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Interaction between components of the human classical complement pathway and immobilized cibacron blue F3GA
Journal of Immunological Methods, 30 (1979) 119--126 © Elsevier/North-Holland Biomedical Press
119
INTERACTION BETWEEN COMPONENTS OF THE HUMAN CLASSICAL COMPLEMENT PATHWAY AND IMMOBILIZED CIBACRON BLUE F3GA
ADRIAN P. GEE, TIBOR BORSOS and MICHAEL D.P. BOYLE Laboratory of Immunobiology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205, U.S.A.
(Received 1 March 1979, accepted 16 May 1979)
The interaction between the complement components in human serum and the dye, Cibacron Blue F3GA, immobilized on cross-linked agarose (Affi-Gel Blue) has been studied. All nine components of the classical complement pathway bound to the dye and could be recovered using a linear salt gradient. With the exception of C5 and C8, all the components were eluted over a narrow NaCl concentration range, with the following yields: C1, 17%; C2, 69%; C3, 92%; C4, 87%; C6, 105%; C7,109%; C9, 128%. C5 and C8 eluted throughout the NaCl gradient with yields of 103% and 14%, respectively. Since all components could be eluted without substantial contamination by albumin or IgG, this procedure may prove valuable as an initial step in the purification of complement components. In addition, the ability of immobilized Cibacron Blue F3GA to physically remove complement components may prove useful for both the decomplementation of serum and in elucidating the role of complement in immunological reactions.
INTRODUCTION A l b u m i n is p r e s e n t as a t r a c e c o n t a m i n a n t o f m a n y o f t h e f u n c t i o n a l l y p u r i f i e d c o m p o n e n t s o f t h e classical c o m p l e m e n t p a t h w a y , e.g. h u m a n C9 at a s e r u m c o n c e n t r a t i o n o f less t h a n 10 p g / m l has p h y s i o c h e m i c a l p r o p e r t i e s similar to t h o s e o f a l b u m i n w h i c h is p r e s e n t in s e r u m a t 3 5 - - 5 5 m g / m l . Although a trace a m o u n t of albumin does n o t appear to affect the functional a c t i v i t y o f t h e c o m p o n e n t s , it will h i n d e r t h e q u a n t i f i c a t i o n o f antigenic a n d h e m o l y t i c a U y - a c t i v e c o m p l e m e n t o n an a b s o l u t e m o l e c u l a r basis. T h e prese n c e Of a l b u m i n m a y also be a p r o b l e m d u r i n g t h e p r e p a r a t i o n o f c o m p o n e n t s o f high p u r i t y , r e q u i r e d f o r radiolabelling a n d t h e p r o d u c t i o n o f m o n o specific antisera. I t has r e c e n t l y b e e n r e p o r t e d t h a t t h e d y e C i b a c r o n Blue F 3 G A i m m o b i l i z e d on cross-linked agarose (Affi-Gel Blue) or S e p h a r o s e CL6B (Blue S e p h a r o s e ) has a s t r o n g a f f i n i t y f o r a l b u m i n (Travis a n d Pannell, 1 9 7 3 ; Angal a n d D e a n , 1 9 7 7 ) , as well as b i n d i n g e n z y m e s w h i c h h a v e a d i n u c l e o t i d e f o l d , e.g. D N A p o l y m e r a s e , a d e n y l a t e kinase a n d c y t o c h r o m e c ( T h o m p s o n et al., 1 9 7 5 ) . We believed t h a t t h e a f f i n i t y o f i m m o b i l i z e d Cibac r o n Blue F 3 G A f o r a l b u m i n c o u l d p o s s i b l y be used t o r e m o v e t h e t r a c e
120
contamination in complement components. Consequently, we examined the interaction between Affi-Gel Blue and the components of the h u m a n classical complement pathway. In this paper we report that all the components (C1--9) b o u n d to immobilized Cibracron blue F3GA and could be recovered with differing yields. The usefulness of this procedure for decomplementation of serum, or as an initial step in the purification of complement components is briefly discussed. M A T E R I ALS AND METHODS Sera
Serum was obtained from normal adult volunteers and stored w i t h o u t preservative at --40°C until use.
