsen Department of Medical and Physiological Chemistry, The Biomedical Center, Uppsala University, Uppsala, Sweden

(Received 18 October 1984, accepted 19 March 1985)

An improved method for simultaneous purification of complement factors C3, C5 and H from human plasma has been developed. Using an initial batch separation technique with QAE-Scphadex, followedby chromatography on SP-Sephadex and gel filtration in Sephadex G-200, 600 nag of highly pure C3 can be prepared from 1600 ml of plasma. Simultaneouslyabout 70 mg of highly pure factor H and 30 mg of C5 are obtained by chromatography of post SP-scphadex material on DEAE-Sephacel. A small amount of C3 in the C5 pool is removed by anti-C3-Sepharose. By maleylation or citraconylafion of reduced and alkylated C3, the constitutive polypeptide chains are modified in a way that made them separable by ion exchange chromatography. Key words: complement - C3 - C5 - factor H

Introduction C3 is the complement component that is present in the highest concentration in h u m a n serum, about 1.2 m g / m l . The relatively high serum level probably reflects the central role of C3 in the expression of complement phenomena. The molecule is composed of 2 polypeptide chains with molecular weights of 115,000 (a-chain) and 75,000 (fl-chain) (Bokisch et al., 1975; Molenaar et al., 1975; Nilsson et al., 1975; Tack and Prahl, 1976). The chains are associated both by cystine bridges and by non-covalent forces. C3 is activated by the C3 convertases C4b,C2a and C3b,Bb. F r o m the amino terminal part of the a-chain, the anaphylatoxin C3a is derived as an activation peptide (Bokisch et al., 1969). The other activation fragment, C3b, contains a metastable binding site for complement-activating particles (Mi'qlerEberhard et al., 1967). It has recently been shown that the metastable binding site is a thiolester (Tack et al., 1980). By a transesterification reaction, C3 is covalently linked to surfaces by way of an ester (Law and Levine, 1977). * Correspondence to present address: Department of Clinical Chemistry, MaimS5 General Hospital, S-214 01 Malm6, Sweden. 0022-1759/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

148

Because of the important role of C3 in the complement system, many efforts have been made to improve the methods for purification of this component as the original methods were both time-consuming and low in yield. One improvement was made by Molenaar et al. (1973) by the introduction of an anti-impurity column to free C3 from contaminating proteins, Methods based on the same idea have since been described by other authors (Harrison and Lachmann, 1979). The drawback of such methods is the difficulty in getting a good anti-impurity column that can be used repeatedly. A real breakthrough in C3 biochemistry occurred in 1976 when Tack and Prahl (1976) described a method of purifying C3 from plasma with a 30% yield. The method was moreover designed in a way that made it easily upscaled to yield gram quantities of highly pure C3. The method has since been improved and extended to include a purification scheme for several other complement proteins (Hammer et al., 1981). The C5 protein was first described and isolated by Nilsson and Mi~ller-Eberhard (1965). The activated form C5b serves as a nucleus for the assembly of the terminal membrane attack complex (MAC). The molecule is very similar in shape to C3, and has the same subunit composition and an almost identical molecular weight. C3 and C5 are usually separated by hydroxyapatite chromatography. The alternative pathway of complement operates mainly through an enzyme complex between the activation products of C3 and factor B (Grtze and Mi~llerEberhard, 1971; Goodkofsky and Lepow, 1971; Alper et al., 1973). The enzyme C3b,Bb is an amplification convertase, that is, by creation of new C3b molecules more convertases can be formed. The activity of the C3b,Bb enzyme is regulated by other complement proteins. It is stabilized by factor P (Fearon and Austen, 1975) and it is inactivated by factor I in the presence of factor H (Lachmann and Mialler-Eberhard, 1968; Ruddy et al., 1972; Whaley and Ruddy, 1976). Factor H, although its existence has long been known, was not described as a complement protein until 1976. This glycoprotein accelerates the proteolytic inactivation of C3b by factor I. It may also serve as a regulatory substance in itself by displacing factor B from the C3 convertase (Weiler et al., 1976). Both C5 and factor H are commonly isolated as by-products of C3 preparation. This report describes a high yield method for preparing C3. In addition, relatively large amounts of C5 and factor H can be obtained simultaneously. A method for separating the polypeptide chains of C3 is also described.

