New Chromogenic Substrates for Rapid Determination of Collagenase Activity

Abstract

Cowhide collagen insolubilized by formaldehyde treatment was grafted with chlorotriazine dyes in order to obtain chromogenic substrates for rapid determination of collagenases. The cross-linking degree and the dyeing procedure were chosen as to permit a maximal sensitivity of the method. Collagenolytic activity was estimated by colorimetric measurement of the coloured soluble split products liberated in supernatant as a result of the enzyme action on the insoluble dyed substrate. Some precision parameters were calculated (cv=6.62%) and proved a satisfactory reproducibility of the method.     

Purification,Characterization, and Structural Studies of a Sulfatase from Pedobacter yulinensis

Sulfatases are ubiquitous enzymes that hydrolyze sulfate from sulfated organic substrates such as carbohydrates, steroids, and flavones. These enzymes can be exploited in the field of biotechnology to analyze sulfated metabolites in humans, such as steroids and drugs of abuse. Because genomic data far outstrip biochemical characterization, the analysis of sulfatases from published sequences can lead to the discovery of new and unique activities advantageous for biotechnological applications. We expressed and characterized a putative sulfatase (PyuS) from the bacterium Pedobacter yulinensis. PyuS contains the (C/S)XPXR sulfatase motif, where the Cys or Ser is post-translationally converted into a formylglycine residue (FGly). His-tagged PyuS was co-expressed in Escherichia coli with a formylglycine-generating enzyme (FGE) from Mycobacterium tuberculosis and purified. We obtained several crystal structures of PyuS, and the FGly modification was detected at the active site. The enzyme has sulfatase activity on aromatic sulfated substrates as well as phosphatase activity on some aromatic phosphates; however, PyuS did not have detectable activity on 17α-estradiol sulfate, cortisol 21-sulfate, or boldenone sulfate.     

Stereoselective hydrolysis of sec-mono-alkyl sulfate esters with retention of configuration

An optimised method for the stereoselective hydrolysis of sec-alkylsulfate monoesters with absolute retention of configuration was developed. Under optimised conditions, clean hydrolysis of (R)-2-octyl sulfate was achieved in aqueous t-butyl methyl ether (3:97) using 0.6 equiv of p-toluenesulfonic acid as catalyst and 0.33 equiv of dioxane as mediator to give (R)-2-octanol as the sole product in the absence of side reactions, such as racemisation or elimination.     

Detection of Sulfatase Enzyme Activity with a CatalyCEST MRI Contrast Agent

A chemical exchange saturation transfer (CEST) MRI contrast agent has been developed that detects sulfatase enzyme activity. The agent produces a CEST signal at δ=5.0 ppm before enzyme activity, and a second CEST signal appears at δ=9.0 ppm after the enzyme cleaves a sulfate group from the agent. The comparison of the two signals improved detection of sulfatase activity.     

Determination of Arteether in Plasma Using a Simple and Rapid High-Performance Liquid Chromatographic Assay

Abstract

A simple and rapid high-performance liquid chromatographic (HPLC) assay for the determination of the antimalarial drug arteether in plasma was developed and validated in this report. Perchloric acid was used in this method as a plasma protein precipitant and to attain an acidic medium suitable for the decomposition of arteether to a derivative possessing UV absorption. This derivative and the internal standard (progesterone) were separated from the plasma on a 10 μm μ-Bondapack C₁₈ reversed-phase column at ambient temperature with a mobile phase composed of acetonitrile:water (60:40 v/v) and at a flow rate of 1.5 ml/min. The effluent was monitored at 254 nm with a UV detector. Linear relation between drug concentrations and peak height ratios of arteether derivative to the internal standard was achieved in the range of 0.25-10 μg/ml arteether with a detection limit of 50 ng/ml arteether in plasma. The within-day and between-days precisions were evaluated using 3 different concentrations of arteether. The values of the coefficients of variation were 1.35-1.68% and 1.65-2.82% for within-day and between-day, respectively. This method was applied to determine some pharmacokinetic parameters of arteether after intramuscular injection of 50 mg/kg arteether oily solution to rabbits.     

An Enzyme Coupling Procedure for the Determination of Lipoprotein Lipase Activity

Abstract

A novel, sensitive enzyme coupling procedure is described for the quantitative determination of both purified bovine milk lipoprotein lipase (LPL) and human post-heparin plasma lipolytic activity (PHLA). The primary assay is carried out with the aid of a triolein emulsion optimized as regards NaCl, bile salt and C II apolipoprotein concentration. The secondary reaction consists of the photometric measurement of a colored adduct formed following enzymatic oxidation of non esterified fatty acids. The use of radiolabels is thus eliminated. The precision of the assay was comparable to other competent methods (CVs obtained are between 2.1 and 9.5). The system described is sensitive enough for detecting post-heparin plasma lipolytic activity in both healthy humans and cancer patients.     