Complement components Functionally pure h u m a n complement components were obtained from the Cordis Labs., Miami, FL. Cells Sheep red blood cells (E) were collected and washed as described by Rapp and Borsos (1970). Cell intermediates (EA, EAC1, EAC14, EAC4) were prepared using an excess concentration of the IgM portion of anti-Forssman a n t i b o d y and excess complement components by the m e t h o d of Rapp and Borsos (1970). EAC1-7 indicator cells were prepared as described by Boyle et al. (1978). Buffers Isotonic veronal buffered saline (VBS), pH 7.4; VBS-gel, p = 0.15, containing 0.1% gelatin, 0.001 M Mg 2÷ and 0.00015 M Ca 2÷ and sucrose buffer, = 0.065, were prepared as reported by Rapp and Borsos (1970). Column methods Three milliliter samples of serum were applied to an Affi-Gel Blue (100-200 mesh, Bin Rad Labs., Richmond, CA) column of about 30 ml settled •bed volume. Non-specific adherence was reduced by pre-washing the resin with two bed volumes of VBS-gel followed by equilibration in 0.15 M NaC1 prior to use. Samples were eluted as described in the Results section. Protein determination Protein levels were determined on a Micromedic MS2 spectrophotometer by measuring the optical density at 280 nm. Conductance measurements The conductance of the fractions obtained during elution with the salt gradient was measured using a Radiometer Copenhagen Type CDM2c conductivity meter. All measurements were made at 0 ° C.
121
Determination of individual complement components The procedures for the titration of whole complement and components C1, C2 and C4 are described by Rapp and Borsos (1970). C3 and C5 through C7 were titrated according to the general procedure of Nelson et al. (1966) using 1.5 X 107 indicator cells in a total volume of 1.5 ml. C8 and C9 were titrated using 108 indicator cells in a final volume of 1.5 ml as described b y Boyle et al. (1978). Relative hemoglobin levels were determined by measuring the absorbance at 412 or 541 nm.
IgG determinations The concentration of IgG was measured using the Protein A radioimmunoassay described b y Langone et al. (1977). RESULTS Three milliliters of normal human serum were applied to a 1.5 X 14 cm column of Affi-Gel Blue and eluted with 0.15 M NaC1. Fractions of 5 ml were collected and the absorbance at 280 nm was followed. The profile obtained is shown in Fig. 1. The material from the high OD280 peak was pooled and tested for whole complement activity as well as for the presence of the individual complement components and albumin. The results indicated that the Affi-Gel Blue column had retained approximately 50% of the total OD2so applied and more than 98% of the hemolytic activity of both 4.0
3.0-
60
g d
g 2.0-
g
30 m
1.0
20 Gradient
0
10
20
30
40 50 60 70 FRACTION NUMBER
80
90
100
110
0
Fig. 1. E l u t i o n profile o f h u m a n s e r u m o n a n Affi-Gel Blue c o l u m n . 3 m l s e r u m was applied t o a 1.5 × 14 c m c o l u m n a n d e l u t e d w i t h 0.15 M NaCI. 5 m l f r a c t i o n s were collected. A g r a d i e n t f r o m 0.25 M NaCI t o 2.5 M NaC1 was s t a r t e d a t f r a c t i o n 31. The high OD280 p o o l ( f r a c t i o n s 2 - - 1 0 ) c o n t a i n e d less t h a n 5% o f t h e h e m o l y t i c activity o f e i t h e r the w h o l e c o m p l e m e n t or a n y individual c o m p o n e n t . Black circles, a b s o r b a n c e a t 2 8 0 n m ; o p e n circles, c o n d u c t a n c e o f g r a d i e n t fractions.
