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A simple two-step procedure for the purification of plasma C1q from different animal species
Immunology Letters, 3 (1981) 303-308
Elsevier/North-HollandBiomedicalPress
A SIMPLE TWO-STEP PROCEDURE FOR THE PURIFICATION OF PLASMA Clq FROM D I F F E R E N T A N I M A L SPECIES E. John McKAY Department of Clinical Chemistry, University of Lund, Maim6 GeneralHospital, S-214 01 Malm6, Sweden
(Received 22 May 1981) (Accepted 11 June 1981)
1. Summary Plasma C1 q from human, rat, rabbit, dog and sheep were isolated by a 2-step affinity chromatography procedure. In the first step the method exploits the affinity of Clq for heparin andin the second the interaction between C 1q and IgG. The precipitation of Clq by the SO42- groups in agarose gels was used as a means to rapidly monitor the elution of Clq. This interaction was found to be non-species specific and therefore obviates the need for immunochemical and/or haemolytic assays. The isolation procedure is rapid, simple and is not confined to one species. Pure functionally active C1 q was obtained from all species in yields of approximately 85-95%.
unusual amino acid composition of human C 1q, its collagen-like tail region and its subunit structure have also been demonstrated in certain other animal species [7,8]. In this report a simple, the isolation procedure for Clq is described that is suitable for several different species, utilizing properties more or less unique for C 1q.
3. Materials and methods 3.1. Samples
Pooled human, rabbit or dog serum, and plasma from sheep or rat were used as starting material. 3.2. Reagents
2. Introduction The binding of Clq to the Fc domain of IgG contained in antigen-antibody aggregates initiates a series of sequential protein-protein interactions collectively known as the classical complement pathway [ 1,2]. C1 q can, however, bind directly to IgG that is not in complex with antigen [3], to various polyanionic compounds [4,5] and precipitates at low ionic concentration [ 1,2,6]. Such properties have been exploited as a means of isolating C 1q from complex biological fluids with widely varied recovery rates and purity [3-6]. In view of the importance complement has as a fundamental biological system in the animal kingdom [2], interest in the biochemistry and structure of Clq has also focused on animals other than man. The
Agarose ME with a sulphate content of 0.15% (batch no. 61639) was supplied from Marine Colloids, Rockland, U.S.A. Heparin-Sepharose 4B, gradient slab PAGE plates (PAA 4/30), CNBr-activated Sepharose 4B and DEAE-Sephadex A50 were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Anti-human C1 q was purchased from Dakopatts, Copenhagen, Denmark. Rabbit anti-serum against whole human and rat sera was available at the laboratory. Monospecific rabbit anti-human Clq, Clr and Cls were kindly supplied by Dr. A.-B. LaureU. Whole rabbit serum was first chromatographed over DEAE-Sephadex A50 equilibrated with 0.05 M Tris-HC1, pH 7.1. The 'break through' IgG-containing fractions were pooled and precipitated with ammonium sulphate (40% saturation) and dialyzed extensively against the conjugation buffer (0.1 M NaHCO3 in 0.5 M NaC1, pH 8.1). An IgG affinity col-
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umn (R-IgG Sepharose) was prepared by coupling rabbit IgG to CNBr-activated Sepharose according to the manufacturers' instructions. The coupling efficiency proved to be approximately 10 mg of IgG bound per 1.0 ml of wet gel. 3.3. Chromatography procedures Heparin-affinity chromatography was performed essentially as described before [9] but in the presence of 0.02 M EDTA. The column was washed with 3 times its bed volume using the equilibration buffer (0.05 M Tris-HC1, 0.15 M NaC1, 0.02 M EDTA). Proteins bound to heparin-Sepharose were eluted by an increasing linear NaC1 gradients. The C1 q-containing fractions were pooled and dialyzed against 0.05 M Tris-HC1, 0.02 M EDTA in 0.1 M NaC1 and applied to a column of R-IgG Sepharose (total IgG bound "~ 250 mg) equilibrated with the same buffer. C1 q was eluted from the IgG column with an increasing linear NaC1 gradient.
