pH dependent conformational changes in the T- and R-states of insulin in solution: circular dichroic studies in the pH range of 6 to 10.
Zinc insulin hexamer has been shown to undergo a phenol-induced T6 to R6 conformational transition in solution. Our circular dichroic (CD) studies demonstrate that insulin undergoes pH-dependent conformational changes over the pH range of 6-10 in the T-state and in the R- state. In order to determine which specific amino acid residues may be responsible for these pH-dependent changes, a series of insulin analogs were utilized. In the T-state, the pH dependent CD changes monitored in the far UV region have a pK of 8.2 and appear to be related to the titration of the A1-Gly amino group. Using the near UV CD a second pH-dependent conformational change was detected with a pK of 7.5 in the T-state. 1H N.M.R. studies suggest that B5-His may be responsible for this conformational transition. In the presence of m-cresol (R-state), the pK value was found to be 6.9. During this titration, the increased ellipticity for the R-state is diminishing as pH decreases from pH 8 to 6, and no difference in ellipticity was observed at 255 nm between T- and R-states at pH 6. Therefore, this may be due to the transition from the R back to the T-state.
Kim
Y
Y
Parenteral Products Research and Development, Eli Lilly and Company, Indianapolis, IN 46285.
Shields
J E
JE
D002942:Circular Dichroism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000031:analogs & derivatives / Q000737:chemistry*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011487:Protein Conformation; D012996:Solutions; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D007328:Insulin; D012996:Solutions