High-performance liquid chromatography of amino acids, peptides and proteins. XCIX. Comparative study of the equilibrium refolding of bovine, porcine and human growth hormone by size-exclusion chromatography.

The equilibrium refolding of bovine, porcine and human growth hormone and ovine prolactin in guanidine hydrochloride has been investigated using high-performance size-exclusion chromatography (HPSEC). It was found that bovine and porcine growth hormones exhibited very similar refolding behaviour. However, the renaturation of human growth hormone followed a different pathway. In particular, the folding transition of human growth hormone occurred at 4.7 M guanidine hydrochloride compared to 3.8 and 3.5 M for the bovine and porcine molecules, respectively, and 3.5 M for ovine prolactin. The refolding mechanism of an internally clipped fragment derived from partial tryptic digestion, exhibited similar folding properties to the corresponding intact molecule. The internally clipped analogue existed as a relatively larger molecule under fully denaturing conditions. Reduction followed by carboxymethylation resulted in growth hormone molecules with significantly reduced stability and altered folding properties. The results have been correlated with differences in structure to further demonstrate the utility of HPSEC in the study of protein folding and stability.