122 whole complement and of the individual components (data not shown). In addition, there was no detectable albumin in the material that passed directly through the column. By contrast, when the h u m a n serum was passed down a Biogel A-15 m column (the support matrix for Cibacron Blue) 95-100% of the OD280 was recovered, with no loss of total complement activity, and less than 37% loss of any individual c o m p o n e n t (data n o t shown). Quantitative immunoprecipitation revealed t h a t the pool from Affi-Gel Blue contained ~ 1 % of the serum albumin applied, while close to 100% recovery was achieved from the Biogel A-15 m column. The recovery of IgG from the AffiGel Blue column ranged from 48% to over 90% of that applied depending upon the batch of serum or resin that was used. Rechromatography of the high OD2s0 pool on a second Affi-Gel Blue column resulted in recoveries of 36--78% of the IgG, suggesting that the original loss was due to nonspecific interactions, rather than the depletion of a specific IgG subclass. From these results it would appear that Affi-Gel Blue selectively removed virtually all the hemolytically active components of the classical complement pathway and all the serum albumin. In addition, up to 50% of the IgG was lost in a nonspecific manner. The next series of experiments tested the feasibility of eluting the various bound complement components from Affi-Gel Blue using a linear gradient of NaC1 from 0.25 M to 2.5 M. Three milliliters of human serum was applied to a 1.5 × 14 cm column of Affi-Gel Blue and then washed with 0.15 M NaC1 until the OD2s0 of the collected material was less than 0.05 units. At this stage a linear salt gradient consisting of 300 ml of 0.25 M NaC1 (12.3 m m h o ) and 300 ml 2.5 M NaC1 (93.0 m m h o ) was applied and 5 ml fractions were collected (Fig. 1). The various fractions were screened for the activity of individual complement components by the appropriate assay described in Materials and Methods. Since these assays were designed to locate, rather than quantify, the components, the conditions used were variable, e.g. the time of incubation was n o t necessarily the end point for the reaction. The results, presented in Fig. 2, demonstrate that the activity of the various components I through 9 was recoverable. With the exception of C5 and C8, all the components were eluted between conductances of 10 and 35 m m h o . C8 was eluted over a greater concentration range as double peaked curve (Fig. 2) indicating that this c o m p o n e n t may n o t be homogeneous. The fractions containing the various c o m p o n e n t activities were pooled on the basis of their hemolytic activity as shown in Fig. 2 and the yield of each c o m p o n e n t estimated by titrating an aliquot of the pool in the appropriate system. To enable measurement of complement activity on a molecular basis the following conditions must be fulfilled: (1) The number of receptor sites on the cell surface that are capable of reacting with the c o m p o n e n t being assayed must be large enough to allow pseudo first-order reaction kinetics. (2) The a m o u n t and rate of lysis must be a function only of the c o m p o n e n t being titrated; therefore, an increase in the concentration of any other c o m p o n e n t should have a negligible effect on the rate and e x t e n t of lysis. Preliminary experi-
a 1.0 = 100% o f the starting activity.
OD280 units recovered % OD2so recovered % Effective molecules recovered Increase in specific c o m p l e m e n t activity/OD280 unit a
Effective molecules applied (3 ml serum) OD280 units applied V o l u m e of pool from c o l u m n (ml) Effective molecules recovered 1.3 X l O 13 201.7 244 2.24 X 1012 4.88 2.4 17.2 7.1
C1 8.5 x l O lo 201.7 244 5.86 X 1010 4.88 2.4 68.9 28.5
C2 5.8 x l O 11 201.7 244 5.34 × 1011 4.88 2.4 92.1 38.0
C3 3.9 ×1013 201.7 244 3.41 × 1013 4.88 2.4 87.2 36.1
C4
RECOVERY OF COMPLEMENT COMPONENTS FROM AFFI-GEL BLUE
TABLE 1
1.2 ×1012 201.7 588 1.23 × 1012 15.28 7.6 102.5 13.5
C5 2.8 ×1012 201.7 244 2.93 × 1012 4.88 2.4 104.6 43.3
C6
2.1 x l O 12 201.7 244 2.29 × 1012 4.88 2.4 109.0 46.9
C7
3.1 ×1013 201.7 588 4.17 × 1012 15.28 7.6 13.5 1.8
C8'
1.9 ×1013 201.7 244 2.44 × 1012 4.88 2.4 128.4 53.1
C9
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Fig. 2. Elution of complement components from Affi-Ge] Blue. Triangles, absorbanee at 9.80 nm; black circles, hemolytic activity of component; open circles, conductance of fractions. For precise experimental details see text.
