Inactivation and reactivation of insulin.

Abstract

That insulin may be inactivated by treatment with reducing and oxidizing agents has been demonstrated by Shonle and Waldo. 1 They found that, when insulin was treated with a dilute solution of hydrogen peroxide or potassium permanganate, its glucopyretic property was entirely destroyed. They likewise observed that the activity was destroyed by action of such reducing agents as sodium bisulphide, sulphur dioxide, hydrogen, and stannous chloride. They were never able to recover the activity after it had once been lost by either oxidation or reduction as indicated. Dodds and Dickens 2 observed that the activity of insulin prepared by the picrate method was reduced and finally lost if subjected to formalin in various concentrations. Complete inactivation resulted from exposure to 10 per cent formalin at 37° for one hour. Destruction was much less rapid at a lower temperature. Dodds and Dickens believe that the inactivation was due to combination of formaldehyde with free NH₂ groups of the insulin protein. However, formaldehyde is a fairly good reducing agent and we suggest that the destruction of the glucopyretic property of insulin may equally well be accounted for by a reducing effect upon the insulin molecule. We have observed that fairly pure insulin products, as prepared by our acid aqueous extraction with heat, precipitation by sodium chloride, and purification by repeated reprecipitation with amyl alcohol 3 is extremely sensitive to oxidation or reduction. This was first noticed quite by accident when a dried amyl alcohol precipitate was closed up in a vial, the stopper of which was covered with tinfoil. The precipitate had been dissolved in weak hydrochloric acid at an earlier stage, and it was noticed that the tinfoil became very black, due to action of the volatile acid.