| Budget Amount *help |
¥5,500,000 (Direct Cost: ¥5,500,000)
Fiscal Year 1986: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1985: ¥4,400,000 (Direct Cost: ¥4,400,000)
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| Research Abstract |
Bovine mercaptalbumin (BMA) has one SH group (Cys-34) and 17 disulfide bonds per molecule. In alkaline pH region, BMA isomerizes to the aged form (A-form) by the intramolecular SH, S-S exchange reaction (N-A isomerization, molecular ageing). Using BMA purified by SE-Sephadex, the present authors studied on the N-A isomerization and obtained the following results.
1. The kinetics of the N-A isomerization (ageing reaction) was studied by using ion-exchange HPLC (TSK SP-5PW, pH 5.00), assuming a reversible first-order reaction of the type N <->!<-> A, where N and A are the N (nonaged)- and A (aged)-forms, respectively. <k_+> and <k_-> are rate constants for N->A and A->N reactions, respectively and Kapp is <k_+> / <k_-> . Values of Kapp for the ageing reaction at pH 8.6 at 20゜, 25゜ and 30゜ in salt-free solution were approximately unity. However, <k_+> and <k_-> were greatly increased with an increase in temperature. Both <k_+> and <k_-> were suppressed, with strong effect on <k_+> , on inc
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reasing ionic strength from 0 to 0.10 M KCl, resulting in the shift of the equilibrium from the A- to N-forms. Both <k_+> and <k_-> were suppressed, with strong effect on <k_+> , on decreasing pH from 8.6 to 7.0, resulting in the shift of the equilibrium from the A- to N-forms. It was reported by Kuwata et al (1985) that global structural fluctuations of helical segments or subdomains are necessary for the ageing reaction of BMA. The global structural fluctuations, corresponding to <k_+> , was strongly affected by salt concentration and pH.
2. <^1H> -NMR spectra and cross-relaxation times ( <T_(IS)> ) between irradiated and observed protein protons of the A-form were similar to those of the N-form, indicating the rigidity in intramolecular structure being not changed in the ageing reaction. <alpha> -helical contents of the N- and A-forms were 70 and 62 % respectively at pH 8.6. Molecular volume of the A-form was larger than that of the N-form. Transition temperature ( <T_m> ) and enthalopy of denaturation ( <DELTA> <H_D> ) of the A-form were lower and smaller than those of the N-form, respectively. These results might suggest that the A-form is more relaxed conformation with increased molecular volume and loss of heat stability but without loss of rigidity of subdomains observed by <T_(IS)> measurements.
3. These results might indicate that the primary structure of BMA dictates at least two or three different three-dimensional structures with different disulfide pairing and different secondary structure. Less
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