Material and Methods

The following materials were purchased: QAE-Sephadex A-50, SP-Sephadex C-50, DEAE-Sephacel, CM-Sepharose CL-6B, Sephadex G-200 and Sepharose 4B from Pharmacia Fine Chemicals, Uppsala; phenylmethylsulfonylfluoride from Sigma; fresh frozen human blood plasma from The Blood Centre, University Hospital, Uppsala; antisera against C1, C9, albumin, IgG, IgA, fibrinogen, haptoglobin, fl-lipoprotein, transferrin, factor B and human serum proteins from Behringwerke; antibody fraction of antisera against IgM and ceruloplasmin from

149 DACO laboratories, Denmark; sheep erythrocytes and rabbit anti-sheep hemolysin from the National Bacteriological Laboratory, Stockholm; all other chemicals were reagent grade or of the highest quality available.

Assay of complement proteins C3 was assayed in column effluent by a hemolytic test with use of sensitized sheep erythrocytes (EA cells) and hydrazine-treated human serum supplemented with C4 as described in detail previously (Lundwall et al., 1981b). Molecular titration of C3 was performed with EAC4, °xyc2 and KSCN/hydrazine-treated guinea-pig serum, according to Cooper and Mi~ller-Eberhard (1974) with modifications by Lundwall et al. (1981b). Hemolytic titration of C5 activity was performed as described by Cooper and Mialler-Eberhard (1974), with modifications according to Nilsson et al. (1974). C3, C5 and H antigens were determined either by the single radial immunodiffusion technique of Mancini et al. (1965), or by rocket immunoelectrophoresis (Laurell, 1972).

Antiserum and immunosorbent Antisera against C3, C5 and factor H were raised in rabbits as previously described (Lundwall et al., 1981b). An immunosorbent column for human C3 was prepared with the IgG fraction of rabbit anti-C3 serum, which was isolated on Protein A-Sepharose (Hjelm et al., 1972) and coupled to CNBr-activated Sepharose 4B. Approximately I0 nag of IgG was bound per ml of gel.

Miscellaneous techniques Polyacrylamide slab gel electrophoresis was performed on 11% gels, using the system described by Neville (1971). The preparation of samples had been described earlier (Lundwall et al., 1981a). Immunoelectrophoresis and immunodiffusion were carded out on microscopic slides as described elsewhere (Lundwall et al., 1981b). Amino acid analysis was performed On a Beckman 121 M amino acid analyzer according to the method of Spackman et al. (1958). Details of the sample preparation have been given in a previous paper (LundwaU et al., 1981a) The protein concentration in pure preparations was based on the specific absorbance at 280 nm. Values of 9.7 for C3 (Tack and Prahl, 1976), 13.1 for H (Whaley et al., 1978) and 10.0 for C5 in 1% solutions were used.

Isolation of complementfactor C3 Six to 8 U of fresh frozen human plasma were thawed in lukewarm water and phenylmethylsulfonylchloride (PMSF) was added to a final concentration of 0.25 mg/ml in plasma. The mixture was chilled to cold-room temperature (6-8°C), whereafter packed QAE-Sephadex was added, giving a 15% suspension of gel in plasma. After stirring for 1 h, the gel was settled and the supernatant was decanted. This procedure was repeated with a second portion of QAE-Sephadex. The 2 plasma-adsorbed gels were layered on top of fresh QAE-Sephadex ,in separate columns. Following a wash with 20 mM Tris-HC1 buffer, pH 7.0, containing 0.11 M NaC1 and 2 mM EDTA, C3 was eluted by increasing the NaC1 concentration to 0.2

