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Treatment of the third component of human complement (C3) with methylamine results in a loss of hemolytic function and the appearance of a thiol group. Studies with [14C]methylamine have indicatd a stoichiometric and covalent reaction with the native protein. Hydrazine-inactivated C3 and C3b prepared with bovine trypsin were unreactive with [14C]-methylamine. Alkylation experiments with [1-14C]iodoacetamide have further established a 1:1 correspondence between methylamine incorporation and expression of the reactive thiol. Autoradiographic analyses of [14C]methylamine-treated C3 and methylamine-inactivated [1-14C]carboxyamidomethylated C3 after NaDodSO4/polyacrylamide gel electrophoresis have shown a specific incorporation of each radiolabel into the alpha polypeptide chain. [14C]Methylamine-treated C3 was immobilized on Sepharose 4B by reaction of the protein thiol with a mixed disulfide. Digestion with bovine trypsin in 4 M urea released 96% of the bound absorbance (at 280 nm) units; the radiolabel remained associated with the Sepharose beads. Peptide material labeled with 14C was eluted with dithiothreitol, carboxymethylated with [3H]iodoacetic acid, and chromatographed on Sephadex G-75. On Edman degradation S-[3H]carboxymethylcysteine was released at step 9 and gamma-glutamyl[14C]-methylamide was released at step 12. We interpret these data to indicate the presence of an internal thiolester bond in native C3. In addition, evidence is presented for an identical reactive site in alpha 2-macroglobulin.

Treatment of human C3 with hydroxylamine or hydrazine at physiological pH and ionic strength totally abrogates the intrinsic ability of this protein to sustain classical pathway induced hemolysis of sheep red blood cells. Concomitant with the loss of this function the appearance of a single sulfhydryl group can be followed by titration with the sulfhydryl-specific reagents p-(chloromercuri)benzoate, [1-14C]iodoacetamide, 2,2'-dipyridyl disulfide, and 5,5'-dithiobis(2-nitrobenzoic acid). These reagents have also been used to follow the appearance of a free sulfhydryl group on conversion of C3 to C3b with bovine trypsin. Autoradiography of the electrophoretogram of separated alpha-, alpha'-, and beta-polypeptide chains of inactivated, [1-14C]carboxamidomethylated C3 samples has shown that the reactive sulfhydryl group is present in the alpha chain of C3 and in the alpha' chain of C3b, respectively. Digestion of the radiolabeled protein with porcine elastase has localized this sulfhydryl group to a 28 000-dalton fragment of the alpha chain with immunochemical and functional reactivities of the C3d domain. Autoradiographic analysis of a hydrolysate prepared from radioalkylated C3 and subjected to high-voltage paper electrophoresis has shown the labeled amino acid to be [1-14C]-S-(carboxymethyl)cysteine. The susceptibility of native C3 to rapid and irreversible inactivation by nitrogen nucleophiles with the parallel appearance of a cysteinyl residue may indicate the presence of an internal thiol ester. The relationship of the proposed thiol ester to the ability of nascent C3b to acylate cell surface components and carbohydrate polymers is discussed within the context of a transesterification reaction.

The third component of complement has been purified from fresh human plasma employing an initial fractionation with poly(ethylene glycol) followed by sequential depletion of plasminogen by affinity adsorbents, chromatography on diethylaminoethylcellulose, gel filtration on agarose, and batch adsorption/desorption on hydroxylapatite. Final recoveries of C3 were 33% of the initial protein, as quantitated by radial immunodiffusion, and 31% of the initial hemolytic activity. Apparent homogeneity is indicated by immunological criteria and by polyacrylamide gel electrophoresis. A partial specific volume of 0.736 +/- 0.003 mlgm-1 was determined for C3 by the mechanical oscillator technique. "Low speed" sedimentation equilibrium yielded an apparent weight average molecular weight for the protein of 187 650 +/- 5650. Based upon this molecular weight, a molar extinction coefficient of 1.82 X 10(5) 1. mole-1 cm-1 at 280 nm was calculated from boundary-spreading experiments in the ultracentrifuge and as assumed refractive index increment. Amino acid analyses revealed no unusual or distinctive characteristics. Automated Edman degradation revealed a double N-terminal sequence, Ser-Val,Pro-Glx,Met-Lee,Tyr-Thr,Ser-Glx,Ile-Lys,Gly-Arg,Thr-Met,Pro-Asx, in agreement with the two chain structure observed on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and revealing both chains are available to degradation. Serine is postulated as the initiating sequence in both chains based upon high recoveries of dinitrophenylserine upon hydrolysis of dinitrophenylated C3, and our inability to identify any other dinitrophenyl or phenylthiohydantoin derivatives in this position. Alanine is the ultimate carboxy-terminal amino acid of at least one of the chains, as indicated by the action of carboxypeptidases on C3 in the presence of sodium dodecyl sulfate.

The sequences of the C-terminal octadecapeptides obtained by cyanogen bromide cleavage of the gamma-chains of myeloma proteins of the four subclasses, and a urinary heavy-chain-disease protein, have been determined. Although the sequences were markedly homologous, unique replacements were identified that distinguished between the gamma(2b), gamma(2c) and gamma(2d) subclasses. The data are in accord with the postulated existence of four genetic loci or cistrons, these having arisen by the process of gene duplication.

The digestion of the Fc fragment of rabbit immunoglobulin IgG by several proteolytic enzymes was investigated by using gel filtration and starch-gel electrophoresis in 8m-urea-formate as criteria of the extent of degradation. Though fragment Fc and mildly reduced fragment Fc proved resistant to tryptic hydrolysis, papain and pepsin cleaved the fragment at acidic pH values and appeared to give rise to a similar spectrum of products. A (limit) peptide comprising the C-terminal 113 residues of the heavy chain was isolated and identified from the pepsin-digest products of fragment Fc. The products of proteolytic digestion of fragment Fc were no longer able to inhibit passive cutaneous anaphylaxis by rabbit anti-(bovine serum albumin) or demonstrate reversed passive cutaneous anaphylaxis in the guinea pig. Nor were they able to inhibit the intestinal absorption of heterologous immunoglobulin IgG in the young mouse. These studies imply that the site or sites responsible for these biological properties of intact fragment Fc reside in the N-terminal 30-40% of the fragment.

The heavy chain of rabbit immunoglobulin G exists in three major allotypic patterns, Aa1-Aa3. A comparison of the amino acid compositions of the heavy chains isolated from immunoglobulin IgG homozygous for each allotypic determinant revealed the presence of an additional methionine residue per chain in the Aa3 allotype relative to the Aa1 and Aa2 allotypes. The position of the additional methionine residue was determined by cyanogen bromide cleavage and by tryptic digestion of the gamma-chains; it coincided with the inter-Fd-Fc area of the chain. Isolation and characterization of the corresponding tryptic peptides of 31 amino acid residues from each of the allotypes showed the presence of a methionine-for-threonine replacement in the Aa3 allotype, but only in about 70-80% of the molecules. No other allotypic variations were seen in this tryptic peptide. Allotypically related variations in composition were also detected in the N-terminal cyanogen bromide-cleavage peptide.