title: 1H NMR studies of insulin: histidine residues, metal binding, and dissociation in alkaline solution.
abstract: The shifts of the H2 histidine B5 and B10 resonances of 2-Zn insulin hexamer were followed in 2H2O by 1H NMR spectroscopy at 270 MHz from pH 9.85 to 7. The two resonances present at high pH, previously assigned to H2 histidine B5 and B10 residues, moved slightly downfield and split into four resonances at pH 8.95 and also at pH 7. By use of a paramagnetic broadening probe (Mn2+) and the addition of Zn2+ to metal-free insulin, it was deduced that the four resonances arose from histidines B10 and B5 in two different magnetic environments, probably either bound to Zn2+ or not bound to Zn2+. The pK' values of the B5 and B10 histidines were determined in 60% 2H2O-40% dioxan, in which insulin was soluble throughout the pH range, to be 7.1 and 6.8, respectively at 37 degrees C. Studies at higher pH indicated that at a concentration level suitable for 1H NMR (approximately 1 mM) at 37 degrees C in 2H2O the 2-Zn hexamer was largely dissociated to dimer at pH 10.3 and to monomer at pH 10.8. Addition of paramagnetic shift probe Ni2+ to metal-free insulin caused changes to the spectrum similar to those produced on addition of diamagnetic Zn2+. Addition of Co2+ gave a different result, but there was no paramagnetic shift of the H2 histidine B10 resonance, probably because of rapid exchange at the binding site. Addition of Cd2+ and of Cd2+ and Ca2+ produced changes that were similar to each other but were different from those observed on addition of Zn2+, probably due to the binding of Cd2+ and Ca2+ at glutamate B13.
authors.affiliation: Chemistry Department, Australian National University, Canberra.
authors.forename: V
authors.initials: V
authors.lastname: Ramesh
authors.forename: J H
authors.initials: JH
authors.lastname: Bradbury
mesh_terms: D002104:Cadmium / Q000378:metabolism / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism / Q000494:pharmacology; D003903:Deuterium; D004146:Dioxanes; D006639:Histidine / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007328:Insulin / Q000378:metabolism*; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism; D008670:Metals / Q000378:metabolism*; D009532:Nickel / Q000378:metabolism / Q000494:pharmacology; D012996:Solutions; D012997:Solvents; D015032:Zinc / Q000378:metabolism
chemical_list: D004146:Dioxanes; D007328:Insulin; D046911:Macromolecular Substances; D008670:Metals; D012996:Solutions; D012997:Solvents; D002104:Cadmium; D003035:Cobalt; D008345:Manganese; D006639:Histidine; D009532:Nickel; D003903:Deuterium; D015032:Zinc; C025223:1,4-dioxane; D002118:Calcium