壳寡糖的酶法制备

通过还原端基、酶解产物聚合度、水解液调碱性后透光率的测定以及酶解产物的TLC、HPLC分析,研究了壳聚糖酶降解壳聚糖过程中壳聚糖浓度、酶用量、反应时间和pH对酶促反应速率和产物聚合度的影响。结果表明:壳聚糖酶在底物浓度0.03 g/mL、壳聚糖酶用量4~6 u/g、pH=5.8和水解200 min时,可得到以聚合度3~7为主的壳寡糖。     

低抗凝肝素寡糖的制备、结构表征及免疫活性评价

以猪肠黏膜来源低抗凝肝素为原料,采用苄酯碱水解法制备了寡糖.通过聚丙烯酰胺凝胶电泳(PAGE)、高效凝胶渗透色谱(HPGPC)-多角激光光散射法(MALLs)联用、核磁共振波谱(NMR)和液相色谱-质谱(LC-MS)联用技术对所制备寡糖的分子量分布及化学结构进行了表征.以该寡糖对活化X因子(FXa)及人凝血酶(FⅡa)活性的抑制作用评价了其抗凝血活性,并通过测定其对RAW264.7巨噬细胞吞噬能力及NO释放量的影响评价其免疫活性.结果表明,所制备寡糖的聚合度分布于2~22之间,平均分子质量为5300,无明显的抗凝血活性,但具有显著的免疫增强活性.     

Synthesis of a Mimicking Hybrid of Annonaceous Acetogenin with Steroid for Antitumoral Activity Investigation

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反相高效液相色谱法同时测定保健口服液中20种甾体激素的研究

建立了利用反相高效液相色谱法同时测定保健口服液中２０种甾体激素的方法,进行了色谱条件的优化和样品预处理方法的探讨,并应用于市售保健口服液中微量激素的测定。最低检出量为０．１６～３．１８ｎｇ,回收率为８０．５％～１０３．１％,相对标准偏差为３．４％～９．２％。     

乙酰胆碱酯酶活性的计时电位法测定

报道了一种采用电化学计时电位法测定乙酰胆碱酯酶活性的方法;以碘化硫代乙酰胆碱作底物,将两支铂电极和一支饱和甘汞电极浸入该底物溶液中,向两支铂电极上施加一个合适的恒电流,记录电位V随时间t的变化曲线;该曲线的斜率ΔV／Δt代表酶催化水解的速度,从而反映出酶活性的大小;对影响测定的重要因素如电流、pH值、温度、底物浓度进行了考察,结果表明：当电流为20μA,pH7.4,温度26.0℃,底物浓度2mmol/L时,可获得满意的测量结果;与其他方法相比,该法更加简便、快速。     

A nonlinear regression model applied to kinetic multi-component analysis of esters with a SAW-impedance sensor

A kinetic nonlinear regression model for multi-component assay of esters was proposed based on their different alkaline-catalysed hydrolysis rate. The reaction rate was determined by monitoring the conductance change in solution with a liquid-purpose surface acoustic wave impedance sensor(SAW). The model was tested theoretically and experimentally with the mixture of methyl acetate and n-propyl acetate. The experimental detection limit of methyl acetate and n-propyl acetate (within 10 min) was 0.5 umol/L and 1.0 umol/L respectively and the recovery of the sensor system ranged from 93% to 106% (n=6).     

木薯淀粉硫酸酯的制备及理化性质

以NaHSO3和NaNO2为酯化剂,采用水相法制备了低取代度的淀粉硫酸酯(SSE),测定了SSE糊的冻融稳定性、凝沉性能、透明度、黏度;利用红外光谱仪分析了产物的化学结构.结果表明,酯化的SSE糊的透光率比淀粉的提高22.4％,透明度稳定性明显增强;SSE糊的黏度冷热差值均比淀粉的低,抗老化性能增强、凝沉性能得以改善,...     

高效液相色谱-串联质谱法同时检测鸡肉和鸡蛋中合成类固醇类激素和糖皮质激素

建立了鸡肉和鸡蛋中合成类固醇类激素(睾酮、甲基睾酮、群勃龙、勃地龙、诺龙、美雄酮、司坦唑醇、丙酸诺龙、丙酸睾酮及苯丙酸诺龙)和糖皮质类激素(泼尼松、泼尼松龙、地塞米松、氟氢可的松、甲基泼尼松、倍氯米松及氢化可的松)多残留的液相色谱-串联质谱(LC-MS/MS)检测方法.样品经乙腈超声提取,正己烷脱脂净化,以甲醇-甲酸水溶液为流动相,经C18柱分离后进行LC-MS/MS选择反应监测模式下的定性及定量分析.合成类固醇类激素采用正离子模式检测,糖皮质激素则采用负离子模式检测,正、负离子化模式一次进样同时检测.类固醇类和糖皮质类激素的定量检出限为0.5 μg/kg.在0.5、 1.0和5.0 μg/kg 3种浓度添加水平,上述激素的平均回收率为73.4%～108.9%;相对标准偏差为3.4%～13.4%.可实现样本灵敏、准确地定性定量分析.     

