Chromatographic Resolution of the First Component of Human Complement Into Three Activities

Abstract

Serum complement has traditionally been defined as a four component system, Cq, C'2, C~3, and C~4. Although several investigators had suggested the existence of additional components (for review of earlier literature, see reference 1), only recently has unequivocal evidence been available supporting the further complexity of complement. The third component, C'3, was shown first to consist of at least two activities (1--4) and then three (5, 6), designated C~3a, C'3b, and C'3c. A highly purified protein,/31o-globulin (7), would appear to be identical with C'3a (5).Miiller-Eberhard and Kunkel (8, 9) and Taranta, Weiss, and Franklin (10) have described a new serum protein, distinct from the classical components of complement and designated the llS component on the basis of its sedimentation constant. In earlier work on the sequence of action of the components of complement (11, 12), the first component, C'I, was the first factor recognized to interact with sensitized erythrocytes. However, evidence has been presented that the I1S component participates in immune hemolysis at a stage even earlier than Ctl (8, 9), an observation which would appear to require revision of previous concepts of sequence.It is the purpose of this paper to report the resolution of Cq into three activities by chromatography on diethylaminoethyl (DEAE) cellulose of a euglobulin fraction of normal human serum. It will be shown that all three factors are required for reconstitution of Cq activity in several hemolytic systems and for generation of Cq esterase in the absence of antigen-antibody complexes (13).

THE INHIBITION OF PLASMIN, PLASMA KALLIKREIN, PLASMA PERMEABILITY FACTOR, AND THE C'1r SUBCOMPONENT OF THE FIRST COMPONENT OF COMPLEMENT BY SERUM C'1 ESTERASE INHIBITOR

“…Briefly, the method consisted of adding ammonium sulfate to human serum to 40% ~turafion, sedimenting the insoluble protein, and dialyzing the supematant solution exhaustively against distilled water. The dialyzed supematant fraction was passed through a column of Dowex 2-X10 and protein containing Cq esterase inhibitor activity was elated by stepwise increases of sodium chloride concentration (3). Fractions rich in C'I esterase inhibitor were pooled, concentrated by pressure dialysis, and applied to a column of DEAE cellulose (Whatman DE-52) equilibrated with 0.06 ~ tris (hydroxymethyl) aminomethane (Tris)-chloride buffer, pH 8.6.…”

The Hemolysis of Red Cells from Patients with Paroxysmal Nocturnal Hemoglobinuria by Partially Purified Subcomponents of the Third Complement Component*

“…The following have all been described previously: the preparation of barbital-buffered saline (BBS) at various pH's (3); the preparation of Na2MgEDTA-BBS containing 1.5 or 2.6 X 10-2 M Na2MgEDTA (3,5); the collection of guinea pig and human blood and the isolation and storage of their respective sera (3,4); the preparation and storage of sera deficient in the various components of C' and RP (4) ; the preparation of sensitized sheep red cells both with and without the use of Na3HEDTA-BBS (3); the preparation of EAhuC'1,4,2 and EAgpC'1,4,2 (3); the collection and storage of NHE and PNHE (4); the adjustment of serum pH (4); the preparation of serum containing EDTA salts (4); the estimation of in vitro acid hemolysis (4) ; the assay for C'1 esterase activity (4). Preparation of C'3a by TEAE chromatography C'3a was prepared as outlined by Mfiller-Eberhard, Nilsson, and Aronsson (6).…”

“…The individual fractions were dialyzed overnight against 0.135 M NaCl containing 10-Na3-HEDTA. The entire isolation procedure was carried out at 40 C. After dialysis, the fractions were analyzed for C'lq, C'lr, and C'ls activities (5), for C'3a activity (5,7), for lytic activity against PNHEC'3a, and for hemolytic activity against EAC'1,4,2,3a. Comparable results were obtained in all four experiments.…”

“…This activity was usually measured with EAgpC'1,4,2. A sample of the fraction to be tested was added to a 5 X 10' cell button of EAgpC'l,4,2 together with 4 ml of a 1: 5,000 dilution of guinea pig C' in Na3HEDTA-BBS, pH 7.4 (5 (8).…”

Biologic Properties of Polynucleotides. IV. Studies on the Mechanism of Complement Inhibition by Polyinosinic Acid Together with Observations on the In Vivo Effect of Polyinosinic Acid on Complement Activity*

“…General considerations. Although recent work indicates that C'1 and C'3 are complex factors each consisting of at least three separate components (9,10), the basic concepts regarding the sequence of action of C' components in the lysis of EA remain unchanged (7a). In the presence of Ca++, EA reacts with C'1 to form EAC'1; at the same time the esterase associated with C'1 is activated.…”