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The chemotactic factor, generated in whole rabbit serum following treatment with immune precipitates, was found to be principally associated with fractions containing the fifth and sixth components of complement (C'5 and C'6) after electrophoretic separation of serum. The chemotactic factor could also be generated in the intact animal, adding credence to its importance in tissue reactions induced by immunological agents. The isolated and activated C'5 and C'6 complex was found to be at least 10-20 times more active in the chemotaxis of rabbit polymorphonuclear leucocytes (PMNs) in vitro than bradykinin, kallidin, histamine, serotonin and extracts of PMN granules. The amino acid derivatives N-CBZ-glycyl-L-phenylalanine and N-CBZ-α glutamyl-L-tyrosine inhibited formation of the chemotactic factor in serum. The latter derivative also caused loss of activity of the preformed chemotactic factor in rabbit serum and in density gradient ultracentrifugation it was found that the C'5 and C'6 complex dissociated in the presence of this inhibitor. Chemotactically active C'5 to C'6 fractions, when added to the suspension of PMNs, prevented the ability of these cells to migrate toward a chemotactic source. It was also found that the ratio of combination of C'5 and C'6 was critical for the full expression of chemotactic activity. Utilizing purified components of human C', very recently obtained data indicate the requirement of the seventh component of C' (C'7) for generation of chemotactic activity. No later reacting components of C' are required. Whether C'7 is incorporated into the chemotactically active complex which sediments rapidly in the ultracentrifuge is not yet established. Similar data with guinea-pig C' complexes were also obtained. [ABSTRACT FROM AUTHOR]

The membrane attack complex of complement (C5b-9) is identical in composition regardless of which pathway of activation was instrumental in its formation. Band V protein was consistently a subunit of the soluble complex. Since band V protein is not required for complement-dependent cytolysis, it probably represents a membrane site equivalent in serum of the nascent C5b-9 complex.

A 10 year old girl presented with recurrent febrile episodes over 2 months. A non-typable strain of Neisseria meningitidis was grown from blood cultures on three occasions. She was found to lack functional C8 activity in serum but material with C8 antigenic activity was present. The opsonic activity of the C8 deficient serum for N. meningitidis was equivalent to that of normal controls.

The fourth component of complement (C4) occurs in human plasma as a protein with a three polypeptide chain structure. Lately a single-chain form of C4 has been purified from human plasma by sequential steps of immunoadsorbent chromatography, gel filtration in 6 M guanidine hydrochloride, and gel filtration in 6 M guanidine hydrochloride and 0.015 M dithiothreitol. Single-chain C4, referred to as pro-C4, behaved as a protein of approximately 200,000 daltons upon gel filtration, and was separated from the three chains of C4 on the basis of molecular size. Pro-C4 could be isolated from each of five human plasmas examined and was found to constitute approximately 2% of total, immunoreactive C4. Replicate amino acid analyses of pro-C4 and C4 agreed within 0.5 mol % for all residues except glycine and threonine, which agreed within 0.7 mol percent. Polyacrylamide gel electrophoresis of pro-C4 in the presence of sodium dodecyl sufate in 5% gels under reducing conditions indicated an approximate molecular weight of 202,000. The sum of the molecular weights of the alpha-, beta-, and gamma-chain of C4 is 205,000. After gel electrophoresis, pro-C4 stained positively with periodic acid-Schiff reagent, suggesting the presence of covalently-bound carbohydrate. Competitive inhibition radioimmune assays with pro-C4, purified alpha-, beta-, or gamma-chain, and chain-specific antisera demonstrated the existence of antigenic sites on pro-C4 that are assignable to common determinants on each of the alpha-, beta-, and gamma-chains of C4.

Two immunoelectrophoretically defined, heretofore unidentified beta(1)-globulins of human serum, provisionally designated beta(1C)- and beta(1A)-globulin, were isolated by means of preparative electrophoresis and chromatography on anion exchange cellulose. The sedimentation coefficient S(0) (20, w) of beta(1C)-globulin was shown to be 9.5 S, and that of beta(1A)-globulin, 6.9 S. Both proteins were found to contain similar amounts of carbohydrate, to be devoid of lipids, and to possess the solubility characteristics of euglobulins. In the Ouchterlony double diffusion test they gave the reaction of partial identity, which revealed beta(1A)-globulin to be anti-genically deficient as compared to beta(1C)-globulin. beta(1A)-globulin could not be detected in fresh sera and beta(1C)-globulin was absent from aged sera. Highly purified beta(1C)-globulin stored at 1 degrees C. was converted to beta(1A)-globulin within 4 to 6 weeks, and at 37 degrees C. was converted within 6 days. The likelihood of a dimer-monomer relationship between these two proteins was discussed.

A technique has been described for the demonstration of a human complement component by an immunofluorescent method. The component detected is beta(1C)-globulin, a moiety of the third complement component, which has previously been obtained in pure form and to which a specific antiserum has been prepared. It has been shown in a model system that the binding of beta(1C)-globulin as shown by immunofluorescence is strictly equivalent to complement fixation as assessed by standard serological methods. This technique has been applied to the detection of in vivo bound complement in pathological human tissues. It was found that in vivo complement binding occurs in the lesions of several human diseases, but not elsewhere in the same tissues. In a rather limited survey of diseases that has been carried out, in vivo complement binding was found particularly in systemic L.E., various nephritides, and amyloidosis, as well as in single cases of some other diseases. The spectrum of in vivo complement binding has been compared with that of gamma-globulin binding (7S and 19S types) and with the demonstration of in vitro complement fixation and rheumatoid factor fixation. It was distinct from each of these. Rheumatoid factor fixation, detected by anti-19S antiserum showed promise as a method for the detection of antigen-antibody complexes and aggregated gamma-globulin in tissue sections. The interpretation of these findings in regard to the nature of the binding sites, and their possible significance in regard to pathogenic mechanisms have been discussed.

In the sera of a number of patients with rheumatoid arthritis an unusual, high molecular weight protein component could be detected by direct ultracentrifuge analysis of whole serum. This material sedimented more rapidly than the normal 19 S component and reached concentrations up to 340 mg. per cent. Similar components were not observed in a limited control series. The high molecular weight material was present in the gamma-globulin fraction of serum and joint fluid. It had an S20,(w) of approximately 22 S and could be dissociated into 2 fractions, one of which had a sedimentation coefficient of approximately 19 S. Evidence for a direct relationship between the 22 S component and the gamma-globulin precipitation test was obtained. The latter reaction was found to occur in the presence of altered, aggregated gamma globulin. Absorption of serum with altered gamma globulin removed the 22 S component. There also appeared to be a connection with the sheep cell agglutination reaction and the latex fixation test. The 22 S fraction was always observed in sera giving the most positive tests. Procedures of density gradient and repeated preparative ultracentrifugation demonstrated that each of these reactions was caused by a high molecular weight fraction. The relationship between the unusual protein complex and various 19 S gamma-globulins and 19 S antibodies is discussed.

Various preparations of gamma-globulin homogeneous in the ultracentrifuge showed a similar content of hexose, hexosamine, fucose, and sialic acid. Subfractionation of Fr. II gamma-globulin by zone electrophoresis revealed multiple components of different mean mobilities but containing similar amounts of carbohydrate. Gamma globulin isolated directly from normal serum by zone electrophoresis showed a heavy component in addition to the usual 7 S material. The heavy component (s(20, w) = 19 S) concentrated by preparative ultracentrifugation was found to be considerably richer in carbohydrate than the rest of the gamma-globulin and accounted for small differences in carbohydrate content between different preparations of gamma-globulin. Pathological sera with marked elevation in gamma-globulin showed a carbohydrate-protein ratio for the gamma-globulin similar to that found for the corresponding 7 S fraction in normal serum. This was only partially true of the myeloma proteins with a mobility in the gamma-globulin region. Certain of these proteins showed slight but significant differences. The myeloma proteins of faster mobility (beta-myelomas) contained considerably more carbohydrate. The possible role of these carbohydrates in accounting for some of the mobility and immunological differences in the myeloma proteins is discussed. The pathological proteins found in two cases of macroglobulinemia showed a high carbohydrate content similar to but slightly lower than the normal 19 S component of gamma-globulin.