Photochemically induced nuclear polarization study of the accessibility of tyrosines in insulin.
The accessible tyrosines of bovine insulin were studied by the photochemically induced dynamic nuclear polarization (photo-CIDNP) method. Tyrosine 1H nuclear polarization is observed in acidic, neutral, and basic solutions at all concentrations studied, in the absence of added salts as well as in the presence of 0.05-0.1 M chloride or phosphate. At pH 2.1 in the presence of chloride, at concentrations of 640 microM and above, most of the nuclear polarization at delta 6.82 originates from one group of tyrosines. On the basis of the crystallographic model, these are assumed to be the A14 tyrosines. We explored the possibility of a genuine concentration dependence of the photo-CIDNP intensity of insulin due to aggregation. In order to discern between such effects and trivial kinetic effects traceable to the optical irradiation method, the effects of concentration changes on polarization were examined in three apparently nonassociating trypsin inhibitor proteins. In insulin, the intensity of Tyr-A 14 polarization changes slowly at concentrations above 1 mM, suggesting that these residues are similarly accessible in all association states. At insulin concentrations below 320 microM, additional tyrosine emission signals were observed. These signals are probably due to B16 and B26 tyrosines of monomers. Polarization transfer effects from Tyr-A14 are evident in the tetramer and hexamer. Enhanced absorption effects in the two histidines (B5 and B10) of the insulin monomer were observed at pH 10 in the presence of 0.1 M phosphate.
D000818:Animals; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007328:Insulin; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008970:Molecular Weight; D010777:Photochemistry; D011487:Protein Conformation; D014443:Tyrosine / Q000032:analysis*
D007328:Insulin; D014443:Tyrosine