Multiple effects of haemin binding on protease susceptibility of bovine serum albumin and a novel isolation procedure for its large fragment.
The effects of ligand binding on the proteolytic susceptibility of BSA were investigated. The rate for proteolytic digestion with either trypsin or chymotrypsin decreased in the presence of bilirubin and fatty acids, suggesting that overall albumin conformation is stabilized by these ligands. In contrast, haemin showed multiple effects on a proteolytic digestion pattern: the rate for the degradation of intact albumin greatly increased, but a large 45 kDa fragment accumulated during proteolytic digestion in the presence of this ligand. This unique fragmentation pattern allowed us to isolate the 45 kDa fragment at a high yield (about 30% on a molar basis) by one-step purification. Sequence analyses indicated that this fragment lies between residues Thr190 and Ala583, which constitutes domains II and III of the albumin molecule. Far-u.v. c.d. spectra strongly suggested that the secondary structure in the intact albumin is almost retained in the 45 kDa fragment. The isolated 45 kDa fragment showed haemin-binding ability, as evaluated by spectroscopic titration; upon re-digestion of the 45 kDa fragment, haemin showed strong protective effects. These results were consistent with the idea that haemin binding to BSA induces an increased protease susceptibility in the loop region between domains I and II, but in the overall conformation of domains II and III, a protease-resistant property.
Shin
W S
WS
Research Institute for Food Science, Kyoto University, Japan.
Yamashita
H
H
Hirose
M
M
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin; D010450:Endopeptidases / Q000737:chemistry*; D006427:Hemin / Q000737:chemistry*; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D017421:Sequence Analysis; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D014357:Trypsin
D010446:Peptide Fragments; D012710:Serum Albumin, Bovine; D006427:Hemin; D010450:Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin