HYDROXYL GROUP OF INSULIN A19TYR IS ESSENTIAL FOR RECEPTOR-BINDING - STUDIES ON (A19PHE)INSULIN

One mutant proinsulin gene was constructed through PCR with the code o f A19Tyr changed into that of Phe. The mutant proinsulin, (A19Phe)-lys proinsulin (F19KPI) was expressed in E. coli and purified. After tryps in and carboxypeptidase B cleavage and Resource(TM) Q separation, (A19 Phe)-human insulin (F19HI) was obtained. With native insulin as standa rd, activity assay and structural analysis were carried out. It was fo und that the conformation of F19HI is very similar to that of native i nsulin according to the data from native PAGE, reverse-phase FPLC and circular dichroism spectrum, which are also in agreement with the resu lt of radioimmune assay. F19HI retains almost full immune activity, bu t displays only 13.3 % of receptor binding activity. These results sug gest that the hydroxyl group of insulin A19Tyr is essential for recept or binding.