150 M. Up to this point the procedure was the same as for the C4 isolation technique previously described (Lundwall et al., 1981a). The C3-containing fractions from the QAE-Sephadex columns were pooled together and allowed to react with 25 mg of PMSF for 1 h. The sample was then dialyzed overnight against 12 liters of 20 mM Na-phosphate buffer, pH 6.0, containing 40 mM NaC1 and 0.02% NaN 3 (SP-Sephadex buffer). Before application on a 5 x 30 cm column of SP-Sephadex, the dialyzed sample was diluted with water to equalize its conductivity with that of the starting buffer. The column was run at a rate of 120 m l / h . Following a l-liter wash with starting buffer, C3 was eluted with a combined salt and pH gradient, composed of 1.5 liters of starting buffer and 1.5 liters of 20 mM Na-phosphate buffer, pH 7.5, containing 0.1 M NaC1 and 0.02% N a N 3. The material was ultrafiltered in Diaflo ® cells to a concentration of 4-5 m g / m l . The sample was then divided into 3 - 4 portions, which were subsequently gel-filtered in 5 x 95 cm columns of Sephadex G-200. Fractions of 10 ml were collected. The highly pure C3 was pooled and stored refrigerated with 0.05% NaN 3 added.

Isolation of complement factors H and C5 After the main C3 peak, a peak containing factors H, C5 and small amounts of C3 was eluted from the SP-Sephadex column. This material was pooled, dialyzed against 2 × 5 liters of 20 mM Tris-HC1, 50 mM NaC1, p H 7.0 and applied at a rate of 70 m l / h on a 5 x 30 cm column of DEAE-Sephacel. Following a 0.5 liter wash with the Tris buffer, the column was eluted with a 2 liter linear salt gradient up to 0.25 M NaC1. The first eluted peak contained highly pure factor H. This material was pooled, dialyzed against phosphate-buffered saline (PBS), concentrated to 3 - 4 m g / m l by ultrafiltration and stored at - 7 0 ° C until used. The second eluted peak contained C5 contaminated with 10-20% C3, as measured by the Mancini technique. To remove the C3, the material was chromatographed on a 2.5 × 25 cm column of CM-Sepharose in 10 mM Na-phosphate, p H 7.0. Following a 350 ml wash, the column was eluted at a rate of 30 m l / h with a 1 liter, linear NaC1 gradient limiting at 0.15 M, whereafter C5-containing fractions were pooled. Alternatively, the post-DEAE-Sephacel C5 pool was concentrated and dialyzed against PBS, whereafter it was passed through an anti-C3 Sepharose column. The final C5 was transferred into PBS containing 0.05% NaN 3 and stored refrigerated at a concentration of 1 mg/ml.

Preparation of the polypeptide chains of C3 C3 was inactivated by treatment with 10 mM methylamine at p H 9.0 as described for C4 (Lundwall et al., 1981b). Five hundred milligrams of inactivated C3 were dialyzed against 10 mM N H 4 H C O 3 and lyophilized. The material was dissolved in 50 ml of 0.2 M Tris-HC1 buffer, p H 8.6, containing 10 mM EDTA and 6 M guanidine chloride. It was reduced with 25 mM D T T at 37°C for 4 h and alkylated at room temperature with 35 mM iodoacetic acid for 1 h, after which it was extensively dialyzed against 1 M acetic acid and lyophilized again.

151 The material was then redissolved in 50 ml of 0.1 M pyrophosphate with 6 M guanidine chloride and then transferred to a beaker. The beaker was placed on a magnetic stirrer in an ice bath. Maleylation was done by drop-wise addition of 7.5 ml of 1 M maleic anhydride in dioxane to the well-stirred solution (Butler et al., 1969). The pH, monitored by a glass electrode, was kept between 8 and 9 with 1 M NaOH during the reaction. The maleylated C3 chains were then dialyzed against 20 mM Tris-HC1 buffer, pH 8.0, containing 0.25 M NaC1, and loaded on a 2.5 × 35 cm column of DEAE-Sephacel at a rate of 25 ml/h. The column was eluted by a 1 liter linear salt gradient limiting a 0.75 M NaC1. The 2 peaks were pooled. Unblocking of the maleyl groups was achieved in 0.1 M glycine buffer, pH 3.0. The extent of modification was calculated from the UV spectra of modified and unblocked or unmodified proteins as described (Butler et al., 1969). The amount of lysine in the samples was determined by amino acid analysis.