3.4. Clq detection Localization of C1 q eluted during chromatography was monitored by electroimmunoassay [10] and/or by precipitation of Clq induced by galactose SO42groups contained in agarose gels. The latter occurs during conventional agarose gel electrophoresis at pH 8.6 and can be visualized immediately after electrophoresis without the need of fixation or staining. The length of the precipitation track produced by the migrating C1 q during electrophoresis was found to be linear with Clq concentrations between 30 and 500 #g/ml [111. 3.5. Characterization of Cl q Agarose gel electrophoresis was performed according to Johansson [12] and SDS/PAGE according to Weber and Osborne [13]. The treatment of samples for PAGE and molecular size estimates were performed as described by Yonemasu et al. [8] using known marker-proteins (thyroglobulin 669,000; ferritin 440,000; catalase 232,000; LDH 140,000; and bovine serum albumin 67,000). Purified Clq recovered from all species was estimated from their absorbance at 280 nm u s i n g A ~ m = 6.8 [7]. The purity of human and rat Clq was additionally checked by crossed immunoelectrophoresis. All preparations were assessed for precipitating activity with rabbit IgG [4] 304
and for immunological cross-reactivity against antihuman C1 q by double immunodiffusion. The functional activity of isolated C1 q preparations was assessed in two ways: (a) whether purified Clq was capable of reforming macromolecular C1 with C1 r-C1 s complexes contained in C 1q-depleted human serum; and (b) whether these assembled preparations were haemolytically active. C 1q was removed from human serum by the addition of Sepharose 4B conjugated specific anti-Clq antibodies (2 mg/ml of gel) in the presence of 0.02 M EDTA and 0.001 M benzamidine. Specific rabbit anti-C1 q IgG was obtained first by solid phase immunoabsorption. This was used to minimize the level of non-anti-C1 q antibody containing rabbit IgG for conjugation to CNBr Sepharose 4B. C1 q-depleted serum was recalcified in the presence of benzamidine. Reassembled C1 was assessed by crossed immunoelectrophoresis using monospecifc rabbit anti-human C1 s antiserum. Haemolytic activity of the reassembled preparations was analyzed by a 'haemolysis in gel' technique [ 15 ]. Amboceptor-sensitized sheep erythrocytes were added to molten agarose (at 45°C) and allowed to gel, forming a 1 mm thick, homogeneous carpet of indicator cells in agarose. Circular wells (4 mm diameter) were punched from the gel and 10 #1 samples applied. Normal serum, by this method, demonstrated decreasing zones of lysis to a dilution of 1:8. Undiluted, Clq-depleted serum, by contrast, failed to produce any lysis. Haemolytic capacity of C1 q-depleted human serum, after addition of C1 q preparations, were compared to that obtained with normal human serum.
4. Results Initial experiments performed demonstrated that all C 1q contained in 3.0 ml of human serum could be removed by 1.0 ml of heparin-Sepharose. This assessment was made by comparing the recovery levels of C1 q eluted from the heparin column with the levels contained in serum before chromatography by EIA. Each ml of rabbit anti-IgG Sepharose was capable of removing all C1 q from 30 ml of pooled human serum, also determined quantitatively by EIA. Fig. 1 illustrates the slightly varying elution patterns of C1 q following chromatography of the different animal
sera over: (a) h e p a r i n - S e p h a r o s e ; and (b) over R-IgG Sepharose. Fig. 2 provides the agarose electrophoretic patterns of the pooled eluted fractions following heparin and IgG affinity chromatography with normal human plasma. C l q , if heated for 30 min at 56°C, no longer bound to heparin or IgG. Euglobulin preparations [6]
(rnm)
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Fig. 2. Agarose gel electrophoresis in barbital buffer containing 0.002 M EDTA, pH 8.6, 20 V/cm for 45 min. 10 pl samples were applied. A: normal human plasma (1:5 dilution). B: the pooled C 1q-rich eluted fractions following heparin affinity chromatography of human serum. C: the pooled Clq-rich eluted fractions following R-IgG affinity chromatography of B.
