变性剂诱导的多结构域蛋白质变性现象的研究

从蛋白质变性的构象渐变模型出发，分析了多结构域蛋白质变性过程的特点，用吸附动力学方法得到了可描述多个结构域变性的关系表示式，特别对典型双结构域蛋白质变性过程予以较详细的讨论。通过对比理论计算结果与实验观测结果，发现在一定参数条件下两者符合较好，从构象渐变的观点，对双结构域蛋白质变性过程中出现双S形变性曲线的原因做出了解释，同时对变性过程中可能出现的一些情况进行了预测和分析。     

Oxidation of Natural and Thermal Denatured Bovine Serum Albumin Hydrazyl Free Radicals in the Presence of Cyclodextrins

The influence of certain thermal treatments on the reactivity of bovine serum albumin (denaturation followed by renaturation in three different cooling conditions) was studied monitoring the kinetics of oxidation of BSA with two water-soluble stable hydrazyl radicals. The results showed that the addition to the reaction mixture of -cyclodextrin and -cyclodextrin decreases the oxidation rate, probably due to the encapsulation of terminal amino acid rests by the cyclodextrins cavity. -Cyclodextrin protects more efficiently the albumin probes than -cyclodextrin. The denatured albumin probes are more reactive than natural albumin as a consequence of the reorienting of the hydrophobic rests of albumin molecule to their surface.     

DSC study of cold and heat denaturation processes of β-lactoglobulin A with guanidine hydrochloride

The cold and heat denaturations of bovine P-lactoglobulin A ((β-lg A) has been studied in solutions of guanidine hydrochloride (GuHCI) by differential scanning calorimetry (DSC). The experimental results are presented and discussed. It is shown that the number of protons bound by the monomeric molecules of β-lg A was unchanged before and after its heat denaturation below pH 3, and that the activation energy of the heat denaturation was depressed owing to the presence of GuHCI. In the solutions with 2.50 and 3.06 mol/L of GuHCI, both the cold and heat denaturations of P-lg A were observed. In comparison with the heat denaturation, the activation energy of cold denaturation was far lower and the number of GuHCl molecules bound by the unfolded polypeptide chains after culd denaturation increased a lot. The absolute value of the enthalpy of cold denaturation was larger than that of heat denaturation. It was found by the analysis that the contribution to the total denaturational enthalpy of conformational change itself of the monomeric molecules of β-lg A was the lowest among the globulins, according to the average of the number of heavy atoms. Project supported by the National Natural Science Foundation of China, and by the fund for excellent items under Director of the Institute of Chemistry.     

Two-state irreversible thermal denaturation of anionic peanut (Arachis hypogaea L.) peroxidase

Detailed differential scanning calorimetry (DSC), steady-state tryptophan fluorescence and far-UV circular dichroism (CD) studies, together with enzymatic assays, were carried out to monitor the thermal stability of anionic peanut peroxidase (aPrx) at pH 3.0. The spectral parameters were seen to be good complements to the highly sensitive but integral method of DSC. Thus, changes in far-UV CD corresponded to changes in the overall secondary structure of the enzyme, while changes in intrinsic tryptophan fluorescence emission corresponded to changes in the tertiary structure of the enzyme. The results, supported with data concerning changes in enzymatic activity with temperature, show that thermally induced transitions for aPrx are irreversible and strongly dependent upon the scan rate, suggesting that denaturation is under kinetic control. It is shown that the process of aPrx denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme, , where k is a first-order kinetic constant that changes with temperature, as given by the Arrhenius equation; N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.     

蛋白质变性机理与变性时的热力学参数研究进展

生物大分子是近年来生命科学的研究热点和难点之一,而对蛋白质变性的研究有助于深刻揭示生命现象的机理.利用光谱学和热力学可以分别从微观和宏观角度对蛋白质变性进行研究,并由此得到表征蛋白质变性的热力学参数.这对深入了解蛋白质的折叠与伸展、变性机理、结构稳定性及生命体的新陈代谢等问题具有很大意义.近年来,国内外学者在此方面做了大量的工作,主要涉及蛋白质在水溶液中的变性机理、在有变性剂存在下水溶液中的变性机理及在含有其它物质水溶液中的变性机理.用来表征蛋白质变性的热力学参数有热容、变性自由能、变性焓和变性熵等.本文对这些研究进行了概述.     