ments were carried o u t to ensure t ha t these conditions were m e t for each of the assay systems used. The n u m b e r of effective molecules was calculated f r o m a log-log plot o f relative c o n c e n t r a t i o n of the pooled material vs. --ln ( l - - y ) , where y is the fraction of cells lysed. In all cases this plot yielded a straight line with a slope of close to 1, indicating t hat under the conditions o f the assays the reactions were c o n f o r m i n g to a one-hit mechanism (Mayer, 1961). The results, presented in Table 1, indicate t hat with the e x c e p t i o n of C1 (17%) and C8 (14%), more than 69% of the ot her c o m p o n e n t s were recovered. Indeed for C5, C6, C7 and C9 recoveries in excess of 100% were observed, possibly indicating t hat during the procedure t h e y had been separated f r o m an inactivator, or some o t h e r inhibitor o f their activity. In all cases, the activity of the c o m p o n e n t s per OD280 unit was increased over the starting serum. In particular C6, C7 and C9 showed a greater than 40-fold increase in activity/OD~80 unit, suggesting t hat this m ay be a useful initial step in the purification of these c o m p o n e n t s . DISCUSSION
In the experiments r e p o r t e d in this paper we f o u n d t hat Affi-Gel Blue b o u n d and removed all of the c o m p o n e n t s of the classical c o m p l e m e n t p a t h w a y and all of the albumin f r o m normal h u m a n serum. Similar results
125
(not shown) have been obtained with guinea pig serum. It is not clear at present what structural feature on each of the components facilitated their binding to Cibacron Blue F3GA. The interaction between a wide range of biologically active molecules (approximately 50% of serum) and the immobilized dye brings into question the selectivity of this material. The specificity of the interaction of the dye with enzymes has been attributed to the presence of a dinucleotide fold, or a super-secondary structure to which the dye binds (Thompson et al., 1975). More recently it has been suggested that Cibacron Blue F3GA and a series of other structurally dissimilar sulphonated aromatic dyes bind by virtue of their ability to act as competitive inhibitors, rather than by specifically binding to the nucleotide-binding site (Ashton and Polya, 1978). The high affinity of the dye for albumin has not been explained, but seems to be characteristic of a series of other dyes, e.g. Bromophenol Blue (Bjerrum, 1968) and drugs, e.g. Phenylbutazone (Dayton et al., 1973). However, the ability of immobilized Cibacron Blue F3GA to remove total complement activity may be of value, since, unlike chemical or thermal inactivation, the complement components are both hemolytically inactivated and physically removed. This may be useful in determining the role of complement in various immunological reactions, e.g. various complement components or their cleavage products have been implicated in the killing of tumor cells by macrophages (Schorlemmer et al., 1978). By contrast, in many of the reactions in which complement was not thought to be involved, the complement was inactivated rather than physically removed from the system under investigation, e.g. antibody-dependent cell mediated cytotoxicity (Gale and Zighelboim, 1975). It is therefore possible that the remaining antigenic non-hemolytically-active components may have had some part in the effects observed. Physical removal of complement components by binding to Affi-Gel Blue would overcome this problem. Affi-Gel Blue may also be of great use as an initial step in the purification of certain late-acting components, particularly C6, C7 and C9 which can be recovered with high yield and show a conciderable increase in their specific activity. This approach is being actively pursued. REFERENCES Angal, S. and P.D.G. Dean, 1977, Biochem. J. 167,301. Ashton, A. R. and G.M. Polya, 1978, Biochem. J. 175,501. Bjerrum, O. J., 1968, Scand. J. Clin. Lab. Invest. 22, 41. Boyle, M.D.P., J.J. Langone and T. Borsos, 1978, J. Immunol. 120, 1271. Dayton, P.G., Z.H. Israili and J.M. Perel, 1973, Ann. N.Y. Acad. Sci. 226, 172. Gale, R.P. and J. Zighelboim, 1975, J.Immunol. 114, 1047. Langone, J.J., M.D.P. Boyle and T. Borsos, 1977, J. Immunol. Methods 18, 281. Mayer, M.M., 1961, in: Immunoehemieal Approaches to Problems in Microbiology, eds. M. Heidelberger and O.J. Plescia (Rutgers University Press, New Brunswick, NJ) p. 268. Nelson, R.J., J. Jensen, I. Gigli and N. Tamura, 1966, Immunochemistry 3, 111. Rapp, H.J. and T. Borsos, 1970, Molecular Basis of Complement Action (AppletonCentury-Crofts, New York).
126 Schorlemmer, H.J., U. Hadding, D. Bitter-Suermann and A.C. Allison, 1978, Proceedings of the European Reticuloendothelial Society Symposium on the Macrophage and Cancer, in press. Thompson, S. T., K.H. Cass and E. Stellwagen, 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 669. Travis, J. and R. Pannell, 1973, Clin. Chim. Acta 49, 49.