(mm)
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Fig. 1. The elution patterns of different Clq from various species following (A) heparin affinity;and (B) rabbit-IgG affinity chromatography, o - - % human Clq; ~ % dog Clq; • •, rabbit Clq; • •, sheep Clq; and D - - D , rat Clq estimated from Clq/agarose gel precipitation reaction during electrophoresis. Rabbit Clq demonstrated the same heparin affinity as human Clq, and sheep Clq the same R-IgG affinity as human Clq and are therefore not illustrated.
of pooled serum, in our hands, contained a significant proportion of C 1q ( 1 5 - 3 0 % ) that did not bind to IgG-Sepharose. Presumably this was due to irreversible aggregation of C1 q and/or complex formation with IgG that may occur during euglobulin precipitation. F r o m these results a simplified step-wise elution was designed, i.e. C1 q was removed from h e p a r i n Sepharose with the equilibration buffer but now containing 1.8 M NaC1. The C1 q-rich fractions were pooled, and dialyzed against 0.05 M Tris-HC1, 0.02 M EDTA in 0.1 M NaC1 before application to R-IgG Sepharose. Elution from the R-IgG column was achieved with a buffer containing 0.05 M Tris, 10% sucrose, 0.05 M EDTA in 1.0 M NaC1, pH 8.0 [20]. The isolated C l q was stored in this buffer at a concentration o f 2--4 mg/ml at 4°C. Table 1 summarizes and compares the purification steps and recovery o f C l q from all species tested. Isolated C1 q invariably produced a single cationic protein band by agarose gel electrophoresis with intense protein trailing from the site of sample appli305
Table 1 Purification of Clq Steps
Volume (ml)
Total protein (mg)
Clq (mg)
Recoverya (%)
A 1 Human serumb 2 Hep-Sepharose 3 R-IgG Sepharose
600 150 30
43,500 675 82
87 84 82
100 96 94
1 Rat plasmac 2 Hep-Sepharose 3 R-IgG Sepharose
150 40 5
10,700 152 12
14 13 12
100 96 85
1 Rabbit serumc 2 Hep-Sepharose 3 R-IgG Sepharose
250 45 15
18,200 162 265
28 25 25
100 89 89
1 Dog serumc 2 Hep-Sepharose 3 R-IgG Sepharose
250 55 15
17,200 87 27
32 28 27
100 87 84
1 Sheep plasmac 2 Hep-Sepharose 3 R-IgG Sepharose
250 65 15
14,800 183 30
35 32 30
100 91 85
aThese figures are the mean of duplicate runs and are approximate (-+10%) as they have been assessed from the values obtained solely by the Clq/agarose gel interaction. bClq measured by EIA and Clq-agarose gel interaction. CClq measured by Clq-agarose gel interaction only.
A
m I
H
D
R
Ra
S
B
H 306
D
R
Ra
S
cation. C1 q precipitation in agarose gels for all 5 species tested is illustrated in Fig. 3 and demonstrates, the similarity in results whether the gels were fixed, pressed and stained (Fig. 3A) or simply pressed and stained without fixation (Fig. 3B). An IgG 'M' component with cathodal migration was included for comparison. S D S - P A G E analysis revealed single protein bands under non-reducing conditions. By comparison with k n o w n marker-proteins, the molecular size of C l q obtained was estimated to be: h u m a n C l q 415,000; dog C l q 405,000; rabbit C l q 395,000;
Fig. 3. Clq/agarose gel electroprecipitation of the purified Clq from different species, run under the same conditions as for Fig. 2. A: the gel was fixed in picric acid, pressed dry and stained in 0.05 % kenacid blue. B: the gel was pressed without fixation and stained demonstrating the loss of IgG control protein only from the gel. I, human IgG myeloma protein (control); H, human Clq; D, dog Clq; R, rat Clq, Ra, rabbit Clq; S, sheep Clq.
sheep Clq 385,000 and rat Clq estimated to be 420,000. All species of Clq isolated, revealed noncovalently linked subunits in the presence of urea and multiple covalently linked peptide chains after reduction and alkylation similar to that described for human Clq [2]. Purified human and rat Clq demonstrated single cationic precipitin arcs when tested by crossed immunoelectrophoresis with rabbit anti-total human and rat serum, respectively. Clq isolated from all species demonstrated an ability to form macromolecular C1 with C l r - C l s contained in Clq-depleted human serum as shown for rat C 1q by crossed immunoelectrophoresis (Fig. 4). The
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|
I
I
F
r
,, f3c
Fig. 4. Crossed irnmunoelectrophoresisdemonstrating the reassembly of C1 following the addition of purified rat Clq (150 t~g/ml)to human Clq-depleted serum using anti-Cls antiserum. A: normal human serum. B: Clq depleted normal human serum A. C: rat Clq added to (B). "r, denotes macromolecular C1 ;#, Clr-C1 s complexes;and ~,, Cls-CT inactivator complexes.