Thermal denaturation of bovine serum albumin and its oligomers and derivativespH dependence

In a previous paper, we report a preliminary DSC study on bovine (BSA) and human (HSA) serum albumins. However, at accurate HPLC analysis the commercial proteins show three peaks: Fraction V-I, probably globulins (as declared by the producers), Fraction V-II (about 15–18% of the product) and Fraction V-III that represents pure BSA or HSA. A hypothesis is that the Fraction II is a covalent dimer, or trimer or a mixture of both, generated during the scalf-life of the commercial product. Denaturation enthalpies of the purified Fraction V-III and Fraction V-II of BSA, have been determined calorimetrically, at changing thepH, and the results of both compared with those obtained on the untreated protein. Few calorimetric experiments have been also carried on a BSA monomer derivative with sulphidril group protected. Computer program have been developed for the deconvolution of exo- and endothermic effects and for the analysis of thermal denaturation profiles.     

大肠杆菌中重组猪生长激素提取方法及变复性的比较研究

通过对基因工程菌pgh／E．coli BL21用酶溶解、超声波处理、超声波处理与液氮冻融相结合三种方法进行破菌，用盐酸胍法进行变复性，提取重组猪生长激素(rpGH)．结果表明用超声波处理与液氮冻融相结合的方法裂解细菌，盐酸胍法变复性，rpGH的提取率最高，达72．16％；而用尿素法对rpGH在大肠杆菌内所形成的包涵体变复性，rpGH的提取率仅为49．82％．     

Antibacterial and Miscibility Properties of Chitosan/Collagen Blends

The miscibility, bioactivity, and antibacterial properties of chitosan/collagen specimens were systematically studied. The specimens were prepared by blending collagen and chitosan with varying deacetylation degrees in solutions; the collagen molecules had been extracted from pigskins using the acid swelling-pepsin digestion method. To understand the miscibility properties of collagen and chitosan molecules, the intrinsic viscosity and differential scanning calorimetry analysis of collagen, chitosan, and collagen/chitosan specimens were performed. The instrinsic viscosity measurements suggested that chitosan and collagen molecules with varying deacetylation degrees were miscible at molecular level for all compositions and degrees of deacetylation of chitosan/collagen mixture solutions prepared in this study. Fourier transform infrared analyses suggested that the percentage of preserved triple helix structures present in collagen molecules in collagen/chitosan specimens decreased with increasing chitosan contents, since the ratios of peak absorbance at 1239 cm^?1 of amide III and 1455 cm^?1 of C?H bending of collagen/chitosan specimens decreased significantly with increase in their chitosan contents. Abnormally high denaturation temperatures (T_d) were observed as the chitosan contents of collagen/chitosan specimens reached 40 wt%, at which T_d of collagen molecules was even higher than that of the corresponding pure chitosan molecules with varying deacetylation degrees. The antibacterial activity of collagen/chitosan blends increased consistently with increasing deacetylation degrees and concentrations of chitosan molecules in collagen/chitosan solutions. Possible explanations for these interesting thermal denaturation, antibacterial, and miscibility properties of chitosan/collagen specimens are reported.     

Single‐Molecule Detection Reveals Knot Sliding in TrmD Denaturation

An increasing number of proteins are found to contain a knot in their polypeptide chain. Although some studies have looked into the folding mechanism of knotted proteins, why and how these complex topologies form are still far from being fully answered. Moreover, no experimental information about how the knot moves during the protein‐folding process is available. Herein, by combining single‐molecule fluorescence resonance energy transfer (smFRET) experiments with molecular dynamics (MD) simulations, we performed a detailed study to characterize the knot in the denatured state of TrmD, a knotted tRNA (guanosine‐1) methyltransferase from Escherichia coli, as a model system. We found that the knot still existed in the unfolded state of TrmD, consistent with the results for two other knotted proteins, YibK and YbeA. More interestingly, both smFRET experiments and MD simulations revealed that the knot slid towards the C‐terminal during the unfolding process, which could be explained by the relatively strong interactions between the β‐sheet core at the N terminal of the native knot region. The size of the knot in the unfolded state is not larger than that in the native state. In addition, the knot slid in a “downhill” mode with simultaneous chain collapse in the denatured state.