119
INTERACTION BETWEEN COMPONENTS OF THE HUMAN CLASSICAL COMPLEMENT PATHWAY AND IMMOBILIZED CIBACRON BLUE F3GA
ADRIAN P. GEE, TIBOR BORSOS and MICHAEL D.P. BOYLE Laboratory of Immunobiology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20205, U.S.A.
(Received 1 March 1979, accepted 16 May 1979)
The interaction between the complement components in human serum and the dye, Cibacron Blue F3GA, immobilized on cross-linked agarose (Affi-Gel Blue) has been studied. All nine components of the classical complement pathway bound to the dye and could be recovered using a linear salt gradient. With the exception of C5 and C8, all the components were eluted over a narrow NaCl concentration range, with the following yields: C1, 17%; C2, 69%; C3, 92%; C4, 87%; C6, 105%; C7,109%; C9, 128%. C5 and C8 eluted throughout the NaCl gradient with yields of 103% and 14%, respectively. Since all components could be eluted without substantial contamination by albumin or IgG, this procedure may prove valuable as an initial step in the purification of complement components. In addition, the ability of immobilized Cibacron Blue F3GA to physically remove complement components may prove useful for both the decomplementation of serum and in elucidating the role of complement in immunological reactions.
INTRODUCTION A l b u m i n is p r e s e n t as a t r a c e c o n t a m i n a n t o f m a n y o f t h e f u n c t i o n a l l y p u r i f i e d c o m p o n e n t s o f t h e classical c o m p l e m e n t p a t h w a y , e.g. h u m a n C9 at a s e r u m c o n c e n t r a t i o n o f less t h a n 10 p g / m l has p h y s i o c h e m i c a l p r o p e r t i e s similar to t h o s e o f a l b u m i n w h i c h is p r e s e n t in s e r u m a t 3 5 - - 5 5 m g / m l . Although a trace a m o u n t of albumin does n o t appear to affect the functional a c t i v i t y o f t h e c o m p o n e n t s , it will h i n d e r t h e q u a n t i f i c a t i o n o f antigenic a n d h e m o l y t i c a U y - a c t i v e c o m p l e m e n t o n an a b s o l u t e m o l e c u l a r basis. T h e prese n c e Of a l b u m i n m a y also be a p r o b l e m d u r i n g t h e p r e p a r a t i o n o f c o m p o n e n t s o f high p u r i t y , r e q u i r e d f o r radiolabelling a n d t h e p r o d u c t i o n o f m o n o specific antisera. I t has r e c e n t l y b e e n r e p o r t e d t h a t t h e d y e C i b a c r o n Blue F 3 G A i m m o b i l i z e d on cross-linked agarose (Affi-Gel Blue) or S e p h a r o s e CL6B (Blue S e p h a r o s e ) has a s t r o n g a f f i n i t y f o r a l b u m i n (Travis a n d Pannell, 1 9 7 3 ; Angal a n d D e a n , 1 9 7 7 ) , as well as b i n d i n g e n z y m e s w h i c h h a v e a d i n u c l e o t i d e f o l d , e.g. D N A p o l y m e r a s e , a d e n y l a t e kinase a n d c y t o c h r o m e c ( T h o m p s o n et al., 1 9 7 5 ) . We believed t h a t t h e a f f i n i t y o f i m m o b i l i z e d Cibac r o n Blue F 3 G A f o r a l b u m i n c o u l d p o s s i b l y be used t o r e m o v e t h e t r a c e
120
contamination in complement components. Consequently, we examined the interaction between Affi-Gel Blue and the components of the h u m a n classical complement pathway. In this paper we report that all the components (C1--9) b o u n d to immobilized Cibracron blue F3GA and could be recovered with differing yields. The usefulness of this procedure for decomplementation of serum, or as an initial step in the purification of complement components is briefly discussed. M A T E R I ALS AND METHODS Sera
Serum was obtained from normal adult volunteers and stored w i t h o u t preservative at --40°C until use.
