Conformational stability of porcine serum transferrin.
The conformation of porcine serum ferric transferrin (Tf) and its stability against denaturation were studied by circular dichroism. Tf was estimated to have 19-24% alpha-helix and 50-55% beta-sheet based on the methods of Chang et al. (Chang, C.T., Wu, C.-S.C., & Yang, J.T., 1978, Anal. Biochem. 91, 13-31) and Provencher and Glöckner (Provencher, S.W. & Glöckner, J., 1981, Biochemistry 20, 33-37). Removal of the bound ferric ions (apo-Tf) did not alter the overall conformation, but there were subtle changes in local conformation based on its near-UV CD spectrum. The Tfs were stable between pH 3.5 and 11. Denaturation by guanidine hydrochloride (Gu-HCl) showed two transitions at 1.6 and 3.4 M denaturant. The process of denaturation by acid and base was reversible, whereas that by Gu-HCl was partially reversible. The irreversible thermal unfolding of Tfs began at temperatures above 60 degrees C and was not complete even at 80 degrees C. The bound irons (based on absorbance at 460 nm) were completely released at pH < 4 or in Gu-HCl solution above 1.7 M, when the protein began to unfold, but they remained intact in neutral solution even at 85 degrees C. The NH2- and COOH-terminal halves of the Tf molecule obtained by limited trypsin digestion had CD spectra similar to the spectrum of native Tf, and the COOH-terminal fragment had more stable secondary structure than the NH2-terminal fragment.
Shen
Z M
ZM
Cardiovascular Research Institute, University of California, San Francisco 94143-0130.
Yang
J T
JT
Feng
Y M
YM
Wu
C S
CS
D000818:Animals; D002942:Circular Dichroism; D004355:Drug Stability; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D013552:Swine; D014168:Transferrin / Q000737:chemistry* / Q000378:metabolism
D006146:Guanidines; D014168:Transferrin; D007501:Iron; D019791:Guanidine