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The reduction and alkylation of the two disulfide bonds in a preparation of human pituitary growth hormone which had been previously modified by limited proteolysis with the enzyme plasmin have been studied. Quantitative and selective reduction of the carboxyl-terminal disulfide, as well as total reduction of both disulfides, has been achieved in the absence of denaturants. Circular dichroism spectra of the various reduced and reduced-alkylated derivatives have provided sufficient information to allow an estimation of the individual contributions of each disulfide bond to the total optical activity of the protein. These contributions were found to represent a significant portion of the total optical activity between 290 and 250 nm. The carboxyl-termimal bond exhibits negative dichroism with an apparent center near 258 nm ([theta]M,258nm = 2100 deg cm2 dmol-1). By comparison, the contribution of the remaining disulfide is red-shifted to 273 nm, is also negative in sign, and somewhat more intense ([theta]M,273nm = 3200 deg cm2 dmol-1). Circular dichroism measurements have also been used to approximate the rate of reduction of the protein.

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The effect of freezing on formation of soluble and insoluble aggregates of human growth hormone (hGH) was studied. The amount of soluble aggregates was affected very little by freezing regardless of the cooling rate. In contrast, the formation of insoluble aggregates (particulates), as determined by light scattering in the 340- to 360-nm range, was found to increase sharply with increasing cooling rates. The amount of these particulates was also dependent on the pH of the solution. Freezing hGH solutions formulated at pH 7.4 resulted in highly scattering solutions, whereas pH 7.8 formulations showed significantly less scattering. These results emphasize the importance of understanding the freezing phenomenon for protein solutions and suggest that the formation of soluble aggregates and insoluble particulates may have different mechanisms.