Complement components Functionally pure h u m a n complement components were obtained from the Cordis Labs., Miami, FL. Cells Sheep red blood cells (E) were collected and washed as described by Rapp and Borsos (1970). Cell intermediates (EA, EAC1, EAC14, EAC4) were prepared using an excess concentration of the IgM portion of anti-Forssman a n t i b o d y and excess complement components by the m e t h o d of Rapp and Borsos (1970). EAC1-7 indicator cells were prepared as described by Boyle et al. (1978). Buffers Isotonic veronal buffered saline (VBS), pH 7.4; VBS-gel, p = 0.15, containing 0.1% gelatin, 0.001 M Mg 2÷ and 0.00015 M Ca 2÷ and sucrose buffer, = 0.065, were prepared as reported by Rapp and Borsos (1970). Column methods Three milliliter samples of serum were applied to an Affi-Gel Blue (100-200 mesh, Bin Rad Labs., Richmond, CA) column of about 30 ml settled •bed volume. Non-specific adherence was reduced by pre-washing the resin with two bed volumes of VBS-gel followed by equilibration in 0.15 M NaC1 prior to use. Samples were eluted as described in the Results section. Protein determination Protein levels were determined on a Micromedic MS2 spectrophotometer by measuring the optical density at 280 nm. Conductance measurements The conductance of the fractions obtained during elution with the salt gradient was measured using a Radiometer Copenhagen Type CDM2c conductivity meter. All measurements were made at 0 ° C.
121
Determination of individual complement components The procedures for the titration of whole complement and components C1, C2 and C4 are described by Rapp and Borsos (1970). C3 and C5 through C7 were titrated according to the general procedure of Nelson et al. (1966) using 1.5 X 107 indicator cells in a total volume of 1.5 ml. C8 and C9 were titrated using 108 indicator cells in a final volume of 1.5 ml as described b y Boyle et al. (1978). Relative hemoglobin levels were determined by measuring the absorbance at 412 or 541 nm.
IgG determinations The concentration of IgG was measured using the Protein A radioimmunoassay described b y Langone et al. (1977). RESULTS Three milliliters of normal human serum were applied to a 1.5 X 14 cm column of Affi-Gel Blue and eluted with 0.15 M NaC1. Fractions of 5 ml were collected and the absorbance at 280 nm was followed. The profile obtained is shown in Fig. 1. The material from the high OD280 peak was pooled and tested for whole complement activity as well as for the presence of the individual complement components and albumin. The results indicated that the Affi-Gel Blue column had retained approximately 50% of the total OD2so applied and more than 98% of the hemolytic activity of both 4.0
3.0-
60
g d
g 2.0-
g
30 m
1.0
20 Gradient
0
10
20
30
40 50 60 70 FRACTION NUMBER
80
90
100
110
0
Fig. 1. E l u t i o n profile o f h u m a n s e r u m o n a n Affi-Gel Blue c o l u m n . 3 m l s e r u m was applied t o a 1.5 × 14 c m c o l u m n a n d e l u t e d w i t h 0.15 M NaCI. 5 m l f r a c t i o n s were collected. A g r a d i e n t f r o m 0.25 M NaCI t o 2.5 M NaC1 was s t a r t e d a t f r a c t i o n 31. The high OD280 p o o l ( f r a c t i o n s 2 - - 1 0 ) c o n t a i n e d less t h a n 5% o f t h e h e m o l y t i c activity o f e i t h e r the w h o l e c o m p l e m e n t or a n y individual c o m p o n e n t . Black circles, a b s o r b a n c e a t 2 8 0 n m ; o p e n circles, c o n d u c t a n c e o f g r a d i e n t fractions.
