Isolation, characterization and effect of acidic pH on the unfoldingrefolding mechanism of serum albumin domains.

Three fragments, viz., BSA-CNBr1 – 183, BSA-CNBr184 – 582, and BSA-T377–582 representing domains I, II + III and III of bovine serum albumin have been isolated and purified. The physicochemical properties have been investigated and compared with their parent albumin molecule. The values of Stokes radii (nm) and intrinsic viscosities (ml/g) have been determined to be 2·36, 3·30; 3·43, 4·36; and 2·40, 3·13 for the fragments BSACNBr1– 183, BSA-CNBr184–582 and BSA-T377–582 respectively. The acid induced unfolding-refolding transitions of intact albumin and the fragment BSA-T 377–582 have been shown to occur in two steps while the fragments BSA-CNBr1 – 183 and BSACNBr184– 582 underwent single step transitions. The formation of the acid denatured states of intact albumin, BSA-CNBr1 – 183 and BSA-CNBr184–582 was accompanied by an increase of about 86, 56 and 44% in the values of intrinsic viscosities respectively. Since all the transitions were reversible, the values of equilibrium constants, KD, were calculated. The analysis of the dependence of KD on pH indicated that the first transition (N–X) of albumin was caused due to the uptake of about 3 protons by the native albumin. ,The intermediate state, X, is converted to acid unfolded state, D, by taking up another two protons. A comparision of the results on intact albumin with that of its fragments revealed that the second transition of the fragment BSA-T377– 582 and the two single step transitions of the fragment BSA-CNBr1–183 and BSA-CNBr184–582 were much closer to the second transition (X-D) of the intact albumin. The first transition of albumin has been attributed to its domain III represented by the fragment BSA-T377–582.

Purification and some properties of buffalo spleen cathepsin Β.

Purification of cathepsin Β from buffalo-spleen, a hitherto unstudied system has been achieved by a simple procedure developed by incorporating suitable modifications in the existing methods for isolation of the enzyme from other sources. The purified enzyme has a molecular weight of 25 KDa and its Stokes radius was found to be 2·24 nm. Effects of several reducing agents, urea and thiol-protease inhibitors such as leupeptin and antipain, have been studied and the data unequivocally support the contention that the buffaloenzyme is similar to cathepsin Β from other tissues with respect to these properties.