Immobilized Firefly Luciferase and its use in Analysis

Abstract

Firefly luciferase immobilized on sepharose 4B and cellophane filmis shown to retain as much as 20% of its initial activity and can be durably stored at 4O°C with no appreciable loss in its enzymatic activity. The application of the immobilized luciferase is described for ATP detection at concentrations ranging from 0.1 pM to 1 mM, as well as for activity determination of enzymes catalyzing ATP synthesis (pyruvatekinase) and ATP hydrolysis (ATP-ase). The same sample of luciferase immobilized on cellophane film was 7%.     

Analysis of Free Fatty Acids on the Fingertips by High Performance Liquid Chromatography

Abstract

This investigation studied the efficiency of high performance liquid chromatography in the determination of free fatty acids present on the fingertips, and assessed the quantitative relationship between skin fatty acids and the degree of microbial contamination. Automated surgical scrub was utilized to eliminate the microbial contamination.

The high performance liquid chromatography provided excellent separation of skin fatty acids for evaluation with the bacterial counts. The fatty acid peaks identified ranged in chain length from C12 through C32. All the fatty acids evaluated showed positive correlation with the bacterial counts with the exception of one acid which had an inverse relationship but none were statistically significant. Finally, the surgical scrub chromatograms showed that the straight chain acids C19 and C21 were lower in concentration than C23 and C25; also, C26 was lower in concentration than C28 and C30.

It was evident from the data that fatty acids which have been shown to be bacteriostatic in vitro do not demonstrate the same property on the fingertips. The finding of lower concentration of C19, C21, and C26 than longer chain acids is inconsistent with simple two carbon addition, and indicates there is possible branching at these points in metabolic pathway of fatty acid synthesis.     

Heterogeneous Solid Acid Catalysts for Esterification of Free Fatty Acids

Use of low-quality (and inexpensive) feedstock such as waste oils and animal fats for biodiesel synthesis has been continuously researched as a prospective means of improving the economic efficiency of the process. Pretreatment of free fatty acids by catalytic esterification is necessary for achieving this purpose. This paper reviews some relevant studies on heterogeneous acid catalysts that have been shown to be effective for this reaction.     

Evolution Analysis of Free Fatty Acids and Aroma-Active Compounds during Tallow Oxidation

To explore the role of fatty acids as flavor precursors in the flavor of oxidized tallow, the volatile flavor compounds and free fatty acid (FFAs) in the four oxidization stages of tallow were analyzed via gas chromatography (GC)–mass spectrometry (MS), the aroma characteristics of them were analyzed by GC–olfactory (GC-O) method combined with sensory analysis and partial least-squares regression (PLSR) analysis. 12 common FFAs and 35 key aroma-active compounds were obtained. Combined with the results of odor activity value (OAV) and FD factor, benzaldehyde was found to be an important component in unoxidized tallow. (E,E)-2,4-Heptadienal, (E,E)-2,4-decadienal, (E)-2-nonenal, octanal, hexanoic acid, hexanal and (E)-2-heptenal were the key compounds involved in the tallow flavor oxidation. The changes in FFAs and volatile flavor compounds during oxidation and the metabolic evolution of key aroma-active compounds are systematically summarized in this study. The paper also provides considerable guidance in oxidation control and meat flavor product development.     

降低生物柴油中自由脂肪酸的吸附剂研究

以甘蔗渣为原材料,通过高温煅烧制备甘蔗渣吸附剂,用于降低生物柴油中的自由脂肪酸含量。采用SEM、热重分析仪对甘蔗渣吸附材料进行表征分析,研究不同煅烧温度对吸附剂吸附性能的影响,以及不同吸附条件对吸附效果的影响。结果表明,在实验条件下,当甘蔗渣吸附材料煅烧温度为600℃、吸附剂用量0.30 g、吸附温度25℃、吸附时间100 min时,可以将生物柴油原料油的酸值降至0.4 mg/g。     

Rapid and Quantitative Blood Analysis for Free Fatty Acids by Chemical Ionization Mass Spectrometry

Abstract

A quantitative analysis of fatty acids in micro-samples of dried blood spots by chemical ionization mass spectrometry has been developed. Isotope determination was used as the quantitating technique using the corresponding deuterium labeled internal standards. The procedure yields excellent precision and accuracy as demonstrated by the analysis of known fatty acid mixtures and of both C_12:0 to C_18:0 acids and phytanic acid in the blood from patients.     

