Separation of fragments from human serum albumin and its charged variants by reversed-phase and cation-exchange high-performance liquid chromatography

Abstract

Reversed-phase high-performance liquid chromatography (RP-HPLC) and ion-exchange chromatography on poly(2-sulphoethylaspartamide)—silica (SCX) were compared as alternative approaches in characterizing charge genetic variants of human serum albumin. The chormatographic behaviour of cyanogen bromide (CNBr), typtic and V8 protease digests from normal and mutant albumins were examined. The results showed that substituted site-containing CNBr fragments are successfully resolved by RP-HPLC; in most instances SCX and RP-HPLC are equally adequate in idenfying the modified tryptic peptides from CNBr fragments; although generally useful, SCX chromatography is specifically needed in all instances where amino acid replacement is occurring in a small hydrophilic tryptic fragment and choosing Staphylococcus aureus V8 protease instead of tryptic digestion is advantageous.