Covalent modification of fourth component of human complement with primary amines. Binding studies with metastable fragment C4b and with C4.
A C4-dependent hemolytic complement assay and competitive binding assays with [1,4(n)-3H]putrescine or [14C]methylamine were used to determine the structural preferences of C4 and of metastable C4 for covalent modification with a series of alkyl primary amines. The pKa values of individual amines did not correspond with their ability to inactivate the hemolytic function of C4. The rank order of effectiveness did correlate with the molecular weight and conformation of the organic amines tested. In contrast to results with C4, metastable C4b displays a general increased susceptibility for modification by C3 and C4 alkyl amines. However, C4b exhibits a distinct preference for diamines, putrescine and 1,3-diaminopropane, over monoamines of the same alkyl chain length, s-butylamine and n-propylamine. Taken together, these studies provide the first direct evidence for a conformational change in the thioester region of C4 upon proteolytic activation to metastable C4b. A model is proposed to explain the results of competitive binding experiments with metastable C4b in terms of two binding sites for amines on C4b, the presumptive thioester and a second site such as the free side chain carboxyl group of glutamic acid. It is suggested that all amines except methylamine bind preferentially to the latter site; once bound, only diamines would still be capable of mounting a nucleophilic attack on the thioester bond with the second amino group.
D002250:Carbon Radioisotopes; D003181:Complement C4 / Q000502:physiology*; D015935:Complement C4b; D004355:Drug Stability; D006461:Hemolysis; D006801:Humans; D007700:Kinetics; D008744:Methylamines / Q000494:pharmacology*; D011485:Protein Binding; D011700:Putrescine / Q000494:pharmacology*; D014316:Tritium; D014357:Trypsin
D002250:Carbon Radioisotopes; D003181:Complement C4; D008744:Methylamines; D014316:Tritium; D015935:Complement C4b; C027451:methylamine; D014357:Trypsin; D011700:Putrescine