脲变性兔脑肌酸激酶在复性过程中活力与构象变化的比较

研究了脲变性兔脑肌酸激酶（ＡＴＰ：Ｃｒｅａｔｉｎｅ Ｎ－ｐｈｏｓｐｈｏｔｒａｎｓｆｅｒａｓｅ ＥＣ２．７．３．２）在复性时的构象变化与活力变化的过程。在适当条件下，酶的活力恢复可达９０％以上。与兔肌肌酸激酶相似，该酶的复活过程也呈现含有三相组成的一级反应过程。在一定的酶浓度范围内，酶活力恢复过程与酶浓度无关。将用荧光、ＣＤ和紫外差吸收监测的构象变化与活力变化的速度常数比较，表明酶活力恢复前期的速度与构象变化速度相当，但在酶分子的总体构象变化完成之后，活力恢复尚未完全。     

混合拥挤试剂对人肌肌酸激酶稳定性的影响作用研究

为了更加真实地模拟细胞内环境,在人肌肌酸激酶(HCK)的去折叠环境中同时加入了大分子拥挤试剂(葡聚糖)和小分子渗透剂(蔗糖),采用酶活性、荧光发射谱和溶液浊度检测等方法研究肌酸激酶在含有蔗糖和葡聚糖的混合拥挤环境中的稳定性,结果表明在肌酸激酶盐酸胍变性过程中,蔗糖不仅抑制肌酸激酶酶活性的丧失,还稳定肌酸激酶的构象,而葡聚糖对肌酸激酶保护能力相对较弱.对于热变性过程,相同质量浓度的蔗糖和葡聚糖能同等程度地减缓肌酸激酶活性的丧失,并抑制聚沉的产生.在葡聚糖和蔗糖同时存在的情况下,两者的保护作用可以累加,并共同维持肌酸激酶的稳定性.     

Activity and Structure Changes of Arginine Kinase from Shrimp Feneropenaeus chinensis Muscle in Trifluoroethanol Solutions

Trifluoroethanol has often been used in protein folding studies. The changes in activity and unfolding of arginine kinase from shrimp Feneropenaeus Ghinensis muscle during denaturation in different concentrations of trifuoroethanol were investigated using far-ultraviolet circular dichroism and fluorescence emission spectra. Arginine kinase was inactivated in trifluoroethanol solutions. The tertiary and secondary structures of arginine kinase were also destroyed in the trifluoroethanol solutions. The unfolding and inactivation courses were measured and compared. Inactivation occurred prior to unfolding, which suggests that the arginine kinase active site is more easily damaged by the denaturant than the enzyme as a whole. The result also indicates that the arginine kinase active site is situated in a limited and flexible region of the enzymemolecule.     

天门冬氨酸对精氨酸激酶的作用研究

应用酶活测定、荧光发射光谱等方法分析了精氨酸激酶在不同浓度天门冬氨酸中活性变化和去折叠动力学。实验结果表明：天门冬氨酸引起精氨酸激酶失活与去折叠,并且过程中没有蛋白质的聚沉;精氨酸激酶的去折叠遵循一级反应动力学的二相过程,包括快相和慢相;失活与天门冬氨酸浓度呈正相关;当去除天门冬氨酸的影响,精氨酸激酶的活力能得到恢复。通过对比实验结果暗示,天门冬氨酸是通过其解离状态的H＋改变了机体pH值而影响精氨酸激酶活性的,并且就天门冬氨酸对精氨酸激酶影响及其意义进行探讨。     

乳酸对肌酸激酶活性抑制作用的研究

应用酶活测定的方法分析了肌酸激酶在不同浓度乳酸中的活性变化．实验结果说明在失活过程中没有蛋白质的聚沉；肌酸激酶的失活遵循一级反应的一相过程；失活与乳酸浓度和作用时间呈正相关．通过对比实验结果显示，乳酸是通过其解离状态的H^ 改变了机体PH值而影响肌酸激酶活性的，并且就乳酸对肌酸激酶影响及其意义进行探讨．     

丙酮酸、乳酸对体外肌酸激酶活性的抑制作用

 应用酶活测定的方法分析了肌酸激酶在不同浓度的乳酸、丙酮酸中的活性变化.实验结果说明在失活过程中没有蛋白质的聚沉,失活与乳酸浓度、丙酮酸的浓度呈正相关.通过对比实验结果提示,丙酮酸离子、乳酸离子对肌酸激酶没有明显影响,丙酮酸、乳酸是通过其解离状态的H⁺改变了机体pH值而影响肌酸激酶活性的,作者就丙酮酸、乳酸对肌酸激酶影响及其意义进行探讨.     

