MAPPING OF RECOMBINANT HEMOGLOBIN USING IMMOBILIZED TRYPSIN CARTRIDGES

A tryptic mapping procedure has been developed for a recombinant hemog lobin (rHb1.1) using an immobilized trypsin cartridge. Apohemoglobin i s passed through the trypsin cartridge and the products of the digesti on are captured directly onto an in-line C18 reversed-phase column. Th e peptides are then separated using a gradient elution. This new proce dure is rapid and reproducible and can be fully automated. The total t ime of analysis is less than 2 h. The mapping of apohemoglobins produc ed an unexpected isomerization of two peptides: beta 8,9 (K-66-K-82) a nd alpha 8,9 (K-61-K-90). It appears that the isomerization may occur through transpeptidation followed by proteolysis at a newly generated site next to the site of ligation. This mapping procedure can be a use ful tool for research and routine analysis of proteins. (C) 1997 Acade mic Press.