Comparison of Biochemical Properties of Human Serum B₁₂ Binders Derived from Sephadex and DEAE‐Cellulose Chromatography

Abstract

Vitamin Biz binding proteins were separated into 2 peaks corresponding to small (TCS) and large (TCL) molecular weight fractions by gel-filtration on Sephadex G-200 using 0.005 M-sodium phosphate buffer, pH 7.4 containing 1 M-NaCI. Each peak, after dialysis and concentration, was chromatographed on DEAE-cellulose. 3 peaks of radioactivity were recovered from both TCS and TCL. Peaks from TCS had an apparent mol. wt. of about 40,000 and thme from TCL about 11O,OOO, as determined by gel-filtration on Sephadex G-200. On electrophoresis, peaks eluted with 0.06 Mphosphate buffer, pH 5.85 from both TCS and TCL moved as ,@-globulins; those eluted with 0.1 M buffer, pH 5.8 between pand a2-globulin and those eluted with 0.25 M buffer, pH 5.4 between q -and a2-globulin. In our assay system, TCS d e livered 57CoB1~ to L-1210 leukaemic lymphoblasts while TCL had no such activity. Of the 6 binders from DEAE-cellulose, only peaks eluted with 0.06 M and 0.1 M buffers from TCS delivered labelled Biz to these cells. Antisera prepared against TC I1 BIZ reacted only with TC I1 BIZ from serum, TCS, and fractions D and E obtained from TCS which were eluted with 0.06 and 0.1 M buffers, respectively.