Macromolecular Crowding Enhances Thermal Stability of Rabbit Muscle Creatine Kinase

The effect of dextran on the conformation (or secondary structure) and thermal stability of creatine kinase (CK) was studied using the far-ultraviolet (UV) circular dichroism (CD) spectra. The results showed that lower concentrations of dextran (less than 60 g/L) induced formation of the secondary CK structures. However, the secondary structure content of CK decreased when the dextran concentrations exceeded 60 g/L. Thermally induced transition curves were measured for CK in the presence of different concentrations of dextran by far-UV CD. The thermal transition curves were fitted to a two-state model by a nonlinear, least-squares method to obtain the transition temperature of the unfolding transition. An increase in the transition temperature was observed with the increase of the dextran concentration. These observations qualitatively accord with predictions of a previously proposed model for the effect of intermolecular excluded volume (macromolecular crowding) on protein stability and conformation. These findings imply that the effects of macromolecular crowding can have an important influence on our understanding of how protein folding occurs in vivo.     

Cause of Irreversible Inactivation of Aminoacylase in Urea Solution

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Comparison of Inactivation and Unfolding of Calf Intestinal Alkaline Phosphatase in Guanidinium Chloride Solution

IntroductionCalf intestinal alkaline phosphatase(CIP,EC3 .1 .3 .1 ) is a metalloenzyme which catalyzes thenonspecific hydrolysis of phosphate monoesters[1 ] .The X-ray crystal structure of bacterial alkalinephosphatase has been reported to0 .2 nm resolutionin the presence of inorganic phosphate[2 ] . Theactive site is a pocket containing a tight cluster oftwo zinc ions (0 .3 9nm separation) and onemagnesium ion (0 . 5 and 0 .7nm from two zincions) .CIP also contains zinc and magnesium ions…     

添加剂促进变性还原溶菌酶复性的作用

研究了多种添加剂促进变性还原溶菌酶复性的作用，考察了添加剂浓度及变性剂盐酸胍浓度对复性收率的影响．结果表明，精氨酸、乙酰胺、丙酮、硫脲及甘油均能有效促进变性溶菌酶复性，并且存在最佳的添加剂浓度使变性溶菌酶的复性收率最大．在促进复性中，乙酰胺等结构类似物与盐酸胍具有相同作用，因此，在降低复性液中盐酸胍浓度的同时，适当提高乙酰胺浓度即可获得较高的复性收率．当盐酸胍浓度为0．2mol／L时，复性收率达到90％时的乙酰胺浓度为2mol／L，但降低盐酸胍浓度至0．06mol／L时，达到相同变性收率的乙酰胺浓度需要4mol／L．甘油与盐酸胍存在着协同作用，在一定浓度的盐酸胍存在下，添加适量的甘油能获得较高的复性收率．     

Characterization of the Partially Folded Monomeric Intermediate of Creatine Kinase

IntroductionRecent studies of the protein folding pathway andintermediate states in vitro and in vivo haveinduced much interest in the importance ofunderstanding the propertiesof partially structuredintermediates[1 7] . Studies have suggested thatintermed…     

油脂一步法合成脂肪腈──工艺条件对甘油回收率的影响

研究了油脂一步法制脂肪腈工艺中，甘油回收技术。选用适当的低温催化剂，在230℃~280℃，氨气流速1000L/hkg左右，采用分段冷凝回收工艺和回流萃取技术可获得浓度85%以上工业粗甘油，总收率达90%，杂质含量很少。     

罗伊乳杆菌转化甘油能力的原位再激活

考察罗伊氏乳杆菌(L.reuteri)静息细胞转化甘油的重复利用,甘油脱水酶的原位再激活,以及补加培养基对再激活甘油脱水酶的变化.结果表明:菌体经二次转化后基本丧失转化能力,甘油对胞内甘油脱水酶具有强烈的抑制灭活作用;确证胞内存在甘油脱水酶再激活体系;不具转化甘油能力的L.reuteri可被补加的培养基在一定程度上再激...     