衍生化毛细管气相色谱法分析血清中的游离脂肪酸

用H2SO4/甲醇对血清中游离脂肪酸进行甲酯化反应,考察了不同温度和时间以及不同的衍生条件对甲酯化完全程度的影响,确定了对血清中游离脂肪酸衍生化完全的条件为：在62℃下用10%H2SO4/甲醇溶液衍生化2h。大部分脂肪酸的衍生反应和色谱分析的相对标准偏差都在3.00%以内。用各脂肪酸和内标色谱峰面积的比值对脂肪酸浓度作标准曲线,线性回归系数在0.999-1之间,定量测定了5个不同地区的250个血清样品中12种游离脂肪酸的含量。     

Highly Sensitive Bioluminescent Assay of Dehydrogenases using Nad (P) H: Fmn Oxidoreductase and Luciferase from Photoeacterium Fischeri

Abstract

A highly sensitive bioluminescent assay of dehydrogenases was performed. NADH was produced by the catalytic action of alcohol and glucose-6-phosphate dehydrogenases and subsequently measured with high sensitivity by a bioluminescent assay using NAD (P) H : FMN oxidoreductase and luciferase from Photobacterium fischeri. The minimal amount of dehydrogenases that could be measured was 0.0055 amol (5.5 × 10^?-²¹ mol).     

基于衍生化技术的游离脂肪酸的色谱-质谱分析方法研究进展

游离脂肪酸在生物体内含量极低,由于结构中缺少易电离的官能团,导致质谱分析时检测灵敏度不高。利用化学衍生化技术对其进行结构修饰是改善色谱行为和提高质谱响应的有效手段。近年来,化学衍生化结合色谱和质谱技术在游离脂肪酸检测中得到广泛应用。该文综述了国内外基于衍生化技术的游离脂肪酸分析方法及其应用研究进展。     

热稳定生物素化荧光素酶的制备及其在焦测序中的应用

新一代大规模焦测序技术需要稳定的可固定化的荧光素酶,为了制备热稳定性好且被生物素化的荧光素酶,本研究采用基因工程方法将生物素羧基载体蛋白C端87个氨基酸残基(BCCP87)与荧光素酶在大肠杆菌E.coli BL21(DE3)中进行融合表达,以实现菌体内直接表达生物素化的荧光素酶(BCCP-LUC);并对北美萤火虫(Photinus pyralis)荧光素酶基因进行了定点突变以增强其热稳定性;用链亲和素包被的磁珠对重组融合蛋白进行固定化,用于焦测序。实验结果表明:突变后的荧光素酶在50℃环境中仍具有活性;在43℃下10min活性保留大于80%,热稳定性明显增强。Western blot分析结果表明,荧光素酶能够在大肠杆菌内生物素化。用链亲和素包被的磁珠结合BCCP-LUC后具有较高活性(2.1×105RLU/μL Beads),经过多次洗涤活性无明显下降。采用微球固定的荧光素酶以及ATP硫酸化酶成功的进行了DNA序列的测定且定量准确,表明固定化的荧光素酶可以应用于焦测序中,为建立高通量大规模芯片焦测序技术提供有效、稳定的工具酶。     

健康人血清中游离脂肪酸含量的测定

应用气相色谱法对６０例健康人血清样品中６种重要脂肪酸定量测定，测定方法简便，获得６个年龄组血清样品游离脂肪酸含量的正常值范围，为临床科学研究及疾病的诊断提供参考依据     

Determination of Free Fatty Acids by Diphasic-Two Dimensional TLC-Fluorescence Spectrodensitometry

Abstract

A sensitive method for the determination of fatty acids is presented. Free fatty acids (20pg-lμg), were first covalently reacted in a displacement reaction with a 70 μM solution of the fluorescent probe, 4-bromomethy 1–6, 7-dimethoxy-coumarin (Br- Mdmc) and the fluorescence-labelled fatty acid esters were separated in a diphasic-two dimensional high performance thin layer chromatographic system (diphasic-2D-TLC). This system consisted of a reversed phase C18 layer (2 × 10 cm) interfaced with a AgNO3-modified silica gel layer (10 × 10 cm). Aliquots of the reeaction mixture were streaked onto the C18 layer, and the plates developed in the first dimension using acetonitrile-acetone-methanol-water (60:20:10:10, v/v/v/v) (solvent 1) as the mobile phase. Development in this dimension gave separation based on the number of carbons. Following the first development, the plates were dried and the silica gel layer impregnated with a saturated solution of AgNO3 in methanol. The plates were then predeveloped in the second dimension in chloroform-ethyl acctate-acetonitrile (90:8:2, v/v/v) (solvent 2) to the plate interphase, and developed in solvent 2 in the second dimension. Development in the AgNO3-modified silica gel, allowed separation based on the number of double bonds. The fluorescent bands were scanned with a Shimadzu CS-9000 spectrodensitometer in the fluorescent mode, using 352 nm as the excitation wavelength and a cut-off filter at 400 nm. Baseline resolution was obtained for all 15 fatty acids tested. The lower linear detector response extended to 0.13 pmol. This method provids a sensitive means of analysis of fatty acids present in biological systems.     

