A fluorimetric method for the determination of pepsin activity

An intramolecularly quenched fluorogenic peptide containing o-aminobenzoyl (Abz) and ethylenediamine 2,4-dinitrophenyl (Eddnp) groups at amino- and carboxyl-terminal amino acid residues, Abz-Lys-Pro-Ile-Glu-Phe-Phe-Arg-Leu-Eddnp, was hydrolyzed by purified human pepsin, gastricsin, and gastric juice uniquely at the Phe-Phe bond. Kinetic parameters determined for purified pepsin were K(m)=0.68+/-0.11 microM; k(cat)=6.3+/-0.16s(-1); k(cat)/K(m)=9.26s(-1) microM(-1); Gastricsin showed K(m)=2.69+/-0.18 microM; k(cat)=0.03+/-0.005s(-1); k(cat)/K(m)=0.011s(-1) microM(-1). Gastric juice (21 samples) from subjects without gastric disorders at endoscopy examination showed activities varying from 0.0008 to 9.72 micromolml(-1)min(-1). Pepstatin A inhibition of gastric juice enzymatic activity was complete at 3.4x10(-5)M (final concentration) inhibitor. In the proposed method the presence of a unique scissile bond in the synthetic substrate provides a direct ratio between enzymatic activity and amount of substrate hydrolyzed, and a unique step reaction facilitates the use of this assay for the determination of the activity of aspartic proteinases in biological fluids and during enzyme purification procedures.     

A fluorimetric method for determination of calcineurin activity

Calcineurin is a calcium-dependent, serine/threonine phosphatase that is involved in a variety of signaling pathways. Calcineurin is distinct among phosphatases because its activity requires calcium and is not sensitive to inhibition by compounds that block the related phosphatases PP1A and PP2A. Therefore, the most common methods to measure calcineurin activity rely on calcium-dependent dephosphorylation of a substrate derived from the RII subunit of protein kinase A in the presence of PP1A/PP2A inhibitors. However, current techniques quantify activity by measurement of released radioactive phosphate or detection of free phosphate with malachite green. Both methods involve technical challenges and have undesirable features. We report a new calcineurin fluorimetric assay that utilizes a fluorescently labeled phosphopeptide substrate and separation of dephosphorylated peptide product by titanium-oxide. The method is rapid, quantitative, involves no radioactivity and is suitable for high throughput assays. Furthermore, with the use of a standard curve, precise measurements of calcineurin activity can be obtained.     

A simple colorimetric method for the determination of tryptophan

A simple, sensitive, and reproducible colorimetric method for the determination of tryptophan in amounts as low as 2 μg is described. It is based on the oxidation of tryptophan by sodium nitrite and the coupling of the oxidized product to the leucodye N-1-(naphthyl)ethylenediamine dihydrochloride. The purple-pink product has an absorption maximum at 550 nm. There is no interference by carbohydrates, other amino acids, neutral salts, or a number of other compounds likely to be found in tissue hydrolysates. A number of indole derivatives including indole-3-acetic acid also react to give a colored product. Dipeptides containing tryptophan are much less reactive than free tryptophan; hence proteins must be hydrolyzed completely for the method to be useful. The assay is carried out at room temperature and can be modified easily to increase or decrease its sensitivity. It has been employed to determine the tryptophan content of a number of proteins following alkaline hydrolysis. Generally, values obtained were in close agreement with values reported in the literature.     

A specific method for determination of free tryptophan and endogenous tryptophan in Escherichia coli.

A sensitive, simple spectrofluorometric technique for determination of tryptophan inamounts as small as 10 pmol is described. It is based on tryptophanase hydrolysis of tryptophan and spectrofluorometric analysis of the resulting indole. The relationship between released indole and fluorescence is linear over three orders of magnitude. The method is free from interference by other amino acids, polar indole derivatives, and a number of other compounds found in cell extracts or used in bacterial growth media. The method is rapid, reproducible, and accurate. A simple method for extraction and measurement of endogenous free tryptophan from bacterial cells is also described.     

A sensitive method for determining quinolinic acid in animal tissues

A sensitive chromatographic method for isolation and measurement of quinolinic acid from rat liver and kidney is described. The method is based on the isolation of quinolinic acid by ion-exchange chromatography. The extraction of quinolinic acid consisted of the freeze clamping of the organ in liquid nitrogen, followed by deproteinization in perchloric acid. The neutralized extract was concentrated by freeze-drying and submitted to the action of concentrated perchloric acid to hydrolyze the nucleotides which interfered in the chromatographic separation of quinolinic acid. The sample was applied to a column of Dowex (HCOO^?) and eluted with a linear gradient of formic acid. The eluted fraction containing quinolinic acid was quantitatively measured by its absorbance at pH 2 and 268 nm in a spectrophotometer.     

A novel inhibitory kinetic fluorimetric method for the determination of trace methomyl in environmental samples

A novel inhibitory kinetic fluorimetric method for the determination of trace methomyl was proposed. It was shown that the Fenton reagent oxidized rhodamine B in acid medium which enabled the fluorescence quenching of the latter. The presence of trace methomyl clearly inhibited the reaction. Upon addition of EDTA, a good linear relationship between the inhibitory effect and the concentration of methomyl was observed, together with improved stabilization and sensitivity. Factors affecting the determination of trace methomyl were investigated systematically. Under the optimum conditions, the linear range for the determination of methomyl was 0.04–2.2 µg/mL; the detection limit and the quantification limit for methomyl were 0.011 and 0.037 µg/mL, respectively. The proposed method was applied to the determination of methomyl in four environmental soil samples, six environmental water samples and one synthetic sample; the results were compared with those determined by the HPLC method. The recoveries and the relative errors were 83.5–101.2 and 0.47–2.02%, respectively. The possible reaction mechanism has also been discussed. Copyright © 2009 John Wiley & Sons, Ltd.     

Adaptation of the phosphotungstate method for the determination of vitamin C contents in animal and human tissues

The usefulness of phosphotungstate reagent for vitamin C determination in tissue homogenates has been confirmed. An optimal homogenization medium was selected: 1.8 M solution of HPO3 in 1.3 M CH3COOH. With this medium the analytical curve (at 700 nm) demonstrated the right linearity, correlation and recovery coefficients were appropriately high (0.999 and 99.8%) and the values of intraserial and interserial variation coefficient were low (< 5% and < 10%, respectively). It makes this method sensitive, easily repeatable, and useful for vitamin C determination in animal and human tissues, including neoplastic ones.     

A new method for isolation of polyamines from animal tissues

A new method for isolation of polyamines from tissues was developed and compared with the butanol extraction method which has been widely used for quantitative determination of polyamines. In the new method protein-free tissue extracts are applied to a small Dowex-50 column. The column is washed with appropriate buffers to remove ninhydrin-positive contaminants and the polyamines are eluted. With this method, the overall recovery of polyamines, after separation by paper electrophoresis and subsequent colorimetric determination with ninhydrin, is always over 90% (average 95%). This method is much better than the butanol method, which gives variable recoveries of 70–90%. The new method also has the advantages over the butanol method that the isolated polyamines are purer and the procedure is simpler.     

分光光度法测定豆类及其粗蛋白质中的色氨酸

建立一种快速、准确测定各种豆类试样及粗蛋白质中的色氨酸含量的分光光度法讨论最佳试验条件及试剂用量,反应物在410nm处具有最大吸收波长ε=1.103×104L·mol-1·cm-1,回收率93.50-110.20%,相对标准偏差RSD＜2.61.