Effect of Urea on Activity and Conformation of a Glycoprotein

Introduction In some enzymes, inactivation occurs before noticeable conformational change can be detected during denaturation by guanidinium chloride or urea[1-6]. Tsou[7] suggested that enzyme active sites are formed by relatively weak molecular interact…     

大熊猫肌酸激酶热变性中活性与构象变化的比较

比较了大熊猫肌酸激酶在各种不同温度下热变性时，活性变化与构象变化的速度常数。结果表明在较低温度下，酶的失活速度与构象变化速度相当．而在较高温度下变性时酶的失活速度快于构象变化速度。这表明酶分子的活性部位的微区构象比整个分子的构象具有更大的易变性或柔性。     

异旋向组合转子混合性能研究

结合实验和数值模拟方法，对异旋向组合转子应用于水-甘油混合体系的混合性能进行了研究，获得水-甘油在异旋向组合转子中混合的动态画面以及甘油在混合流体中的浓度场分布图。研究结果表明，实验结果与数值模拟结果具有很好的一致性，数值模拟方法具有合理性；水-甘油流经异旋向组合转子过程中，受到转子的分割-分隔作用，使甘油的聚集状态被迅速打破；异旋向转子转动方向相反，对混合流体产生方向相反的强烈的扰动作用，促进甘油的分散；与Ll=100mm左旋转子组合的5种不同导程右旋转子中，当右旋转子的Lr=150mm时混合流体达到最好的混合均匀性。     

Lysozyme refolding at high concentration by dilution and size—exclusion chromatography

This study of renaturation by dilution and size exclusion chromatography (SEC) addition of urea to improve yield as well as the initial and final protein concentrations showed that although urea decreased the rate of lysozyme refolding, it could suppress protein aggregation to sustain the pathway of correct refolding at high protein concentration; and that there existed an optimum urea concentration in renaturation buffer. Under the above conditions, lysozyme was successfully refolded from initial concentration of up to 40 mg/mL by dilution and 100 mg/mL by SEC, with the yield of the former being more than 40% and that of the latter being 34.8%. Especially, under the condition of 30 min interval time, i.e. tau > 2(t(R2) - t(R1)), the efficiency was increased by 25% and the renaturation buffer could be recycled for SEC refolding in continuous operation of downstream process.     

拥挤环境对碱性磷酸酶去折叠过程的影响作用研究

为了更好地模拟细胞内的真实环境，在碱性磷酸酶的变性环境中分别加入小分子渗透剂（蔗糖）和大分子拥挤试剂（葡聚糖），创造拥挤环境，利用酶活力测定和荧光光谱等方法，研究该拥挤环境对碱性磷酸酶稳定性的影响作用．实验结果表明:在碱性磷酸酶由盐酸胍导致的变性过程中，蔗糖与葡聚糖可抑制碱性磷酸酶活力的丧失，随着蔗糖与葡聚糖的质量浓度增加，其抑制效果随之增强；荧光光谱的检测结果也表明蔗糖和葡聚糖对碱性磷酸酶的去折叠具有一定的抑制作用．在2种试剂中，蔗糖对碱性磷酸酶活力与构象稳定性的保护能力要强于葡聚糖．      

甘油检测技术研究进展

甘油是重要的工业原料,在国防、化工、油漆、食品、日化、医药、农药及生物制品等领域有着广泛的应用,对发酵产品产量的控制、柴油的质量控制、化妆品的保鲜性及冷冻血液处理后的可用性等具有重要的作用。本文综述了高碘酸氧化法、Cu²⁺比色法和酶催化反应 分光光度法等近年来测定甘油含量方法的应用进展,认为甘油测定方法的未来发展趋势是传统检测技术与新纳米材料、新检测技术的融合,应进一步提高对甘油检测的专一性、灵敏性,实现甘油含量检测的仪器化、自动化、微型化以及快速化,并借助互联网和云技术,实现对甘油含量的连续、遥控、实时与在线监测。     

甘油保护剂对冷冻干燥保存红细胞作用的实验研究

应用甘油作红细胞冷冻干燥保存的保护剂,研究甘油对红细胞的保护机制.红细胞用不同质量分数(0,20%,40%)的甘油前处理后,冻干保存前后分别检查红细胞的相关指标.结果显示,40%甘油前处理的红细胞冻干保存效果最佳,红细胞回收率和血红蛋白回收率分别达到55.3%和63.4%;红细胞渗透脆性和超氧化物歧化酶(SOD)活力与冻干保存前无明显差别.该研究为使用甘油作红细胞冻干保存的保护剂提供了实验基础.