关于酶促反应速度与酶反应的初速度的教学

酶促反应速度是酶促反应动力学中的一个基本概念。一定条件下 ,酶催化反应的活力大小要由该反应条件下酶促反应速度来反映和确定 :酶促反应速度越小 ,酶活力越小 ;反之 ,酶促反应速度越大 ,酶活力也越大。在研究底物浓度、酶浓度、温度、ｐＨ、激活剂、抑制剂等因素对酶活力的影响时 ,都必须测定酶促反应的速度。为了排除实验中存在的一些干扰 ,一个酶的酶促反应的速度要在酶促反应进行的初期测定 ,即测定所谓的酶促反应的初速度。在教学过程中 ,学生不易弄清楚酶促反应速度与酶促反应的初速度之间的关系。我们发现 :当一般地问起酶反应速度…     

苯丙氨酸解氨酶

苯丙氨酸解氨酶由四个亚基组成,含两个脱氢丙氨酸残基。植物酶具有内在不稳性,由多种同工酶组成。酶催化过程中发生构象变化,底物经过负碳离子中间体完成反应。该酶并非是二单体负协同变构酶。一级结构表明,酶以无规则卷曲结构为主。酵母基因约2.7kb,有六个内含子,编码75kD_a肽。植物酶由多基因编码,有一个内含子,编码78kD_a肽。启动子部位有两个富含A、C碱基的序列,为胁迫作用基因活化因子结合部位。     

放线菌果胶酶产酶条件及酶促反应条件的研究

从埋麻土壤中分离到放线菌２９８株,经初筛和复筛得到产酶活性较高的一株放线菌５－７１。最适产酶条件是：果胶０．５％,葡萄糖０．５％,蛋白胨０．５％,酵母粉０．１％,Ｋ２ＨＰＯ４ ０．１％,ＫＨ２ＰＯ４ ０．１％,ＮａＣｌ０．１％,ＭｇＳＯ４·７Ｈ２Ｏ０．０５％,ｐＨ８．０。２５℃培养３天达产酶高峰。通气量对产酶影响不大。酶促反应最适条件是：ｐＨ９．６,４５℃,底物果胶浓度０．７５％,作用时间９０ｍｉ     

大鼠甘氨肽酰化单氨酶基因的克隆与表达

本文采用原位杂交及ＰＣＲ方法,从大鼠脑ｃＤＮＡ库中,筛选到３个大鼠甘氨肽酰化单氧酶基因片段。经ＤＮＡ全序列分析表明,它们跨越ｒＰＡＭ－２全部编码区。通过点突变,ＰＣＲ重组技术等,分别拼接出此双功能酶的ｒＰＨＭ,ｒＰＳＡＬ及其ｒＰＡＭ全构建了多种大肠杆菌表达质粒。     

香蕉低温酶促褐变

7℃低温导致香蕉果皮变褐,褐变程度可用提取液的OD_(450)值表示。随低温处理时间的延长,OD_(450)增大;还原性物质抗坏血酸、谷胱甘肽含量迅速下降;多酚氧化酶底物多巴胺先有增加,接着降低。 低温下,多酚氧化酶(PPO)、过氧化物酶(POD)和过氧化氢酶(CAT)活性较常温低,PPO和POD的游离态酶活性增高。此外,H_2O_2处理加速果皮变褐,刺激PPO及POD活性;棓酸丙酯处理起相反的效应。故认为香蕉低温褐变是低温损伤、酶促褐变引起的一个复杂过程。     

微生物降解有机磷农药酶促机制

有机磷农药污染严重,微生物有机磷农药是治理有机磷农药残留的新技术,综述有机磷农药降解酶的研究现状、酶促作用机理、基因工程等方面的研究现状。     

An Enzymatic Fluorometric Assay for Pyridoxal with High Specificity and Sensitivity

An enzymatic fluorometric assay for pyridoxal with pyridoxal dehydrogenase was developed. The detection limit was about 10 pmol: the calibration curve of pyridoxal showed high linearity (r=0.993). The values obtained by this method correlated well with those by the HPLC method. The enzyme had a high specificity for pyridoxal, and thus animal samples could be directly analyzed without separation of pyridoxal 5′-phosphate by column chromatography.     

酶法合成黑色素的稳定性研究

运用紫外分光光度计对以酪氨酸为底物酶法合成的黑色素进行稳定性分析,结果表明:该黑色素的最大吸收峰为212 nm,不溶于水和酸,易溶于碱,在碱性条件下颜色稳定。热、自然光、紫外光、蔗糖、葡萄糖对色素稳定性无影响;氧化剂H2O2和还原剂Na2SO3对其影响不大;几种金属离子(除Fe2+)对黑色素吸光值及颜色无影响。     

A Novel Amplified Enzymatic Assay Method for Hypoxanthine

A sensitive and rapid enzymatic assay for hypoxanthine, using a Clark oxygen electrode as sensor, is proposed. In the presence of sodium sulfite, oxidation of hypoxanthine by milk xanthine oxidase caused very rapid oxygen consumption in excess of the stoichiometric requirement for hypoxanthine oxidation. Hypoxanthine from 0.5 to 10 μM can be assayed within a few minutes by addition of 25 mM sodium sulfite to the reaction mixture. This assay proved to be over 10-times more sensitive and much more rapid than the control method without sulfite.     

A Novel Amplified Enzymatic Assay Method for Hypoxanthine

A sensitive and rapid enzymatic assay for hypoxanthine, using a Clark oxygen electrode as sensor, is proposed. In the presence of sodium sulfite, oxidation of hypoxanthine by milk xanthine oxidase caused very rapid oxygen consumption in excess of the stoichiometric requirement for hypoxanthine oxidation. Hypoxanthine from 0.5 to 10 μM can be assayed within a few minutes by addition of 25 mM sodium sulfite to the reaction mixture. This assay proved to be over 10-times more sensitive and much more rapid than the control method without sulfite.     

