STUDIES OF THE MOLECULAR MECHANISMS OF C3B INACTIVATION AND A SIMPLIFIED ASSAY OF BETA-1H AND THE C3B INACTIVATOR (C3BINA)

A sensitive radio assay was developed to evaluate and quantitate the function of the control proteins important in the cleavage of [human] cell-bound C3b [b fragment of complement component 3] and in the release of the subfragement C3c. The effects of the control proteins on C3b functional hemolytic activity and on C3b immune adherence reactivity were measured. The cellular C intermediate EA[sheep erythrocyte-antibody]C43b was prepared with highly purified radiolabeled C3. The effects of incubation with .beta.1H and the C3b inactivator were followed over a wide range of protein concentrations and the kinetics of the interaction was measured. Incubation of EAC43b* with .beta.1H led to some decrease in immune adherence activity but no loss in C3b functional hemolytic activity and no change in the integrity of the .alpha.- or .beta.-chains. Addition of C3b inactivator to the membrane-bound .beta.1H-C3b complex led to a single celavage of the C3b .alpha.-chain without release of the C3c fragment from the cellular site. This led to complete loss of C3b functional hemolytic activity. The .alpha.-chain cleavage led to loss but not complete disappearance of immune adherence reactivity. The action of .beta.1H and the C3b inactivator on cell-bound C3b led to the appearance of a trypsin-sensitive bond within the .alpha.-chain. Addition of trypsin led to a 2nd cleavage of the .alpha.-chain and the release of the radiolabeled C3c fragment. In kinetic studies the appearance of the trypsin-sensitive bond paralleled the loss of C3b functional hemolytic activity. Kinetic studies demonstrated that plasma and serum contain all of the ingredients for a C3b-cleaving system. By utilizing this sensitive radio-release assay, the functional titers of C3b inactivator in the serum of 15 normal individuals were 11,310 .+-. 666 SE. The mean .beta.1H titer for 12 normal individuals was 20,600 .+-. 873. This assay can be used to quantitate the level of the trypsin-like enzyme. The assay provides a simple method to quantitate and evaluate the function of the C3b control proteins.