Acid denaturation of recombinant porcine growth hormone: Formation and self-association of folding intermediates

We have investigated the conformational changes incurred during the acid-in duced unfolding and self-association of recombinant porcine growth hormone (pGH), Acidification (pH 8 to pH 2) of PGH resulted in intrinsic fluorescen ce, UV absorbance, and near-UV CD transitions centered at pH 4.10. At pH 2. 0, a red shift in the fluorescence emission maximum of similar to 3 nm and a 15% loss of the far-UV CD signal at 222 nm imply that the protein did not become extensively unfolded. Acidification in the presence of 4 M urea res ulted in similar pH-dependent transitions. However, these occurred at a hig her pH (similar to 5.2). At pH 2.0 + 4 M urea, an 8 nm red shift in the flu orescence emission maximum suggests that unfolding was greater than in the absence of urea. The presence of a prominent peak centered at 298 nm in the near-UV CD spectrum, which is absent without urea, signifies further diffe rences in the intermediates generated at pH 2. Sedimentation equilibrium ex periments in the analytical ultracentrifuge showed that native pGH and the partially unfolded intermediates reversibly self-associate. Self-associatio n was strongly promoted at pH 2 while urea reduced self-association at both pH 8 and pH 2. These results demonstrate that acidification of pGH in the absence or presence of 4 M urea induced the formation of molten globule-lik e states with measurable differences in conformation. Similarities and diff erences in these structural conformations with respect to other growth horm ones are discussed.