Analysis of the Triglycerides and the Free and Derivatized Fatty Acids in Fish Oil by Capillary Electrochromatography

Capillary ElectroChromatography (CEC) on a fused silica capillary column (40 cm L × 100 μm i.d.) packed with 3 μm octadecylsilica (ODS) was evaluated for the analysis of the triglycerides and their fatty acids in fish oils, and more especially in the oil of Moroccan Sardinia pilchardus. The very high complexity of the lipids in fish oil is well illustrated by CEC with a nonaqueous mobile phase consisting of acetonitrile/isopropanol/n-hexane in the ratio 57/38/5 to which 50 mM ammonium acetate has been added. In order to unravel the complexity, the oil was hydrolyzed and the free fatty acids (FFAs), the methyl esters (FAMEs), and the phenacyl esters (FAPEs) were analyzed by CEC on the same column used for the analysis of the triglycerides. Isocratic elution was achieved with the mobile phase acetonitrile/50 mM MES pH 6 in ratio 9/1. The migration characteristics of FFAs, FAMEs, and FAPEs are compared. The analysis of FAPE derivatives has the advantage that quantification applying UV detection is possible and moreover that the number of double bonds in the fatty acid chains can be elucidated by measuring the UV abundance ratio 240/210 nm.     

虾蛄脂肪酸分析

用快速脂肪提取器对雄、雌虾蛄肉及虾蛄头中的脂肪进行了提取和测定。脂肪经KOH CH3OH溶液水解并甲酯化后 ,采用 60m× 0 .3 2mmi.d.交联PEG 2 0M弹性石英毛细管柱 ,FID检测器 ,结合GC MS ,对脂肪酸进行了分析。结果表明 :雄虾蛄肉脂肪含量为 5 .1 4 % ,不饱和脂肪酸占脂肪酸总量的 74 .7% ,其中多不饱和脂肪酸 (PUFA)为 4 9.3 % ,EPA +DHA为 3 0 .2 % ;雌虾蛄肉脂肪含量为 6.0 2 % ,不饱和脂肪酸占脂肪酸总量的 71 .5 % ,其中多不饱和脂肪酸 (PUFA)为 4 2 .1 % ,EPA +DHA为 2 5 .2 % ;虾蛄头脂肪含量为 5 .65 % ,不饱和脂肪酸占脂肪酸总量的 74 .3 % ,其中多不饱和脂肪酸 (PUFA)为 5 1 .5 % ,EPA +DHA为 2 9.7%。     

Determination of 13 Free Fatty Acids in Pheretima Using Ultra-Performance LC-ESI-MS

A simple and rapid ultra-performance liquid chromatography-electrospray ionization mass spectrometry method for the simultaneous determination of thirteen free fatty acids (FFAs) in Pheretima has been developed and validated. Measurements for each FFA were linear over a wide range (0.05–3.95 μg mL^?1) with good correlation coefficients (>0.99). The limit of detection and limit of quantification for all the fatty acids were below 26 and 78 ng mL^?1, respectively. The intra- and inter-assay precision and accuracy for the thirteen FFAs fell well within the predefined limits of acceptability. Satisfactory recoveries were in the range of 96–103%.     

啤酒风味物质及游离脂肪酸的气相色谱研究

啤酒中含量极低、极不稳定的游离脂肪酸(FFAs)是影响啤酒风味的主要因素。实验采用气相色谱技术,对形成啤酒的主要风味(如药物味、水果味、酒精味和哈喇味等),重点是不良风味的风味物质进行了检测。啤酒样品经两次酸化处理,酯化后薄层分离的方法测得游离脂肪酸的含量具有较好的重复性和较高的回收率,以癸酸和亚油酸为代表测得回收率分别为85%和114%。     

Nitro Fatty Acids (NO2-FAs): An Emerging Class of Bioactive Fatty Acids

Unsaturated nitro fatty acids (NO₂-FAs) constitute a category of molecules that may be formed endogenously by the reaction of unsaturated fatty acids (UFAs) with secondary species of nitrogen monoxide and nitrite anions. The warhead of NO₂-FAs is a nitroalkene moiety, which is a potent Michael acceptor and can undergo nucleophilic attack from thiol groups of biologically relevant proteins, showcasing the value of these molecules regarding their therapeutic potential against many diseases. In general, NO₂-FAs inhibit nuclear factorκ-B (NF-κB), and simultaneously they activate nuclear factor (erythroid derived)-like 2 (Nrf2), which activates an antioxidant signaling pathway. NO₂-FAs can be synthesized not only endogenously in the organism, but in a synthetic laboratory as well, either by a step-by-step synthesis or by a direct nitration of UFAs. The step-by-step synthesis requires specific precursor compounds and is in position to afford the desired NO₂-FAs with a certain position of the nitro group. On the contrary, the direct nitration of UFAs is not a selective methodology; thus, it affords a mixture of all possible nitro isomers.