Direct insight into insulin aggregation by 2D NMR complemented by PFGSE NMR.
The aggregation of Zn(II)-bound and zinc-free human insulin was studied in solution using the H(beta)-CH(3) crosspeaks of threonine residues in 2D COSY, TOCSY, and NOESY NMR spectra which allow viewing of the oligomers in equilibrium. This is complemented by PFGSE measurements of the translational diffusion coefficient, D(i), used for monitoring the changes in equilibrium composition of aggregates on dilution of both insulins in physiological medium. The back calculation of the dilution isotherm allows establishing the association constants for oligomeric equilibria in solution and discussion of the models of association.
Bocian
Wojciech
W
National Medicines Institute, Warsaw, Poland.
Sitkowski
Jerzy
J
Tarnowska
Anna
A
Bednarek
Elzbieta
E
Kawecki
Robert
R
Koźmiński
Wiktor
W
Kozerski
Lech
L
D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods; D006801:Humans; D007328:Insulin / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D008956:Models, Chemical; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011485:Protein Binding; D015032:Zinc / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
D007328:Insulin; D015032:Zinc