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Comparison of Biochemical Properties of Human Serum B 12 Binders Derived from Sephadex and DEAE‐Cellulose Chromatography
Author(s) -
Kumar Sudhir,
Meyer Leo M.,
Gams Richard A.
Publication year - 1977
Publication title -
scandinavian journal of haematology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.904
H-Index - 84
eISSN - 1600-0609
pISSN - 0036-553X
DOI - 10.1111/j.1600-0609.1977.tb02094.x
Subject(s) - sephadex , chromatography , chemistry , size exclusion chromatography , elution , cellulose , sodium , biochemistry , enzyme , organic chemistry
Vitamin B 12 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 ‐NaCl. 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 those from TCL about 110,000, as determined by gel‐filtration on Sephadex G‐200. On electrophoresis, peaks eluted with 0.06 M ‐phosphate buffer, pH 5.85 from both TCS and TCL moved as β‐globulins; those eluted with 0.1 M buffer, pH 5.8 between β‐ and α 2 ‐globulin and those eluted with 0.25 M buffer, pH 5.4 between α 1 ‐ and α 2 ‐globulin. In our assay system, TCS delivered 57 CoB 12 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 B 12 to these cells. Antisera prepared against TC II B 12 reacted only with TC II B 12 from serum, TCS, and fractions D and E obtained from TCS which were eluted with 0.06 and 0.1 M buffers, respectively.