Acid denaturation of recombinant porcine growth hormone: formation and self-association of folding intermediates.
We have investigated the conformational changes incurred during the acid-induced unfolding and self-association of recombinant porcine growth hormone (pGH). Acidification (pH 8 to pH 2) of pGH resulted in intrinsic fluorescence, 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 approximately 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 resulted in similar pH-dependent transitions. However, these occurred at a higher pH (approximately 5.2). At pH 2.0 + 4 M urea, an 8 nm red shift in the fluorescence 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 differences in the intermediates generated at pH 2. Sedimentation equilibrium experiments in the analytical ultracentrifuge showed that native pGH and the partially unfolded intermediates reversibly self-associate. Self-association 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-like states with measurable differences in conformation. Similarities and differences in these structural conformations with respect to other growth hormones are discussed.
Parkinson
E J
EJ
BresaGen Ltd., P.O. Box 259, Rundle Mall Adelaide, SA 5000, Australia.
Morris
M B
MB
Bastiras
S
S
D000143:Acids; D000818:Animals; D002021:Buffers; D002850:Chromatography, Gel; D002942:Circular Dichroism; D013006:Growth Hormone / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D014461:Ultracentrifugation; D014508:Urea
D000143:Acids; D002021:Buffers; D014508:Urea; D013006:Growth Hormone