I am working on human insulin and need to start experimenting with 1) monomeric insulin and 2) hexameric insulin. The insulin is known to form hexamer in the presence of zinc ions. To obtain monomeric insulin, lyophilized zinc free powder was dissolved in acidified MilliQ (pH 2.5) and dialyzed extensively against 50 mM HEPES buffer (pH 7.2), containing 100 mM NaCl and 5 mM EDTA. The EDTA was completely removed by a second round of dialysis against HEPES NaCl buffer. Initially, there was precipitate formation which subsequently disappeared after overnight incubation. To this solution (1 mg/ml insulin), stepwise addition of zinc chloride was carried out and scattering was monitored at 350 nm. Initially, no change in scattering was observed. But after few addition steps, a continuous enhancement in the scattering was observed and solution became turbid. How to obtain clear hexameric insulin solution (Zn-coordinated) and what are the possible experiments that could give a concrete determination of its hexameric state?
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