Results

Preparation of complementfactors C3, C5 and H When developing the method for C4 isolation (Lundwall et al., 1981b), we noticed that C3 could also be recovered in a high yield by adsorption to QAE-Sephadex. In contrast to C4, however, substantial amounts of C3 were left in the plasma after 1 adsorption to 15% QAE-Sephadex. By a second adsorption step this material was recovered and almost no C3 antigen could thereafter be detected in the plasma. The use of a batch adsorption technique allowed the handling of rather large amounts of plasma in an easy way. A 40-fold purification with simultaneous removal of C3 convertase components such as C2 and factor B, which did not adsorb to the ion exchanger, and C4 which was eluted at a higher salt concentration than C3, demonstrated the benefits of the batch technique. At chromatography on SP-Sephadex, the next step, a more than 90% pure preparation of C3 was obtained. Furthermore, this step separated C5 and factor H from C3. As the chromatography was performed at a rather, low ionic strenght, close to pI for C3 and C5, there was a slight tendency for ~these proteins to form precipitates during dialysis and application to the column. To avoid this, the post-QAE-Sephadex pool was kept in the SP-Sephadex buffer for as short a time as possible. The material was therefore dialyzed against only 1 vol. of buffer, followed by an adjustment of the sample conductivity with distilled water prior to application. Occasionally precipitates still formed during handling, but these did not disturb the resolution of the chromatogram or significantly lower the yields. Initially we eluted the SP-Sephadex cohmm with a salt gradient only. Although this procedure produced a smooth C3 peak, the C 5 / H peak was irregular in shape, with pronounced tailing phenomena. We considered this to be a consequence of gel shrinkage. In order to avoid these effects, we introduced a pH gradient in combination with a minor salt gradient. By this procedure we obtained a smooth C 5 / H peak well separated from the C3 peak (Fig 1). On analysis of the post-SP-Sephadex C3 pool by polyacrylamide gel electrophoresis, only a few faint bands at the top of the gel - - in

152 A280

C3 I

I

t

C5,H

rnSIcm pH _.Y 1-y 15.0

- 1.5

7.G i ) 10.0

6.5

t.O

:

z

o 0.5

6.0

,]

0

5.0

50

100

150

200

Fraction

Fig. 1. Chromatography on SP-Sephadex. Pooled C3 from QAE-Sephadex was chromatographed on a 5 × 30 cm column of SP-Sephadex. Elution was performed with a combined pH and salt gradient constructed of 1.5 liter of 20 mM Na-phosphate, 40 mM NaC1, pH 6.0 and 1.5 liter of 20 mM Na-phosphate, 100 mM NaC1, pH 7.5. Absorbance at 280 nm ( ), C3 hemolytic activity expressed as ~ (y denotes the number of lysed cells relative to the total number of cells) ( e e), C5 antigen (zx zx), H antigen (© shown. Pooling was as indicated.

0 ) , specific conductivity (n

[2) and pH

(11

II) are

addition to the C3 band - - were seen. These unidentified contaminating proteins were removed by gel filtration in Sephadex G-200. Prior to gel filtration, the post-SP-Sephadex pool was concentrated by ultrafiltration. To avoid precipitation during concentration, the material was first dialyzed into PBS and only 'fresh' membranes allowing high flow rates were used in the concentration cell. By this procedure C3 was concentrated to 5 mg/ml. If, however, the time for concentration was extended by clogged membranes, C3 started to form precipitate at a concentration of as low as 2 mg/ml. The protein peak which eluted late from the SP-Sephadex column contained, in addition to factors C5 and H, small amounts of C3. By subjecting this material to chromatography on DEAE-Sephacel, factor H was obtained in an apparently pure form, well separated from C5, which eluted later (Fig. 2). Contaminating C3 could be removed by CM-Sepharose chromatography in 10 mM phosphate buffer at pH 7.0. Under these conditions C5 adsorbed to the gel, while hemolytically active C3 passed through the column (Fig. 3). Occasionally small amounts of hemolytically

153 A280

0.75

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0.50

1.0 E

0.251

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~ t A

50

150

100

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Fig. 2. Chromatography on DEAE-Sephacel. Factors H and C5 were separated on a 5 x 30 cm column of DEAE-Scphacel. The column was eluted by a 2 liter salt gradient in 20 mM Tris-buffer, pH 7.0, ranging from 50 to 250 mM NaC1. Absorbance at 280 n m ( ), C5 antigen (A a), H antigen (© O), C3 antigen (e e) and specific conductance (ll I ) are shown.

inactive C3 could still be detected in the C5 pool. We therefore found it preferable to remove C3 antigen from the post DEAE-Sephacel pool with anti-C3-Sepharose. This procedure also gave a slight increase in C5 yields. When the immunosorbent was regenerated by acid or chaotropic ions, a decrease in capacity was noted. By instead using 6 M guanidine hydrochloride to desorb C3, the capacity of the anti-C3 column remained unchanged and it could be used repeatedly. The yields of the factors are given in Table I.