reassembled C1 preparations were haemolytically active when tested by the 'haemolysin in gel' technique. Antiserum, monospecific for human C1 q, was produced in rabbits within 6 weeks and did not require absorption. A single precipitin line of complete immunological identity with C1 q in human serum and the purified component was obtained by immunoelectrophoresis and immunodiffusion. This antiC 1q produced a reaction of Complete identity with known anti-Clq antisera from two other sources. Clq preparations from the 4 other species also demonstrated weak but definite precipitin lines of partial identity with anti-human C1 q.
5. Discussion 'i
This procedure appears to be both specific and gentle for the isolation of C1 q as both ligands used apparently bound only functionally active Clq. The utilization of excess ligand over the amount of Clq to be applied in both affinity steps ensures high recovery of Clq from the species tested (85-94%). Heparin-affinity chromatography appears to be a more efficient and selective first step in the isolation of Clq than euglobulin precipitation. Clr and Cls are dissociated from Clq in the presence of EDTA and pass out in the void volume [21,22], Clt does not bind heparin in the presence of EDTA [23], and IgG does not bind to heparin at 0.15 M NaC1 [11]. An interaction between Clq and agarose has been previously recognized [17,18]. However, Assimeh et al. [19] considered it to be caused by contaminating IgG while Pohl et al. [20] considered it to represent some form of altered C1 q. Our results indicate that the C1 q-gel interaction is limited to a biologically active component as heat-treated Clq and the nonIgG-binding C1 q fraction in euglobulin preparations remained at the site of application without cathodal migration or precipitation. Monitoring of eluted C1 q from the affinity columns by its precipitation during electrophoresis in agarose gels appears to have some advantages over the more commonly used immunochemical and C1 qspecific haemolytic assays. Although much less sensitive, it is rapid since results can be obtained within 1 h. This agarose-gel interaction appears selective for Clq but not to be species specific. Haemolytic assays 307
specific for C l q can apparently be misleading because o f the presence o f inhibitory substances [ 16]. S D S - P A G E electrophoresis demonstrated that C 1 q isolated from all species studied were homogeneous with molecular sizes ranging from 385,000 to 421,000 indicating there was little significant difference between the species. C 1 q assembly experiments followed by haemolytic assays indicated that C1 q preparations from all 5 species studied were able complex with human C l r / C l s and form haemolyticaUy active C1.
Acknowledgements This work was supported by the Swedish Medical Research Council (Project No. B81-03X-00581-17A). The author is supported by a post-graduate scholarship from the New Zealand Medical Research Council. I wish to thank Inger Bomark for typing this manuscript.
References [1] Porter, R. R. and Reid, K. B. M. (1979) Adv. Protein Chem. 33, 1-71. [2] Gigli, I. and Austin, K. F. (1971) Ann. Rev. Microbiol. 25,309-332. [3] Stedge, C. R. and Bing, D. H. (1973) J. Immunol. 108, 661-665. [4] Agnello, V., Caro, R. I., Koffler, D. and Kunkel, H. G. (1969) Fed. Proc. 28, 2447.
308
[5] Raepple, E., Hill, H.-U. and Loos, M. (1976) Immunochemistry 13, 251-255. [6] Calcott, M. A. and Miiller-Eberhard, H. (1972) Biochemistry 18, 3443-3450. [7] Reid, K. B. M., Lowe, D. M. and Porter, R. R. (1972) Biochem. J. 130,749-763. [8] Yonemasu, K., Sasaki, T. and Shinkai, H. (1980) J. Biochem. 88, 1545-1554. [9] McKay, E. J. and Laurell, C.-B. (1980) J. Lab. Clin. Med. 95, 69-78. [10] Sj6holm, A. G. (1975) Scand. J. Immunol. 4, 25-30. [11] McKay, E. J. and Laurell, C.-B. (1981) Anal. Biochem. (submitted). [12] Johansson, B. G. (1972) Scand. J. Clin. Lab. Invest. 29 (Suppl. 124), 7-15. [13] Weber, K. and Osborne, M. (1969) J. Biol. Chem. 244, 4406-4412. [14] Ganrot, P.-O. (1972) Scand. J. Clin. Lab. Invest. 29 (Suppl. 124), 39-45. [15] Truedsson, L., SjSholm, A. G. and Laurell, A.-B. (1981) Acta Pathol. Microbiol. Scand. [16] Conradie, J. D., Volnakis, J. E. and Stroud, R. M. (1975) Immunoehemistry 12,967-971. [17] Paul, W. E. and Benacerraf, B. (1965) J. Immunol. 99, 1067-1072. [18] Agnello, V., Winchester, R. J. and Kunkel, H. G. (1970) Immunology 19,909-919. [19] Assimeh, S. N., Bing, D. H. and Painter, R. H. (1974) J. Immunol. 113,225-234. [20] Pohl, D. A., Gibbons, J. J., Tsai, C. C. and Roudman, S. T. (1980) J. Immunol. Meth. 36, 13-27. [21] Ziccardi, R. J. and Cooper, N. R. (1979) J. Immunol. 123,788-792. [22] McKay, E. J., LaureU, A.-B., MSrtensson, U. and Sj6holm, A. (1981) Mol. Immunol. in press. [23] Thompson, R. A. (1977) Biochemistry 17, 4304-4311.
Elsevier/North-HollandBiomedicalPress
A SIMPLE TWO-STEP PROCEDURE FOR THE PURIFICATION OF PLASMA Clq FROM D I F F E R E N T A N I M A L SPECIES E. John McKAY Department of Clinical Chemistry, University of Lund, Maim6 GeneralHospital, S-214 01 Malm6, Sweden
(Received 22 May 1981) (Accepted 11 June 1981)
1. Summary Plasma C1 q from human, rat, rabbit, dog and sheep were isolated by a 2-step affinity chromatography procedure. In the first step the method exploits the affinity of Clq for heparin andin the second the interaction between C 1q and IgG. The precipitation of Clq by the SO42- groups in agarose gels was used as a means to rapidly monitor the elution of Clq. This interaction was found to be non-species specific and therefore obviates the need for immunochemical and/or haemolytic assays. The isolation procedure is rapid, simple and is not confined to one species. Pure functionally active C1 q was obtained from all species in yields of approximately 85-95%.
unusual amino acid composition of human C 1q, its collagen-like tail region and its subunit structure have also been demonstrated in certain other animal species [7,8]. In this report a simple, the isolation procedure for Clq is described that is suitable for several different species, utilizing properties more or less unique for C 1q.
3. Materials and methods 3.1. Samples
Pooled human, rabbit or dog serum, and plasma from sheep or rat were used as starting material. 3.2. Reagents
2. Introduction The binding of Clq to the Fc domain of IgG contained in antigen-antibody aggregates initiates a series of sequential protein-protein interactions collectively known as the classical complement pathway [ 1,2]. C1 q can, however, bind directly to IgG that is not in complex with antigen [3], to various polyanionic compounds [4,5] and precipitates at low ionic concentration [ 1,2,6]. Such properties have been exploited as a means of isolating C 1q from complex biological fluids with widely varied recovery rates and purity [3-6]. In view of the importance complement has as a fundamental biological system in the animal kingdom [2], interest in the biochemistry and structure of Clq has also focused on animals other than man. The
Agarose ME with a sulphate content of 0.15% (batch no. 61639) was supplied from Marine Colloids, Rockland, U.S.A. Heparin-Sepharose 4B, gradient slab PAGE plates (PAA 4/30), CNBr-activated Sepharose 4B and DEAE-Sephadex A50 were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Anti-human C1 q was purchased from Dakopatts, Copenhagen, Denmark. Rabbit anti-serum against whole human and rat sera was available at the laboratory. Monospecific rabbit anti-human Clq, Clr and Cls were kindly supplied by Dr. A.-B. LaureU. Whole rabbit serum was first chromatographed over DEAE-Sephadex A50 equilibrated with 0.05 M Tris-HC1, pH 7.1. The 'break through' IgG-containing fractions were pooled and precipitated with ammonium sulphate (40% saturation) and dialyzed extensively against the conjugation buffer (0.1 M NaHCO3 in 0.5 M NaC1, pH 8.1). An IgG affinity col-
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303
umn (R-IgG Sepharose) was prepared by coupling rabbit IgG to CNBr-activated Sepharose according to the manufacturers' instructions. The coupling efficiency proved to be approximately 10 mg of IgG bound per 1.0 ml of wet gel. 3.3. Chromatography procedures Heparin-affinity chromatography was performed essentially as described before [9] but in the presence of 0.02 M EDTA. The column was washed with 3 times its bed volume using the equilibration buffer (0.05 M Tris-HC1, 0.15 M NaC1, 0.02 M EDTA). Proteins bound to heparin-Sepharose were eluted by an increasing linear NaC1 gradients. The C1 q-containing fractions were pooled and dialyzed against 0.05 M Tris-HC1, 0.02 M EDTA in 0.1 M NaC1 and applied to a column of R-IgG Sepharose (total IgG bound "~ 250 mg) equilibrated with the same buffer. C1 q was eluted from the IgG column with an increasing linear NaC1 gradient.
