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The effect of iodine on the hemolytic activity of the second component of human complement
Complement Workshop: Abstracts
501
ELECTROPHORESIS OF NINE COMPONENTS OF GUINEA-PIG C O M P L E M E N T ON P O L Y A C R Y L A M I D E G E L K. NISHIOKA National Cancer Center Research Institute, Tokyo, Japan T h i n layer electrophoresis of each complement component or of serum was carried out in a polyacrylamide gel using various buffers. The gel was then overlayed with agar containing sensitized sheep erythrocytes and a mixture of complement components lacking the test component. The hemolytic patterns were developed by incubation in a moist chamber at 37 °. C'3c, C'3b, C'3e, C'3f, C'3a and C'3d activites were found to have characteristic mobilities at pH 8.3 in Tris-borate buffer. C'2 and C'4 could also be detected in this assay system. T h e hemolytic activity of C'1 of the euglobulin fraction of the serum of a single guinea-pig was found in three distinct positions after electrophoresis in Tris-citrate buffer at pH 8"8. T h e possible significance of the electrophoretic behavior of C'1 was discussed. Purification and electrophoresis of each component were carried out in collaboration with Drs Tachibana, Takahashi, Yoshida, Sekine, Mayumi, Okada, Torisu and Arata.
THE EFFECT OF IODINE ON THE HEMOLYTIC ACTIVITY OF THE SECOND COMPONENT OF HUMAN COMPLEMENT M. J. POLLEY and H. J. MOLLER-EBERHARD Scripps Clinic and Research Foundation, La Jolla, California It has now been recognized that the enhancement of C'2 hemolytic activity previously attributed to iodoacetamide is due to the action of iodine which is present in crystalline iodoacetamide. Treatment of C'2 with 10 -4 M Is in 5 × 10 -~ M K I at pH 6"0 resulted in an apparent 6 to 10-fold increase in hemolytic activity and an increase in half-life of EAC'la,4,2a from 8 rain to approximately 120 min. Whereas treatment with iodine prevented subsequent inactivation by p-chloromercuribenzoate (p-CMB), treatment with p-CMB did not prevent subsequent activation by iodine. Experiments performed with C'3 using 14C-labeled p-CMB showed that protein-bound p - C M B was totally removed by treatment of C'3 with iodine under conditions similar to those employed for C'2. From experiments using radioactively labeled iodine (1251) it was calculated that at maximal enhancement only 1 in more than 100 C'2 molecules had been iodinated. This finding, combined with the results of the experiments with p-CMB, suggest that the enhancement effect on C'2 produced by the iodine is due not to uptake of iodine, but to oxidation of some group, probably a sulfhydryl group, which takes place prior to iodination of the molecule.
S T U D I E S ON C'4 W I T H A N T I - C O M P L E M E N T S E R U M K. W. PONDMAN, J. F. BLEUMERSand M. VAN DER VEER Centraal LaboratoHum van de Bloedtransfusiedienst van het Nederlandsche Roode Kruis, Amsterdam, T h e Netherlands Anti-complement serum contains anti-ill c and anti-/31 ~. as well as other antibodies which can be readily demonstrated when aged serum is analyzed by immunoelectrophoresis. Incubation of fresh serum at 37 ° for 2 hr resulted in the appearance of an al/a~ antigen. It was found that the ~1/a2 antigen was a complex of two molecules, which were called /31• x and /31~v. One of these (/31~z) carries /31~ antigenic determinants. Immunoelectrophoresis data demonstrated that/31 ~ ~ and/31 Ev have common antigenic determinants unrelated to/31 E. This was further substantiated by experiments in which the/31E xu complex was preferentially and completely absent in immunoelectrophoresis patterns from aged serum and anti-complement after absorption of this antiserum with E/31Ev cells. Immune hemolysis studies showed that the/31 gzv complex is hemolytically inactive. 131Eand the/31B xv complex were separated from pseudo-globulins of aged human serum by repeated chromatography on DEAE cellulose columns and preparative acrylamide gel electrophoresis. T h e separated components were used in agglutination inhibition studies with two varieties of EC' cells. Complete inhibition of EC'4 agglutination occurred upon addition of 131~. as well as/31~zu to anti-C' serum. Complete agglutination inhibition occurred with E/31~v upon/31 Ezv addition. Addition of/31 E to anti-complement serum did not diminish the agglutination titer with these cells (E/31~ ) .
