The purification and characterization of Clr, a subcomponent of the first component of bovine complement
Fresh bovine blood was collected and allowed to clot, and centrifuged and then the resulting serum was subjected to dialysis against low ionic strength buffer to precipitate the euglobulin fraction. This was then subjected to ion exchange chromatography, followed by gel filtration, resulting in purification of the ClrCls complex.In the presence of Ca2+, the Clr-Cls complex consisted of two moles each of Clr and Cls, and in the presence of EDTA it consisted of one mole of each. The complex, in the presence of EDTA and incubated at 37oC, displayed an ability to activate both the Cir and the Cis in the complex. The complex could be dialysed against the buffer containing 1.0M-K1, followed by ion exchange chromatography to produce a good yield of Clr. All steps after the initial dialysis were carried out in the presence of the inhibitor benzamidine to ensure purification of the proenzyme.Once separated from Cls, Clr was characterized. In the proenzymic state, Clr had a molecular weight of 85,000, and in the activated state displayed a heavy chain of 65,000 molecular weight, and a light chain of 33,000 molecular weight. Both the heavy and the light chains contained carbohydrate, and the light chain alone was able to incorporate DFP, indicating the presence of the active serine residue on this chain.The Clr produced could cleave bovine Cls, and could also undergo self-activation at 37oC in EDTA-containing buffer. Possible reasons for this occurrence, and its implications with regard to the mechanism of Cl activation are discussed.The N-terminal sequence of the CIF light chain was obtained by automatic sequencing_ and showed good homology with the corresponding sequences of human Clr and other proteinases.