3.4. Clq detection Localization of C1 q eluted during chromatography was monitored by electroimmunoassay [10] and/or by precipitation of Clq induced by galactose SO42groups contained in agarose gels. The latter occurs during conventional agarose gel electrophoresis at pH 8.6 and can be visualized immediately after electrophoresis without the need of fixation or staining. The length of the precipitation track produced by the migrating C1 q during electrophoresis was found to be linear with Clq concentrations between 30 and 500 #g/ml [111. 3.5. Characterization of Cl q Agarose gel electrophoresis was performed according to Johansson [12] and SDS/PAGE according to Weber and Osborne [13]. The treatment of samples for PAGE and molecular size estimates were performed as described by Yonemasu et al. [8] using known marker-proteins (thyroglobulin 669,000; ferritin 440,000; catalase 232,000; LDH 140,000; and bovine serum albumin 67,000). Purified Clq recovered from all species was estimated from their absorbance at 280 nm u s i n g A ~ m = 6.8 [7]. The purity of human and rat Clq was additionally checked by crossed immunoelectrophoresis. All preparations were assessed for precipitating activity with rabbit IgG [4] 304
and for immunological cross-reactivity against antihuman C1 q by double immunodiffusion. The functional activity of isolated C1 q preparations was assessed in two ways: (a) whether purified Clq was capable of reforming macromolecular C1 with C1 r-C1 s complexes contained in C 1q-depleted human serum; and (b) whether these assembled preparations were haemolytically active. C 1q was removed from human serum by the addition of Sepharose 4B conjugated specific anti-Clq antibodies (2 mg/ml of gel) in the presence of 0.02 M EDTA and 0.001 M benzamidine. Specific rabbit anti-C1 q IgG was obtained first by solid phase immunoabsorption. This was used to minimize the level of non-anti-C1 q antibody containing rabbit IgG for conjugation to CNBr Sepharose 4B. C1 q-depleted serum was recalcified in the presence of benzamidine. Reassembled C1 was assessed by crossed immunoelectrophoresis using monospecifc rabbit anti-human C1 s antiserum. Haemolytic activity of the reassembled preparations was analyzed by a 'haemolysis in gel' technique [ 15 ]. Amboceptor-sensitized sheep erythrocytes were added to molten agarose (at 45°C) and allowed to gel, forming a 1 mm thick, homogeneous carpet of indicator cells in agarose. Circular wells (4 mm diameter) were punched from the gel and 10 #1 samples applied. Normal serum, by this method, demonstrated decreasing zones of lysis to a dilution of 1:8. Undiluted, Clq-depleted serum, by contrast, failed to produce any lysis. Haemolytic capacity of C1 q-depleted human serum, after addition of C1 q preparations, were compared to that obtained with normal human serum.
