Characterization of sulfhydryl heterogeneity in human serum albumin and recombinant human serum albumin for clinical use.

Since human serum albumin has one sulfhydryl group and 17 disulfides, reactive sulfhydryl groups give rise to heterogeneity. The present paper presents a comparison of sulfhydryl heterogeneity in human serum albumin and recombinant human serum albumin for clinical use. Low molecular weight sulfhydryl compounds were identified from both sources. The recombinant albumin had a much higher sulfhydryl content than plasma serum albumin.

Kinetic analysis about the effects of neutral salts on the thermal stability of yeast alcohol dehydrogenase.

The effects of salts on the rate constants of inactivation by heat of yeast alcohol dehydrogenase (YADH) at 60.0 degrees C were measured. Different effects were observed at low and high salt concentrations. At high concentrations, some salts had stabilizing effects, while others were destabilizing. The effects of salts in the high concentration range examined can be described as follows: (decreased thermal stability) NaClO(4) < NaI = (C(2)H(5))(4)NBr < NH(4)Br < NaBr = KBr = CsBr = (no addition) < (CH(3))(4)NBr < KCl < KF < Na(2)SO(4) (increased thermal stability). The decreasing effect of NaClO(4) on YADH controlled the thermal stability of the enzyme absolutely and was not compensated by the addition of Na(2)SO(4), a salt which stabilized the enzyme. However, Na(2)SO(4) compensation did occur in response to the decrease in thermal stability caused by (C(2)H(5))(4)NBr. The rate constants of inactivation by heat (k (in)) of the enzyme were measured at various temperatures. Effective values of the thermodynamic activation parameters of thermal inactivation, activation of free energy (DeltaG (double dagger)), activation enthalpy (DeltaH (double dagger)), and activation entropy (DeltaS (double dagger)), were determined. The thermal stability of YADH in 0.8 M Na(2)SO(4) increased more than that of pyruvate kinase from Bacillus stearothermophilus, a moderate thermophile. The changes in the values of DeltaH (double dagger) and DeltaS (double dagger) were great and showed a general compensatory tendency, with the exception of in the case of NaClO(4). The temperature for the general compensation effect (T (c)) was approximately 123 degrees C. With Na(2)SO(4), the thermal stability of YADH at a temperature below T (c) was greater than that in the absence of salt due to the higher values of DeltaH (double dagger) and DeltaS (double dagger), respectively, and thus was an example of low-temperature enzymatic stabilization. With (C(2)H(5))(4)NBr, the thermal stability of YADH at a temperature below T (c) was lower than that in the absence of salt due to the lower values of DeltaH (double dagger) and DeltaS (double dagger), respectively, and thus was an example of low-temperature enzymatic destabilization. But with NaClO(4), the changes in the values of DeltaH (double dagger) and DeltaS (double dagger) were small and the thermal stability of YADH was thus an example of high-temperature enzymatic destabilization.

Kinetic analysis of enhanced thermal stability of an alkaline protease with engineered twin disulfide bridges and calcium-dependent stability.

The thermal stability of a cysteine-free alkaline protease (Alp) secreted by the eukaryote Aspergillus oryzae was improved both by the introduction of engineered twin disulfide bridges (Cys-69/Cys-101 and Cys-169/Cys-200), newly constructed as part of this study, and by the addition of calcium ions. We performed an extensive kinetic analysis of the increased thermal stability of the mutants as well as the role of calcium dependence. The thermodynamic activation parameters for irreversible thermal inactivation, the activation free energy (deltaG), the activation enthalpy (deltaH), and the activation entropy (deltaS) were determined from absolute reaction rate theory. The values of deltaH and deltaS were significantly and concomitantly increased as a result of introducing the twin disulfide bridges, for which the increase in the value of deltaH outweighed that of deltaS, resulting in significant increases in the value of deltaG. The enhancement of the thermal stability obtained by introducing the twin disulfide bridges is an example of the so-called low-temperature stabilization of enzymes. The stabilizing effect of calcium ions on wild-type Alp is similar to the results we obtained by introducing the engineered twin disulfide bridges.