Purity and stability of the products The C3, C5 and H preparations were tested by Ouchterlony analysis. There was no cross-reactivity between the end-products when tested with antisera against C3, C5 and factor H. In none of the preparations could C4, C1, C6, C9, C4Bp, albumin, IgG, IgA, IgM, transferrin, ceruloplasmin, fibrinogen, factor B, haptoglobin or fl-lipoprotein antigen be detected by specific antibodies. Furthermore, with an antiserum against human serum proteins ony 1 precipitate was formed with each preparation, both at immunodiffusion and at immunoclectrophoresis. The preparations also produced a monospecific antibody response when injected into rabbits. When analyzed by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, the preparations were apparently homogeneous, although occasionally small

154 mS/cm

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mm

0.50

5.0--

0.25

7.5

\ ~--o 100

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Fig. 3. Chromatography on CM-Sepharose. The C5 preparation was freed from small amounts of contaminating C3 on a 2.5 × 35 cm column of CM-Sepharose in 10 mM Na-phosphate buffer, pH 7.0. Elution was performed with a 600 ml linear salt gradient, ending at 0.15 M NaCI. Absorbance at 280 nm ( ) and specific conductivity (m B) are shown. C5 (© ©) and C3 (e o) antigens are expressed as diameter (ram) on Mancini plates.

TABLE

I

CALCULATION OF YIELDS Volume

mg of antigen"

Yield (%)

Hemolytic units b

Yield (%)

(.4) C3 Plasma pool QAE-Scphadex pool SP-Scphadex pool Sephadex G-200 pool

1660 840 340 315

1 488 1 099 704 641

100 74 47 43

2.9X1013 2.2 × 1013 1.3×1013 1.2×1013

100 76 46 40

(B) C5 Plasma pool QAE-Sephadex pool SP-Sephadex pool DEAE-Scphacel pool CM-Sepharose pool

1660 840 910 228 103

174 76 55 45 32

100 45 31 26 18

(C) Factor H Plasma pool QAE-Sephadex pool SP-Sephadex pool DEAE Scphacel pool

1660 840 910 222

441 144 89 70

100 33 20 16

a Determined by the single radial immunodiffusion technique. b Determined by hemolytic titration.

155

C3 j.

C5

Factor H

_.

4

Fig. 4. Polyacrylamide gel electrophoresis. The purified components were examined by polyacrylamide gel electrophoresis on 11% gels. From left to right are shown C3, C5 and factor H. Positions of factor H degradation products are indicated by the arrows.

amounts of breakdown products were seen in the factor H pools (Fig. 4). Furthermore, the amino acid compositions (not shown) were in good agreement with previous reports (Tack and Prahl, 1976; Harrison and Lachmann, 1979; Tack et al., 1979; Sire and DiScipio, 1982). The hemolytic yield of C3 is approximately the same as the yield of C3 antigen. It may therefore be concluded that the C3 preparation is functionally intact. This was also true for C5, although its purification was not followed by hemolytical yields. However, the final product had a hemolytic activity of 1.1 × 1013 hemolytic units per mg, or 100-120~ of the specific hemolytic activity of standard serum. The C3 and C5 pools were stored refrigerated in PBS containing 0.05~ NaN a. In this buffer no degradation was detected at polyacrylamide gel electrophoresis even after 6 months of storage. Storage of C5 for longer periods of time was preferably done at - 70°C, where no change in activity was observed, while a decrease could be found after prolonged storage at -20°C. Factor H stored at - 7 0 ° C did not show any degradation, when stored refrigerated, slow degradation of the protein chain, yielding fragments with molecular weights of 120,000 and 35,000, occurred. The

156 A280

mS/cm

0.75

40

0.50

30

0.25

20

50

I

I

100

150

10

200

Fig. 5. Separation of the polypeptide chains of C3. Reduced, alkylated and maleylated C3 was chromatographed on a 2.5 x 35 cm column of DEAE-Sephacel in 20 m M Tris buffer, p H 8.0, containing 0.25 M NaC1. The column was eluted by a 1.5 liter linear salt gradient, ending at 0.75 M NaC1. Absorbance at 280 n m ( ) and specific conductance (11 II) are shown.

half-life of the degradation was estimated at 5-6 months by visual inspection of polyacrylamide gels run after varying periods of storage.