4. Results Initial experiments performed demonstrated that all C 1q contained in 3.0 ml of human serum could be removed by 1.0 ml of heparin-Sepharose. This assessment was made by comparing the recovery levels of C1 q eluted from the heparin column with the levels contained in serum before chromatography by EIA. Each ml of rabbit anti-IgG Sepharose was capable of removing all C1 q from 30 ml of pooled human serum, also determined quantitatively by EIA. Fig. 1 illustrates the slightly varying elution patterns of C1 q following chromatography of the different animal
sera over: (a) h e p a r i n - S e p h a r o s e ; and (b) over R-IgG Sepharose. Fig. 2 provides the agarose electrophoretic patterns of the pooled eluted fractions following heparin and IgG affinity chromatography with normal human plasma. C l q , if heated for 30 min at 56°C, no longer bound to heparin or IgG. Euglobulin preparations [6]
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.
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.
.
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Fig. 2. Agarose gel electrophoresis in barbital buffer containing 0.002 M EDTA, pH 8.6, 20 V/cm for 45 min. 10 pl samples were applied. A: normal human plasma (1:5 dilution). B: the pooled C 1q-rich eluted fractions following heparin affinity chromatography of human serum. C: the pooled Clq-rich eluted fractions following R-IgG affinity chromatography of B.
(mm)
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Fig. 1. The elution patterns of different Clq from various species following (A) heparin affinity;and (B) rabbit-IgG affinity chromatography, o - - % human Clq; ~ % dog Clq; • •, rabbit Clq; • •, sheep Clq; and D - - D , rat Clq estimated from Clq/agarose gel precipitation reaction during electrophoresis. Rabbit Clq demonstrated the same heparin affinity as human Clq, and sheep Clq the same R-IgG affinity as human Clq and are therefore not illustrated.
of pooled serum, in our hands, contained a significant proportion of C 1q ( 1 5 - 3 0 % ) that did not bind to IgG-Sepharose. Presumably this was due to irreversible aggregation of C1 q and/or complex formation with IgG that may occur during euglobulin precipitation. F r o m these results a simplified step-wise elution was designed, i.e. C1 q was removed from h e p a r i n Sepharose with the equilibration buffer but now containing 1.8 M NaC1. The C1 q-rich fractions were pooled, and dialyzed against 0.05 M Tris-HC1, 0.02 M EDTA in 0.1 M NaC1 before application to R-IgG Sepharose. Elution from the R-IgG column was achieved with a buffer containing 0.05 M Tris, 10% sucrose, 0.05 M EDTA in 1.0 M NaC1, pH 8.0 [20]. The isolated C l q was stored in this buffer at a concentration o f 2--4 mg/ml at 4°C. Table 1 summarizes and compares the purification steps and recovery o f C l q from all species tested. Isolated C1 q invariably produced a single cationic protein band by agarose gel electrophoresis with intense protein trailing from the site of sample appli305
Table 1 Purification of Clq Steps
Volume (ml)
Total protein (mg)
Clq (mg)
Recoverya (%)
A 1 Human serumb 2 Hep-Sepharose 3 R-IgG Sepharose
600 150 30
43,500 675 82
87 84 82
100 96 94
1 Rat plasmac 2 Hep-Sepharose 3 R-IgG Sepharose
150 40 5
10,700 152 12
14 13 12
100 96 85
1 Rabbit serumc 2 Hep-Sepharose 3 R-IgG Sepharose
250 45 15
18,200 162 265
28 25 25
100 89 89
1 Dog serumc 2 Hep-Sepharose 3 R-IgG Sepharose
250 55 15
17,200 87 27
32 28 27
100 87 84
1 Sheep plasmac 2 Hep-Sepharose 3 R-IgG Sepharose
250 65 15
14,800 183 30
35 32 30
100 91 85
aThese figures are the mean of duplicate runs and are approximate (-+10%) as they have been assessed from the values obtained solely by the Clq/agarose gel interaction. bClq measured by EIA and Clq-agarose gel interaction. CClq measured by Clq-agarose gel interaction only.