122 whole complement and of the individual components (data not shown). In addition, there was no detectable albumin in the material that passed directly through the column. By contrast, when the h u m a n serum was passed down a Biogel A-15 m column (the support matrix for Cibacron Blue) 95-100% of the OD280 was recovered, with no loss of total complement activity, and less than 37% loss of any individual c o m p o n e n t (data n o t shown). Quantitative immunoprecipitation revealed t h a t the pool from Affi-Gel Blue contained ~ 1 % of the serum albumin applied, while close to 100% recovery was achieved from the Biogel A-15 m column. The recovery of IgG from the AffiGel Blue column ranged from 48% to over 90% of that applied depending upon the batch of serum or resin that was used. Rechromatography of the high OD2s0 pool on a second Affi-Gel Blue column resulted in recoveries of 36--78% of the IgG, suggesting that the original loss was due to nonspecific interactions, rather than the depletion of a specific IgG subclass. From these results it would appear that Affi-Gel Blue selectively removed virtually all the hemolytically active components of the classical complement pathway and all the serum albumin. In addition, up to 50% of the IgG was lost in a nonspecific manner. The next series of experiments tested the feasibility of eluting the various bound complement components from Affi-Gel Blue using a linear gradient of NaC1 from 0.25 M to 2.5 M. Three milliliters of human serum was applied to a 1.5 × 14 cm column of Affi-Gel Blue and then washed with 0.15 M NaC1 until the OD2s0 of the collected material was less than 0.05 units. At this stage a linear salt gradient consisting of 300 ml of 0.25 M NaC1 (12.3 m m h o ) and 300 ml 2.5 M NaC1 (93.0 m m h o ) was applied and 5 ml fractions were collected (Fig. 1). The various fractions were screened for the activity of individual complement components by the appropriate assay described in Materials and Methods. Since these assays were designed to locate, rather than quantify, the components, the conditions used were variable, e.g. the time of incubation was n o t necessarily the end point for the reaction. The results, presented in Fig. 2, demonstrate that the activity of the various components I through 9 was recoverable. With the exception of C5 and C8, all the components were eluted between conductances of 10 and 35 m m h o . C8 was eluted over a greater concentration range as double peaked curve (Fig. 2) indicating that this c o m p o n e n t may n o t be homogeneous. The fractions containing the various c o m p o n e n t activities were pooled on the basis of their hemolytic activity as shown in Fig. 2 and the yield of each c o m p o n e n t estimated by titrating an aliquot of the pool in the appropriate system. To enable measurement of complement activity on a molecular basis the following conditions must be fulfilled: (1) The number of receptor sites on the cell surface that are capable of reacting with the c o m p o n e n t being assayed must be large enough to allow pseudo first-order reaction kinetics. (2) The a m o u n t and rate of lysis must be a function only of the c o m p o n e n t being titrated; therefore, an increase in the concentration of any other c o m p o n e n t should have a negligible effect on the rate and e x t e n t of lysis. Preliminary experi-
a 1.0 = 100% o f the starting activity.
OD280 units recovered % OD2so recovered % Effective molecules recovered Increase in specific c o m p l e m e n t activity/OD280 unit a
Effective molecules applied (3 ml serum) OD280 units applied V o l u m e of pool from c o l u m n (ml) Effective molecules recovered 1.3 X l O 13 201.7 244 2.24 X 1012 4.88 2.4 17.2 7.1
C1 8.5 x l O lo 201.7 244 5.86 X 1010 4.88 2.4 68.9 28.5
C2 5.8 x l O 11 201.7 244 5.34 × 1011 4.88 2.4 92.1 38.0
C3 3.9 ×1013 201.7 244 3.41 × 1013 4.88 2.4 87.2 36.1
C4
RECOVERY OF COMPLEMENT COMPONENTS FROM AFFI-GEL BLUE
TABLE 1
1.2 ×1012 201.7 588 1.23 × 1012 15.28 7.6 102.5 13.5
C5 2.8 ×1012 201.7 244 2.93 × 1012 4.88 2.4 104.6 43.3
C6
2.1 x l O 12 201.7 244 2.29 × 1012 4.88 2.4 109.0 46.9
C7
3.1 ×1013 201.7 588 4.17 × 1012 15.28 7.6 13.5 1.8
C8'
1.9 ×1013 201.7 244 2.44 × 1012 4.88 2.4 128.4 53.1
C9
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Fig. 2. Elution of complement components from Affi-Ge] Blue. Triangles, absorbanee at 9.80 nm; black circles, hemolytic activity of component; open circles, conductance of fractions. For precise experimental details see text.
