11] Isolation and characterization of Vitamin B12-binding proteins from human fluids

Abstract

Publisher Summary This chapter discusses the isolation and characterization of vitamin B 12 -binding proteins from human fluids. Many different methods have been described for the measurement of vitamin B 12 -binding proteins in human serum. The techniques of protein separation that have been used include paper and starch–gel electrophoresis, Geon-block electrophoresis, column chromatography and immunodiffusion techniques of Ouchterlony, DEAE-cellulose and CM-cellulose ion-exchange chromatography, Sephadex G-200, affinity chromatography, and isoelectric focusing. The presence of at least 3 vitamin B 12 -binding proteins in human serum has been demonstrated. These binders have been designated as TC I, TC II, and TC III. This chapter describes the methods for isolation and characterization of B 12 -binding proteins from human serum. However, these methods can be used also for the isolation of binding proteins from human saliva, as well as other biological fluids. Gel filtration on Sephadex G-200 Column is described. Pattern of distribution of radioactivity of vitamin B 12 -binding proteins of normal human serum on a Sephadex G-200 column is illustrated. Pattern of distribution of radioactivity of vitamin B12 binders from human amniotic fluid on large DEAE-cellulose columns is also explained.