Preparation of polypeptide chains The polypeptide chains of reduced and alkylated C3 are strongly associated by non-covalent forces. These forces can, however, be broken by chemical modifications of the lysine residues with maleic or citraconic anhydride (Dixon and Perham, 1968; Butler et al., 1969). By the introduction of the negatively charged maleyle group, the chains will repulse each other. The increase in negative charge also makes the polypeptide chains very soluble in ordinary buffers, By ion exchange chromatography gram quantities of C3 can be handled so as to yield large amounts of chain material (Fig. 5). A curve similar to that in Fig. 5 is obtained with citraconylated C3, but because of the lability of the citraconyl-lysine derivative, the chromatography is performed at pH 8.5. From the pattern on polyacrylamide gels after electrophoresis in dodecyl sulfate (Fig. 6) it is evident that although a pure fl-chain preparation can be obtained, the a-chain is still contaminated by fl-chain material. From amino acid analyses the molar yield of C3fl was estimated at 50-60%. By comparing the ratio of alanine to leucine in our preparation with reported values (Tack et al., 1979), it was calculated that the fl-chain contamination in the a:chain pool was 30%. The presence of fl-chain material in the a-chain pool is probably a result of an unequal modification of C3. The modification of the lysines was calculated to 100%,

157

Fig. 6. Identificationof the polypeptidechains of C3. The fractionsof the DEAE-Sephacelchromatogram in Fig. 5 were examined by polyacrylamidegel electrophoresisin sodium dodecyl sulfate. From left to right are shown each fifth fraction from fraction 80 to fraction 145. The maleylatedchains had a lower electrophoreticmobility than unmodifiedmaterial but owing to the uncertainty in the method there may, however, be a small amount of unmodified lysines, and if these are in a critical position the chains may reassociate. A low modification of a certain population of the molecules may be due to dilution of the sample with the N a O H solution. The concentration of guanidine is only 4 M when the reaction is completed, which perhaps allows a certain proportion of the molecules to reassociate. An increase in the reagent concentrations or adding solid maleic anhydride, making the dilution factor almost nil, did not, however, significantly alter the elution pattern from the DEAE-Sephacel column, neither did a change in the molar amount of reagent from 50 to 200%. Another conceivable reason for the tailing phenomena is an O-acylation of tyrosines by the maleic anhydride. Such derivatives are labile, however, and should hydrolyze spontaneously at alkaline pH. There is also a possibility of maleylation of serine and threonine residues. In this case the maleyl group can be taken off by 1.0 M hydroxylamine (Freedman et al., 1968). Such treatment of maleylated C3 had no effect. As the main purpose was to obtain C3fl (Lundwall et al., 1984), the reason for the contamination of the a-chain pool by fl-chain material was not further investigated.

Discussion The method of Tack and Prahl (1976) with initial precipitation of plasma proteins with polyethylene glycol is at present the standard technique for purification of C3