ments were carried o u t to ensure t ha t these conditions were m e t for each of the assay systems used. The n u m b e r of effective molecules was calculated f r o m a log-log plot o f relative c o n c e n t r a t i o n of the pooled material vs. --ln ( l - - y ) , where y is the fraction of cells lysed. In all cases this plot yielded a straight line with a slope of close to 1, indicating t hat under the conditions o f the assays the reactions were c o n f o r m i n g to a one-hit mechanism (Mayer, 1961). The results, presented in Table 1, indicate t hat with the e x c e p t i o n of C1 (17%) and C8 (14%), more than 69% of the ot her c o m p o n e n t s were recovered. Indeed for C5, C6, C7 and C9 recoveries in excess of 100% were observed, possibly indicating t hat during the procedure t h e y had been separated f r o m an inactivator, or some o t h e r inhibitor o f their activity. In all cases, the activity of the c o m p o n e n t s per OD280 unit was increased over the starting serum. In particular C6, C7 and C9 showed a greater than 40-fold increase in activity/OD~80 unit, suggesting t hat this m ay be a useful initial step in the purification of these c o m p o n e n t s . DISCUSSION
In the experiments r e p o r t e d in this paper we f o u n d t hat Affi-Gel Blue b o u n d and removed all of the c o m p o n e n t s of the classical c o m p l e m e n t p a t h w a y and all of the albumin f r o m normal h u m a n serum. Similar results
125
(not shown) have been obtained with guinea pig serum. It is not clear at present what structural feature on each of the components facilitated their binding to Cibacron Blue F3GA. The interaction between a wide range of biologically active molecules (approximately 50% of serum) and the immobilized dye brings into question the selectivity of this material. The specificity of the interaction of the dye with enzymes has been attributed to the presence of a dinucleotide fold, or a super-secondary structure to which the dye binds (Thompson et al., 1975). More recently it has been suggested that Cibacron Blue F3GA and a series of other structurally dissimilar sulphonated aromatic dyes bind by virtue of their ability to act as competitive inhibitors, rather than by specifically binding to the nucleotide-binding site (Ashton and Polya, 1978). The high affinity of the dye for albumin has not been explained, but seems to be characteristic of a series of other dyes, e.g. Bromophenol Blue (Bjerrum, 1968) and drugs, e.g. Phenylbutazone (Dayton et al., 1973). However, the ability of immobilized Cibacron Blue F3GA to remove total complement activity may be of value, since, unlike chemical or thermal inactivation, the complement components are both hemolytically inactivated and physically removed. This may be useful in determining the role of complement in various immunological reactions, e.g. various complement components or their cleavage products have been implicated in the killing of tumor cells by macrophages (Schorlemmer et al., 1978). By contrast, in many of the reactions in which complement was not thought to be involved, the complement was inactivated rather than physically removed from the system under investigation, e.g. antibody-dependent cell mediated cytotoxicity (Gale and Zighelboim, 1975). It is therefore possible that the remaining antigenic non-hemolytically-active components may have had some part in the effects observed. Physical removal of complement components by binding to Affi-Gel Blue would overcome this problem. Affi-Gel Blue may also be of great use as an initial step in the purification of certain late-acting components, particularly C6, C7 and C9 which can be recovered with high yield and show a conciderable increase in their specific activity. This approach is being actively pursued. REFERENCES Angal, S. and P.D.G. Dean, 1977, Biochem. J. 167,301. Ashton, A. R. and G.M. Polya, 1978, Biochem. J. 175,501. Bjerrum, O. J., 1968, Scand. J. Clin. Lab. Invest. 22, 41. Boyle, M.D.P., J.J. Langone and T. Borsos, 1978, J. Immunol. 120, 1271. Dayton, P.G., Z.H. Israili and J.M. Perel, 1973, Ann. N.Y. Acad. Sci. 226, 172. Gale, R.P. and J. Zighelboim, 1975, J.Immunol. 114, 1047. Langone, J.J., M.D.P. Boyle and T. Borsos, 1977, J. Immunol. Methods 18, 281. Mayer, M.M., 1961, in: Immunoehemieal Approaches to Problems in Microbiology, eds. M. Heidelberger and O.J. Plescia (Rutgers University Press, New Brunswick, NJ) p. 268. Nelson, R.J., J. Jensen, I. Gigli and N. Tamura, 1966, Immunochemistry 3, 111. Rapp, H.J. and T. Borsos, 1970, Molecular Basis of Complement Action (AppletonCentury-Crofts, New York).
126 Schorlemmer, H.J., U. Hadding, D. Bitter-Suermann and A.C. Allison, 1978, Proceedings of the European Reticuloendothelial Society Symposium on the Macrophage and Cancer, in press. Thompson, S. T., K.H. Cass and E. Stellwagen, 1975, Proc. Natl. Acad. Sci. U.S.A. 72, 669. Travis, J. and R. Pannell, 1973, Clin. Chim. Acta 49, 49.