Purification of pro enzymic and activated human complement c 1s free of complement c 1r effects of calcium and ionic strength on activated complement c 1s
Arlaud, G.J.; Reboul, A.; Meyer, C.M.; Colomb, M.G.
Biochimica et Biophysica Acta 485(1): 215-226
1977
ISSN/ISBN: 0005-2728 Accession: 006233414
A rapid method for the purification of the proenzymic and activated forms of C[complement]1s is presented. In the case of proenzymic C1s, di-isopropyl phosphorofluoridate (0.5-5 mM) is added at all stages of the purification procedure, which includes euglobulin precipitation followed by DEAE-cellulose chromatography and affinity chromatography on anti-C.hivin.1r Ig[immunoglobulin]G-Sepharose 6B. The final step completely removes contaminant traces of C.hivin.1r and/or C.hivin.1r, ensuring that the final preparation of C1s is stable in the proenzyme form and suitable for activation studies. The apparent MW of C1s and C.hivin.1s determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis is 85,000 .+-. 2000. Reduction followed by alkylation of C.hivin.1s gives 2 fragments of apparent MW 57,000 and 28,000. Results of N-terminal amino acid determination and labeling with di-iso[3H]propyl phosphorofluoridate are consistent with previous reports. The influence of Ca and ionic strength on the structure and activity of C.hivin.1s has been investigated. Ca leads to a shift of the sedimentation coefficient from 4.3 to 5.6 S [Svelberg], whereas variation in ionic strength has no effect on this parameter. The thermal inactivation curve is profoundly modified both by Ca and ionic strength. The esterase activity is only slightly influenced as judged from the absence of gross modification of Km and V [velocity].