Studies on Angiotensin-Converting Enzyme Inhibitors: Protease Catalyzed Resolution of Aryl 3-Mercapto-2-Methylpropionic Ester

Optically active 3-benzylthio-2-methylpropionic acid and 3-benzoylthio-2-methylpropionic acid have been prepared via protease-catalyzed hydrolysis of their corresponding ester and thioester. Subtilisin catalyzed the hydrolysis of the thioester of methyl 3-benzoylthio-2-methylpropionate twice as fast as that of the methyl ester derivative. The stability of the enzyme in organic cosolvents was studied. Immobilization of subtilisin on the solid support XAD-8 improved the stability of the enzyme. A practical preparative resolution of the title compound is reported.     

Fluorometric Method for Determination of Transketolase Activity

Abstract

A new method for rapid and accurate determination of transketolase is studied. The reaction is monitored by coupling the system with the glyceraldehyde-3-phosphate dehydrogenase system and flurometrically measureing the production of NADH. The assay is rapid and convenient. It responds linearly to transketolase activity as low as 0.1 units in three ml of reaction mixture.     

Preparation of Polyporus Albicans Teng Sulfate and Its Anti-coagulation Activity

More studies have indicated that polysaccharide sulfate has anti-coagulant activity.Now,heparin is the most popular anticoagulant used in clinic,however,its side effects have also caused highly concern.It is still under intensive investigations to synthesize effective safe polysaccharide sulfate as heparin substitute.We extracted water-soluble polysaccharide from fermented mycelium of edible polyporus albicans(Imaz.) teng,and got the water-soluble polyporus albicans teng sulfate(PATS) by modifying the water-solubility polyose with the method of chlorosulfonic acid-pyridine.The anti-coagulant assay of PATS in vitro towards normal human plasma indicates its remarkable anticoagulant activity,while the dose could be as low as 5 mg/L for anticoagulation.The anti-coagulant effect was equivalent to that of heparin about 150 U when the concentration of PATS was 10 mg/L.The study on anti-coagulation mechanism suggests that PATS got involved in the intrinsic pathway.The anti-coagulation activity of PATS was due to the inhibition of the coagulation factors IIa and Xa activities mediated by antithrombin Ⅲ(ATIII).The anti-coagulation mechanism of PATS is absolutely identical to that of heparin.In conclusion,we suggest that PATS has the similar anti-coagulation characteristic to heparin,but with a better anti-coagulation effect.Meanwhile,derived from edible fungus-polysaccharide,PATS has more bio-safety advantage.Therefore,PATS has promising future to be developed and used as an ideal substitute for heparin in clinic.     

气相色谱-质谱法检测动物肌肉组织中残留的甾类同化激素

建立了不同动物肌肉中甾类同化激素残留的气相色谱/质谱（GC/MS）检测方法。样品经酶解、组织捣碎和超声提取后,用叔丁基甲醚萃取和反相固相萃取柱净化,进一步衍生后进行GC/MS分析,分别测定雄激素（表睾酮（ETS）、丙酸睾酮（PTS）、19-去甲基睾酮（17β-NT）、甲基睾酮（MTS）)和雌激素（雌二醇（17β-ES）、雌三醇（EST）、炔雌醇（EES）、雌酮（ESN）、苯甲酸雌二醇（BES)）。实验结果表明,ETS、17β-NT、MTS、PTS、BES、ESN、17β-ES、EES和EST的定量检测限为1.0~2.0 μg/kg。在2.0 μg/kg的定量检测低限添加水平,上述9种激素的平均回收率为62.5%~80.5%,相对标准偏差为12.5%~26.8%。该法可实现对样品进行灵敏、准确的定性定量分析。     

Analysis of Erythromycin II. Determination of Erythromycin in Human Blood Serum by Competitive Displacement of [14C]-Erythromycin from E. Coli Ribosomes

Abstract

A sensitive and selective method for the determination of erythromycin in human blood serum, in the range of 0.1 to 1.0 μg/ml, is described. The procedure is based on extraction of drug into ether and competitive displacement of [¹⁴C]-erythromycin from E. coli ribosomes. The method provides accurate, precise and rapid analyses and should be readily adaptable to bioavailability studies with erythromycin and its salts in man.