Human peripheral blood phagocytes ingest Escherichia coli 026:B6 lipopolysaccharide (LPS)-coated paraffin oil droplets containing Oil red O only if fresh serum deposits C3 on the surfaces of the particles (opsonizes them), by reactions involving the properdin system. The rate of binding of purified [125-I]C3 in serum to LPS-coated particles correlated precisely with the rate of acquisition of ingestibility assayed spectrophotometrically. Once opsonized, LPS-coated particles remained fully ingestible and retained fixed [125-I]C3 radioactivity even after exposure to extremes of temperature, pH, ionic strength, phospholipases, urea or guanidine, some nonionic and ionic detergents, and organic solvents. Trypsin, human conglutinogen-activating factor, another heat-stable activity found in human serum, and sodium dodecyl sulfate removed radioactivity and diminished ingestibility of the opsonized particles. Alkylation, reduction plus alkylation and F(ab')2 from anti-C3 blocked ingestibility but did not alter particle-bound radioactivitymelectrophoretic and tryptic peptide autoradiographic analysis of dodecyl sulfate eluates of opsonized particles, cleansed of many contaminating proteins by boiling with 2 M NaCl (yet still opsonized), revealed that the polypeptide with C3-derived radioactivity had a mol wt of approximately 140,000 and was composed of 70,000 mol wt subunits linked by disulfide bonds. Immunochemical analysis and comparison of the peptide structure of the eluate with that of C3 indicated that the opsonic fragment is not the fragment defined as C3b but a smaller derivative of C3.

The fixation of the third component of complement (C3) results in many important biological phenomena, among which are (a) immune adherence (1), (b) enhancement of phagocytosis (2, 3), (c) the release of an anaphylatoxin which is a potent releaser of histamine (4), and (d) the feedback activation of the alternative pathway (5, 6) . The physiological mechanisms involving C3 fixation require the generation of a C3 convertase which may occur by two separate pathways . C3 convertase can be generated, in the form of C42, by the so-called classical pathway of activation or in the form C3b,B by the alternative or properdin pathway (7). In both cases, C3 is converted to C3b by cleavage of a small peptide, C3a. Normal human serum contains an inactivator of activated C3b. This C3b inactivator or conglutinogen-activating factor (KAF) has been shown to inhibit both immune hemolysis and the immune adherence properties of C3b and to cause cleavage of C3b in the fixed and fluidphase .stages (8-11) .Although it is known that the Cab inactivator is not depleted during its reaction with Cab and that C3b treated with the Cab inactivator becomes extremely sensitive to proteolytic digestion by trypsin and "trypsin-like" enzymes (9), the exact molecular nature of the action of the C3b inactivator on C3b has not been studied. In an effort to delineate the products of this interaction, purified Cab and Cab inactivator were allowed to react for various specific lengths of time and the products of these reactions were then analyzed . Materials and MethodsPurified Serum Proteins. C3 was purified by a modification of the method of Nilsson and Miiller-Eberhard (12) and subsequently labeled with"I by the iodine monochloride technique (13) . C3b inactivator was also purified from normal human serum (14) . 20 mg of cobra venom factor (CVF) was isolated from Naja naja venom (Sigma Chemical Co ., St . Louis, Mo .) by DEAE-cellulose chromatography and gel filtration on Sephadex G-200 (15) and subsequently conjugated to 2 ml of packed cyanogen bromide-treated 413 Sepharose beads (Pharmacia Fine Chemicals, Inc., Uppsala, Sweden) . 0 .1 ml of CVF-Sepharose was reacted with 0.4 ml of fresh normal human serum at 37*C for 15 min in order to generate C3 convertase . The CVF-Sepharose beads were then centrifuged and washed with chilled phosphate-buffered saline . C3b was generated by incubation of purified C3 with serum-exposed, washed CVF-Sepharose beads for 45 min at 37*C which results in the loss of hemolytic C3 activity (L . Halbachs and P. J. Lachmann, unpublished observations).Fresh human serum was saturated to 20% with sodium sulphate . The precipitate was discarded, and the supernate was chromatographed on 0-(carboxymethyl)cellulose as previously described to

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