A sensitive spectrophotometric method for determination of Tris

The paper describes a sensitive spectrophotometric method for qualitative and quantitative determination of Tris, in the 0.1–1.0 μmole range. The procedure is almost identical with that used for histidine. The green-colored reaction product has two absorption peaks with maxima at 422 nm and 640 nm, in contrast to the single peak (540 nm) in the case of histidine. Absorbance and Tris concentration are mutually linear at both wavelengths, but reading conditions are more favorable at 640 nm.     

A rapid spectrophotometric method for the determination of esterase activity

We have developed a spectrophotometric assay method which continuously records esterase activity at 510 nm by monitoring absorbance changes due to the formation of a diazo dye complex. In our method, α-naphthyl ester substrates are hydrolyzed by enzymatic action to α-naphthol which couples to Fast Blue RR salt (a diazonium salt) forming a diazo dye complex. Our method is unique in directly monitoring the formation of the diazo dye complex without extracting the color of the complex as in other methods that use naphthyl esters and diazo coupling of reaction products. The method appears to be limited to α-naphthyl ester substrates, however, since β-naphthyl esters did not give a linear change in absorbance in the enzymatic reactions tested. With this assay method, one can use a single substrate both to determine esterase units quantitatively in solution and to detect esterase staining activity on gel electrophoresis.     

A new spectrophotometric method for the determination of cresolase activity of epidermis tyrosinase.

A sensitive, rapid, quantitative method for the determination of the activities of the bifunctional frog epidermis enzyme, tyrosinase (E.C. 1.14.18.1), has been developed. It is a spectrophotometric method using p-coumaric acid and caffeic acid as substrates at pH 7.0. Lineweaver-Burk plots yielded straight lines and the initial velocities for the reactions were proportional to enzyme concentrations. It is simpler, faster, and more economical than radiometric methods and, furthermore, permits continuous monitoring.     

A spectrophotometric method for the determination of glucose-6-phosphatase activity

A sensitive and rapid spectrophotometric method for determination of glucose-6-phosphatase activity is described. Glucose formed by the enzyme is oxidized by glucose oxidase to gluconolactone and hydrogen peroxide. The latter, phenol, and 4-aminoantipyrine are converted by peroxidase to quinoneimine. The formation of quinoneimine is followed directly on a spectrophotometer at 510 nm. The method described is as sensitive and accurate as conventional assays based on determination of phosphate released, and also works well in phosphate buffers.     

A new enzyme-coupled spectrophotometric method for the determination of arginase activity

A spectrophotometric method for the determination of arginase (EC 3.5.3.1) is presented. Arginase is coupled to urease and glutamate dehydrogenase and the decrease in absorbance at 340 nm due to the oxidation of NADPH is followed. The method is rapid, is sensitive, is economical and permits continuous monitoring. The initial velocities were directly proportional to the enzyme concentrations between 0.06 and 0.30 units per 0.5 ml. The Lineweaver-Burk plot yielded positive allosteric behavior for the tetrameric enzyme. The K' and the Hill coefficient, n, calculated from Hill plot were found to be 4.7 mM and 1.26 (r = 1.00), respectively. These values are in good agreement with the literature.     

A novel spectrophotometric method for determination of kinetic constants of aldehyde oxidase using multivariate calibration method

Although phenanthridine has been frequently used as a specific substrate for the assessment of aldehyde oxidase activity, the use of this method is questionable due to a lower limit of detection and its validity for kinetic studies. In the present study, a novel sensitive multivariate calibration method based on partial least squares (PLS) has been developed for the measurement of aldehyde oxidase activity using phenanthridine as a substrate. Phenanthridine and phenanthridinone binary mixtures were prepared in a dynamic linear range of 0.1–30.0 μM and the absorption spectra of the solutions were recorded in the range of 210–280 nm in Sorenson's phosphate buffer (pH 7.0) containing EDTA (0.1 mM). The optimized PLS calibration model was used to calculate the concentration of each chemical in the prediction set. Hepatic rat aldehyde oxidase was partially purified and the initial oxidation rates of different concentrations of phenanthridine were calculated using the PLS method. The values were used for calculating Michaelis–Menten constants from a Lineweaver–Burk double reciprocal plot of initial velocity against the substrate concentration. The limits of detection for phenanthridine and phenanthridinone were found to be 0.04 ± 0.01 and 0.03 ± 0.01 μM (mean ± SD, n = 5), respectively. Using this method, the K_m value for the oxidation of phenanthridine was calculated as 1.72 ± 0.09 μM (mean ± SD, n = 3). Thus, this study describes a novel spectrophotometric method that provides a suitable, sensitive and easily applicable means of measuring the kinetics of phenanthridine oxidation by aldehyde oxidase without the need for expensive instrumentation.     

A continuous spectrophotometric method for the determination of diphenolase activity of tyrosinase using 3,4-dihydroxymandelic acid.

A continuous spectrophotometric method for the rapid determination of diphenolase activity of tyrosinase is described. It uses 3,4-dihydroxymandelic acid (DOMA) as the substrate of tyrosinase and measures the final product, 3,4-dihydroxybenzaldehyde (DOBA). The spectrum of this product shows a bathochromic displacement of its absorbance maximum when the pH increases. The optimization of the method is described by using tyrosinase from several biological sources, whose enzymatic activities show different optimal pH. Thus, the enzymatic activity of mushroom tyrosinase was assayed at pH 7.5 and monitored at 350 nm (epsilon 350 pH 7.5 (DOBA) = 15,200 M-1 cm-1), whereas the spectrophotometric experiments with grape tyrosinase were carried out at pH 3.0 and monitored at 310 nm (epsilon 310 pH 3.0 (DOBA) = 9200 M-1 cm-1). The method for mushroom tyrosinase was found to be 50-fold more sensitive than the commonly used dopachrome assay, whereas for grape tyrosinase the method was found to be threefold more sensitive than the commonly used o-quinone production assay. The great solubility and stability of the chromophoric product, DOBA, as well as its high molar absorptivities at any pH, enable the method to be employed to determine the diphenolase activity of tyrosinase from different biological sources.     

A spectrophotometric method for determination of bacterial biomass in the presence of a polymer

A simple and rapid spectrophotometric method based on a single optical density measurement was developed to quantify biomass in the presence of a growth - associated exopolymer. It allowed the determination of both cell and polymer concentrations in a cultivation broth without the need for separation of these two elements. Cultivations of Pseudomonas fluorescens NCIMB 11671 in a fermenter were taken as examples to demonstrate the validity of this approach. © Rapid Science Ltd. 1998