Picó G A
Departamento de Química-Física, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina.
Int J Biol Macromol. 1997 Feb;20(1):63-73. doi: 10.1016/s0141-8130(96)01153-1.
The unfolding process of human serum albumin (HSA) was studied by thermal effect on the native fluorescence of the protein, thermal inactivation of the hydrolase activity of albumin and differential scanning calorimetry using the high sensitive calorimeter developed by Privalov. The denaturation process can be described by an approximation of the model of Eyring and Lumry: native [symbol: see text] unfolded reversible [symbol: see text] unfolded irreversible. It was found that the rate of irreversible step was very slow (at temperatures below 74 degrees C), allowing the resolution of the denaturation process as a reversible one on the basis of two states approximation. However, the presence of intramolecular cooperation in the thermal denaturation process at temperatures above 74 degrees C cannot be discarded, which might be favoring the aggregation of albumin molecules. The midpoint temperature of unfolding obtained by differential scanning calorimetry was of 63.1 degrees C +/- 0.4 at pH 7.4. This value was independent of the rate of scanning and it is in agreement with those obtained by techniques such as thermal effect on the protein fluorescence and on the hydrolase activity of albumin. The enthalpy of unfolding at pH 7.4 was 88.9 +/- 4 Kcal/mol. This value was low compared with those obtained for other proteins, suggesting the presence of a molten globule in the unfolding pathway of albumin. The neutral-basic conformational change (pH 7.4) of albumin did not modify the thermal stability and the enthalpy of denaturation of the protein. A pH below 4.3 (transition acid-neutral) the presence of a second peak in the thermogram of albumin with a TM of 46.2 degrees C +/- 0.9 would be suggesting a lost of cooperativity between the various domains of albumin in the unfolding.
通过蛋白质天然荧光的热效应、白蛋白水解酶活性的热失活以及使用普里瓦洛夫开发的高灵敏度量热仪进行差示扫描量热法，研究了人血清白蛋白（HSA）的展开过程。变性过程可用艾林和卢姆里模型近似描述：天然态[符号：见原文]可逆展开态[符号：见原文]不可逆展开态。发现不可逆步骤的速率非常缓慢（在低于74℃的温度下），这使得在双态近似的基础上可将变性过程解析为可逆过程。然而，不能排除在74℃以上温度下热变性过程中存在分子内协同作用，这可能有利于白蛋白分子的聚集。在pH 7.4时，通过差示扫描量热法获得的展开中点温度为63.1℃±0.4。该值与扫描速率无关，并且与通过蛋白质荧光热效应和白蛋白水解酶活性等技术获得的值一致。在pH 7.4时的展开焓为88.9±4千卡/摩尔。与其他蛋白质获得的值相比，该值较低，表明白蛋白展开途径中存在熔球态。白蛋白的中性-碱性构象变化（pH 7.4）并未改变蛋白质的热稳定性和变性焓。在pH低于4.3（酸性-中性转变）时，白蛋白热谱图中出现第二个峰，其熔解温度为46.2℃±0.9，这表明白蛋白在展开过程中各个结构域之间失去了协同性。