501
ELECTROPHORESIS OF NINE COMPONENTS OF GUINEA-PIG C O M P L E M E N T ON P O L Y A C R Y L A M I D E G E L K. NISHIOKA National Cancer Center Research Institute, Tokyo, Japan T h i n layer electrophoresis of each complement component or of serum was carried out in a polyacrylamide gel using various buffers. The gel was then overlayed with agar containing sensitized sheep erythrocytes and a mixture of complement components lacking the test component. The hemolytic patterns were developed by incubation in a moist chamber at 37 °. C'3c, C'3b, C'3e, C'3f, C'3a and C'3d activites were found to have characteristic mobilities at pH 8.3 in Tris-borate buffer. C'2 and C'4 could also be detected in this assay system. T h e hemolytic activity of C'1 of the euglobulin fraction of the serum of a single guinea-pig was found in three distinct positions after electrophoresis in Tris-citrate buffer at pH 8"8. T h e possible significance of the electrophoretic behavior of C'1 was discussed. Purification and electrophoresis of each component were carried out in collaboration with Drs Tachibana, Takahashi, Yoshida, Sekine, Mayumi, Okada, Torisu and Arata.
THE EFFECT OF IODINE ON THE HEMOLYTIC ACTIVITY OF THE SECOND COMPONENT OF HUMAN COMPLEMENT M. J. POLLEY and H. J. MOLLER-EBERHARD Scripps Clinic and Research Foundation, La Jolla, California It has now been recognized that the enhancement of C'2 hemolytic activity previously attributed to iodoacetamide is due to the action of iodine which is present in crystalline iodoacetamide. Treatment of C'2 with 10 -4 M Is in 5 × 10 -~ M K I at pH 6"0 resulted in an apparent 6 to 10-fold increase in hemolytic activity and an increase in half-life of EAC'la,4,2a from 8 rain to approximately 120 min. Whereas treatment with iodine prevented subsequent inactivation by p-chloromercuribenzoate (p-CMB), treatment with p-CMB did not prevent subsequent activation by iodine. Experiments performed with C'3 using 14C-labeled p-CMB showed that protein-bound p - C M B was totally removed by treatment of C'3 with iodine under conditions similar to those employed for C'2. From experiments using radioactively labeled iodine (1251) it was calculated that at maximal enhancement only 1 in more than 100 C'2 molecules had been iodinated. This finding, combined with the results of the experiments with p-CMB, suggest that the enhancement effect on C'2 produced by the iodine is due not to uptake of iodine, but to oxidation of some group, probably a sulfhydryl group, which takes place prior to iodination of the molecule.
S T U D I E S ON C'4 W I T H A N T I - C O M P L E M E N T S E R U M K. W. PONDMAN, J. F. BLEUMERSand M. VAN DER VEER Centraal LaboratoHum van de Bloedtransfusiedienst van het Nederlandsche Roode Kruis, Amsterdam, T h e Netherlands Anti-complement serum contains anti-ill c and anti-/31 ~. as well as other antibodies which can be readily demonstrated when aged serum is analyzed by immunoelectrophoresis. Incubation of fresh serum at 37 ° for 2 hr resulted in the appearance of an al/a~ antigen. It was found that the ~1/a2 antigen was a complex of two molecules, which were called /31• x and /31~v. One of these (/31~z) carries /31~ antigenic determinants. Immunoelectrophoresis data demonstrated that/31 ~ ~ and/31 Ev have common antigenic determinants unrelated to/31 E. This was further substantiated by experiments in which the/31E xu complex was preferentially and completely absent in immunoelectrophoresis patterns from aged serum and anti-complement after absorption of this antiserum with E/31Ev cells. Immune hemolysis studies showed that the/31 gzv complex is hemolytically inactive. 131Eand the/31B xv complex were separated from pseudo-globulins of aged human serum by repeated chromatography on DEAE cellulose columns and preparative acrylamide gel electrophoresis. T h e separated components were used in agglutination inhibition studies with two varieties of EC' cells. Complete inhibition of EC'4 agglutination occurred upon addition of 131~. as well as/31~zu to anti-C' serum. Complete agglutination inhibition occurred with E/31~v upon/31 Ezv addition. Addition of/31 E to anti-complement serum did not diminish the agglutination titer with these cells (E/31~ ) .