A
m I
H
D
R
Ra
S
B
H 306
D
R
Ra
S
cation. C1 q precipitation in agarose gels for all 5 species tested is illustrated in Fig. 3 and demonstrates, the similarity in results whether the gels were fixed, pressed and stained (Fig. 3A) or simply pressed and stained without fixation (Fig. 3B). An IgG 'M' component with cathodal migration was included for comparison. S D S - P A G E analysis revealed single protein bands under non-reducing conditions. By comparison with k n o w n marker-proteins, the molecular size of C l q obtained was estimated to be: h u m a n C l q 415,000; dog C l q 405,000; rabbit C l q 395,000;
Fig. 3. Clq/agarose gel electroprecipitation of the purified Clq from different species, run under the same conditions as for Fig. 2. A: the gel was fixed in picric acid, pressed dry and stained in 0.05 % kenacid blue. B: the gel was pressed without fixation and stained demonstrating the loss of IgG control protein only from the gel. I, human IgG myeloma protein (control); H, human Clq; D, dog Clq; R, rat Clq, Ra, rabbit Clq; S, sheep Clq.
sheep Clq 385,000 and rat Clq estimated to be 420,000. All species of Clq isolated, revealed noncovalently linked subunits in the presence of urea and multiple covalently linked peptide chains after reduction and alkylation similar to that described for human Clq [2]. Purified human and rat Clq demonstrated single cationic precipitin arcs when tested by crossed immunoelectrophoresis with rabbit anti-total human and rat serum, respectively. Clq isolated from all species demonstrated an ability to form macromolecular C1 with C l r - C l s contained in Clq-depleted human serum as shown for rat C 1q by crossed immunoelectrophoresis (Fig. 4). The
I
I i
|
I
I
F
r
,, f3c
Fig. 4. Crossed irnmunoelectrophoresisdemonstrating the reassembly of C1 following the addition of purified rat Clq (150 t~g/ml)to human Clq-depleted serum using anti-Cls antiserum. A: normal human serum. B: Clq depleted normal human serum A. C: rat Clq added to (B). "r, denotes macromolecular C1 ;#, Clr-C1 s complexes;and ~,, Cls-CT inactivator complexes.
reassembled C1 preparations were haemolytically active when tested by the 'haemolysin in gel' technique. Antiserum, monospecific for human C1 q, was produced in rabbits within 6 weeks and did not require absorption. A single precipitin line of complete immunological identity with C1 q in human serum and the purified component was obtained by immunoelectrophoresis and immunodiffusion. This antiC 1q produced a reaction of Complete identity with known anti-Clq antisera from two other sources. Clq preparations from the 4 other species also demonstrated weak but definite precipitin lines of partial identity with anti-human C1 q.
5. Discussion 'i
This procedure appears to be both specific and gentle for the isolation of C1 q as both ligands used apparently bound only functionally active Clq. The utilization of excess ligand over the amount of Clq to be applied in both affinity steps ensures high recovery of Clq from the species tested (85-94%). Heparin-affinity chromatography appears to be a more efficient and selective first step in the isolation of Clq than euglobulin precipitation. Clr and Cls are dissociated from Clq in the presence of EDTA and pass out in the void volume [21,22], Clt does not bind heparin in the presence of EDTA [23], and IgG does not bind to heparin at 0.15 M NaC1 [11]. An interaction between Clq and agarose has been previously recognized [17,18]. However, Assimeh et al. [19] considered it to be caused by contaminating IgG while Pohl et al. [20] considered it to represent some form of altered C1 q. Our results indicate that the C1 q-gel interaction is limited to a biologically active component as heat-treated Clq and the nonIgG-binding C1 q fraction in euglobulin preparations remained at the site of application without cathodal migration or precipitation. Monitoring of eluted C1 q from the affinity columns by its precipitation during electrophoresis in agarose gels appears to have some advantages over the more commonly used immunochemical and C1 qspecific haemolytic assays. Although much less sensitive, it is rapid since results can be obtained within 1 h. This agarose-gel interaction appears selective for Clq but not to be species specific. Haemolytic assays 307
specific for C l q can apparently be misleading because o f the presence o f inhibitory substances [ 16]. S D S - P A G E electrophoresis demonstrated that C 1 q isolated from all species studied were homogeneous with molecular sizes ranging from 385,000 to 421,000 indicating there was little significant difference between the species. C 1 q assembly experiments followed by haemolytic assays indicated that C1 q preparations from all 5 species studied were able complex with human C l r / C l s and form haemolyticaUy active C1.
Acknowledgements This work was supported by the Swedish Medical Research Council (Project No. B81-03X-00581-17A). The author is supported by a post-graduate scholarship from the New Zealand Medical Research Council. I wish to thank Inger Bomark for typing this manuscript.
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