Sensitive Enzymatic Assay for Glucose Determination in Natural Waters

A new enzymatic method for glucose determination is described. It allows measurement of glucose concentration as low as 10^-7 M. Such sensitivity makes this method particularly appropriate for estimation of glucose in natural-water bodies, generally without prior concentration or extraction. The method is based on the reaction between glucose and adenosine 5′-triphosphate, catalyzed by hexokinase to form glucose-6-phosphate. The amount of adenosine 5′-triphosphate consumed in this reaction, which is directly proportional to the amount of glucose present in the sample, is measured by the luciferin-luciferase assay. The optimal conditions for glucose determination by this method have been defined as follows: 20 min of incubation at 30°C, magnesium concentration of 10^-3 M, and pH in the range of 7.5 to 10.5. The specificity of the assay to different carbohydrates has also been studied. Recovery of known amounts of glucose added to Lake Kinneret water was in the range of 80 to 114%. Application of this method is demonstrated in eight monthly profiles of the glucose content in Lake Kinneret.     

兔肝细胞膜高密度脂蛋白受体酶联免疫检测法的研究

用辣根过氧化物酶标记羊抗人apoAI-IgG,建立了检测兔肝细胞膜HDL受体的酶联免疫吸附检测法.测定时, HDL结合量按抗体-配体-抗配体抗体酶交联物反应制作的标准曲线确定;膜蛋白非特异吸附则用与酶交联的抗体来源相同的同种动物血浆HDL平行抑制试验消除.实验测得正常家兔肝细胞膜HDL受体K_d值为7.17±1.18 mg/L,B_max值为(622.5±146.1)mg/g(n=7).     

北方冬小麦/夏玉米轮作体系土壤氨挥发的原位测定

采用通气法测定了北方冬小麦／夏玉米轮作体系田间土壤的原位氨挥发。结果表明,与冬小麦施用基肥相比,夏玉米追肥后土壤的氨挥发速率很快升高,但军发高峰期持续时间较短,最大氨挥发速率亦低于冬小麦,冬小麦拨节期追肥,氨挥发速率低且呈波动变化,未出现高峰值,从整个生长季节来看,冬小麦不施氮和每公顷施氮120、240、360kg时的累计挥发量分别为4．4、6．9、13．0、38．4kgN/hm^2,夏玉米为8．4、15．1、20．0、26．1kgN/hm^2。按我国北方冬小麦／夏玉米播种面积1864．4万hm^2计,每年由氨挥发向大气排放的氨素达23．8－120．2万t,其中17．2－96．4万t来自氮肥,相当于氮肥投入的2．1％－9．5％。     

Vero细胞法检测白喉抗毒素的研究

参照Ｍｉｙａｍｕｒａ等报道,建立了微量细胞培养检测白喉抗毒素的方法。以家兔皮肤试验为参照,Ｖｅｒｏ细胞培养法敏感度为９８．１１％,特异度为８４．００％,符合率为９６．２０％,相关系数ｒ＝０．９３,在白喉血清抗毒素测定中值得推广     

Measurement of Factor V Activity in Human Plasma Using a Microplate Coagulation Assay

In response to injury, blood coagulation is activated and results in generation of the clotting protease, thrombin. Thrombin cleaves fibrinogen to fibrin which forms an insoluble clot that stops hemorrhage. Factor V (FV) in its activated form, FVa, is a critical cofactor for the protease FXa and accelerator of thrombin generation during fibrin clot formation as part of prothrombinase ^{1, 2}. Manual FV assays have been described ^{3, 4}, but they are time consuming and subjective. Automated FV assays have been reported ^5-7, but the analyzer and reagents are expensive and generally provide only the clot time, not the rate and extent of fibrin formation. The microplate platform is preferred for measuring enzyme-catalyzed events because of convenience, time, cost, small volume, continuous monitoring, and high-throughput ^{8, 9}. Microplate assays have been reported for clot lysis ¹⁰, platelet aggregation ¹¹, and coagulation Factors ¹², but not for FV activity in human plasma. The goal of the method was to develop a microplate assay that measures FV activity during fibrin formation in human plasma.This novel microplate method outlines a simple, inexpensive, and rapid assay of FV activity in human plasma. The assay utilizes a kinetic microplate reader to monitor the absorbance change at 405nm during fibrin formation in human plasma (Figure 1) ¹³. The assay accurately measures the time, initial rate, and extent of fibrin clot formation. It requires only μl quantities of plasma, is complete in 6 min, has high-throughput, is sensitive to 24-80pM FV, and measures the amount of unintentionally activated (1-stage activity) and thrombin-activated FV (2-stage activity) to obtain a complete assessment of its total functional activity (2-stage activity - 1-stage activity).Disseminated intravascular coagulation (DIC) is an acquired coagulopathy that most often develops from pre-existing infections ¹⁴. DIC is associated with a poor prognosis and increases mortality above the pre-existing pathology ¹⁵. The assay was used to show that in 9 patients with DIC, the FV 1-stage, 2-stage, and total activities were decreased, on average, by 54%, 44%, and 42%, respectively, compared with normal pooled human reference plasma (NHP).The FV microplate assay is easily adaptable to measure the activity of any coagulation factor. This assay will increase our understanding of FV biochemistry through a more accurate and complete measurement of its activity in research and clinical settings. This information will positively impact healthcare environments through earlier diagnosis and development of more effective treatments for coagulation disorders, such as DIC.