Evidence for an Ester Linkage between the Labile Binding Site of C3b and Receptive Surfaces

Abstract

The labile binding site of C3b interacts with receptive surfaces by way of a covalent bond. We report here experiments directed toward elucidating the nature of the linkage. ¹²⁵I-C3b was bound to zymosan (Z) carrying C3-convertase activity on its surface. The resulting zymosan-C3b particles (Z-C3b) were washed free of noncovalently bound proteins with 1% SDS. The Z-C3b particles were incubated at different pH's, and it was found that C3b can be released under alkaline conditions but not acidic conditions. Release of C3b can also be observed at alkaline pH in the presence of 1 M hydroxylamine. The kinetics of the release of C3b from Z were studied at pH 7.5 in 1 M hydroxylamine and at pH 10 in 0.1 M NaHCO₃. The second order rate constants for the release of C3b from Z were found to be 0.0026 ± 0.003 M^-1 min^-1 in hydroxylamine at neutral pH, and 37 ± 3 M^-1 min^-1 at pH 10. These values are comparable to the rate constants for the hydroxylaminolysis and hydrolysis of compounds containing esters such as benzoyl glycine ethyl ester, acetylcholine, and N,O-diacetylserinamide. They are significantly different, however, from the values obtained for the hydroxylaminolysis and hydrolysis of compounds containing thioesters, tyrosyl esters, and acylimidazole. The results of these experiments are consistent with the hypothesis that the bond between C3b and receptive surfaces is an ester. Further evidence for an ester linkage between C3b and receptive surfaces was obtained by analyzing the products of the release of C3d from zymosan by hydroxylamine. If the bond is indeed an ester, the release of C3d from Z with hydroxylamine will yield an hydroxamate that either associates with C3d or with Z. We were able to detect 0.66 ± 0.12 moles of hydroxamate per mole of C3d peptide, suggesting that the bond between C3b and receptive surfaces (RS) takes the form of RS-O-CO-C3b.