158 in most laboratories. A method giving a similar yield has been presented by Harrison and Lachmann (1979). Other interesting techniques have also been described, for instance by Nagasawa and Stroud (1977). Their technique, which was very briefly reported without graphs or yields, also starts with a batch adsorption to QAE-Sephadex. The use of QAE-Sephadex is beneficial, as this ion exchanger provides good separation of C3 and C4. When we developed the method for C4 purification, we noticed that C3 could be eluted separately from C4 by a batch procedure. As pointed out in that report, a batch procedure as an initial protein fractionating step is very valuable if it, as in this case, gives a rapid accumulation of interesting proteins. The purification scheme can also be extended to include other complement factors. In the C4 pool from QAE-Sephadex we detected C9 and Cls. Among the unbound proteins are factors B, I, P, Clq and C2, although the latter usually is inactivated during handling. Although the yield of C3 was good, considerable amounts of C5 (about 30%) and factor H (about 50%) remained in the plasma after the QAE-Sephadex adsorption. This material can probably be collected by the ion exchanger at a lower ionic strength, something which may double the yield of these factors. We made no attempts in that direction, however, as the main objective was to purify C3. Traditionally C3 and C5 have been separated by hydroxyapatite chromatography (Nilsson and MOller-Eberhard, 1965). It is difficult, however, at least in our hands, to get good resolution with high amounts of protein. The modification introduced by Tack and Prahl (1976) has partly solved these problems. Nevertheless, we have had some trouble with the reproducibility of the method. For example, occasionally the hemolytic yield has been low and C3 has leaked from the column after the desorption of C5 with 2 M KCI. By introducing SP-Sephadex chromatography we have arrived at a reliable method for separation of large amounts of C3 and C5. The method is not without problems, however, as precipitates easily form during dialyses and application. This of course lowers the yields, but the overall yield is still good. We have not found any solubility problems with the eluted material, but if the material is to be concentrated by ultrafiltration, the pH and ionic strength must be increased to avoid precipitation. The post-SP-Sephadex C3 pool is highly pure; but there are traces of high molecular weight contaminants, which are easily removed by gel filtration. With its ease of handling and high yields, the present method is a distinct improvement. From SP-Sephadex a pool containing a mixture of C5, H and C3 is obtained. The C3 molecules are mainly in a hemolytically inactive state. By subjecting this material to chromatography on DEAE-Sephacel, a very symmetrical factor H peak well separated from C5 and C3 is yielded. Although the material is pure as judged by various criteria, some protease is probably still present. There have been reports demonstrating the presence of plasmin or plasminogen in C5 preparations (Nilsson et al., 1972). From this it may be assumed that this protease also is present in our preparation. With specific antibody no plasminogen antigen could be detected in an immunodiffusion test. If still present, plasminogen should be easy to remove by a column with immobilized lysine, but we have not tested this possibility. When the post-DEAE-Sephacel C5 pool was run on a column of CM-Sepharose,

159

hemolytically active C3 passed through, while C5 and hemolytically inactive C3 antigen were retained by the gel. It has previously been shown that C3 can alter its chromatographic behavior as a result of conformational changes (Janatova et al., 1980; Parkes et al., 1981). We have detected an even higher degree of C3 heterogeneity by CM-scpharose chromatography, probably due to the low ionic strength, which may facilitate aggregation of C3 into complexes of varying size (Lundwall, unpublished results). Although this hemolytically inactive C3 usually elutes after C5, C3 antigen occasionally was detected in the C5 preparation. F o r this reason, C3 was preferably removed with an anti-C3-Sepharose column. About 40 ml of anti-C3 gel prepared from an antiserum with ordinary titers was sufficient to remove all C3 antigen from the DEAE-Sephacel pool. As pointed out previously, there are strong forces, in addition to the disulfide bridges, which keep the polypeptide chains of C3 associated. The dimer can not be broken by 1 M acetic acid or 8 M urea. The associating forces can, however, be broken by 0.1~ dodecyl sulfate - - indicating a possible hydrophobic nature - - or by 6 M guanidine. These agents preclude the use of common separation methods such as ion exchange chromatography. We considered it valuable to have a method which in a simple way provided us with large quantities of chain material, for structural analysis. As we felt that these requirements were not offered by hitherto reported methods (Tack et al., 1979; Nilsson et al., 1980), we searched for new approaches. Our requirements were partly fulfilled, as large amounts of maleylated C3fl could be obtained by ion exchange chromatography. It should also be possible to obtain a pure a-chain preparation by optimizing the conditions for maleylation. At the moment, however, it is not necessary from a structural point of view to have large amounts of the a-chain, as it is probably better to produce large a-chain fragments by partial tryptic digestion of C3.

Acknowledgements We like to thank professor John Sj6quist for help and support. The exellent technical assistance of Lena M611er is gratefully acknowledged. We are also in great debt to Dr. Ulf Nilsson, Dep. of Clinical Chemistry, University Hospital, Uppsala, for help with the C5 assay and valuable discussions. Thanks also go to Ludmilla Nirk for performing the amino acid analyses and to Ingeg'ard Schiller for typing the manuscript. This work was supported by a grant from the Swedish Medical Research Council (project number 13X-2518).

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