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 sphingomyelinase.

A colored derivative of sphingomyelin was synthesized and used as substrate for several sphingomyelinases. The compound is N-omega-trinitrophenyl-aminolaurylsphingosylphosphorylcholine. The rate of hydrolysis of this substrate was compared to that of bovine brain sphingomyelin, labelled with tritium in the choline moiety. The following enzyme preparations were used: homogenate-less debris of brain, assayed at pH 5.0 or 7.4; a solubilized preparation derived from rat brain lysosomes, assayed at pH 5.0 and a purified enzyme of Staphylococcus aureus. With all preparations, the rates of hydrolysis of the yellow derivative were very similar to those of the brain sphingomyelin. Extracts of skin fibroblasts of normal and Niemann-Pick patients as well as amniotic cells were also used. Again, the rates of hydrolysis of the yellow derivative practically equalled those using brain sphingomyelin.     

A coupled spectrophotometric enzyme assay for the determination of pectin methylesterase activity and its inhibition by proteinaceous inhibitors

Pectin methylesterase (PME; EC 3.1.1.11) activities are widespread in bacteria, fungi, and plants. PME-mediated changes in cell wall pectin structure play important roles in plant development. Genome sequencing projects have revealed the existence of large PME multigene families in higher plants. Additional complexity for PME regulation arises from the presence of specific PME inhibitor proteins (PMEI) in plant cells. Several assay procedures for the determination of PME activity have been reported. However, previous protocols suffered from various limitations. Here we report a protocol for a coupled enzyme assay based on methanol oxidation via alcohol oxidase (AO; EC 1.1.3.13) and subsequent oxidation of formaldehyde by formaldehyde dehydrogenase (FDH; EC 1.2.1.3). This simple and robust assay allows the continuous monitoring of PME activity in the neutral pH range. Furthermore, as plant PMEIs do not interfer with AO and FDH activities, this assay is suitable for the characterization of the inhibition kinetics of PMEI.     

Selective labeling of alpha- or epsilon-amino groups in peptides by the Bolton-Hunter reagent.

Incorporation of N-succinimidyl-3(4-hydroxyphenyl) propionate (Bolton-Hunter reagent) or its 125I-labeled derivative into peptides can be selectively directed towards either alpha- or epsilon-amine functions by modifying the pH of the reaction. Acylation of alpha-amino groups is favored at pH 6.5 whereas epsilon-amino groups react more readily at pH 8.5. We have taken advantage of this result to prepare two new 125I-labeled analogues of substance P and neurotensin that bind selectively and reversibly to their respective receptors. The method described here is of general interest and can be used to incorporate various reporter groups into peptide structures.     

A spectrophotometric determination of cyanate using reaction with 2-aminobenzoic acid

A specific method has been devised for the assay of cyanate, based on the reaction with 2-aminobenzoic acid. Cyclization of the product in 6 N HCl results in the formation of 2,4(1H,3H)-quinazolinedione. Cyanate content of the samples can be measured by their absorbances at 310 nm. Alternatively, the second derivatives of the spectra can be recorded; the peak-to-peak height between the first maximum (330 nm) and the first minimum (317 nm) was shown to be proportional to the cyanate content. This method is suitable for the estimation of cyanate in aqueous solutions in the concentration range 0.01 to 2 mM. When added to blood plasma, cyanate could be detected down to 0.1 mM.     

The preparation of magnetic proteinaceous microspheres using the sonochemical method

Using high-intensity ultrasound, we have developed a method for the synthesis of magnetic microspheres. The microspheres are composed of iron oxide-filled and coated globular bovine serum albumin (BSA). The magnetic microspheres are prepared from BSA and iron pentacarbonyl, or from BSA and iron acetate. Transmission electron microscopy and scanning electron microscopy show spherical particles. The particle size distributions are gaussian, with a mean diameter of a few micrometers. Using chemical analysis, it was found that the total percentage of iron oxide in the microspheres is between 39% and 42%. M?ssbauer measurements were also performed.     

A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups

A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986) J. Biochem. Biophys. Methods 12, 349-354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5'-O-(4,4'-dimethoxytrityl)-thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4,4'-dimethoxytrityl cation into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption at 498 nm (epsilon 498 = 70,000), is then determined spectrophotometrically. A comparative study of DTDS, N-succinimidyl-3-(2-pyridyldithio)propionate and 4,4-dimethoxytrityl chloride is also included. The method was found to be very useful to determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionalization of solid supports for affinity chromatography and synthesis of biopolymers.     

Spectrophotometric determination of hydrazine, hydrazides, and their mixtures with trinitrobenzenesulfonic acid

A method has been developed to measure hydrazine, hydrazides, and their mixtures using a modification of the trinitrobenzenesulfonic acid method [T. Okuyama and K. Satake (1960) J. Biochem. (Tokyo) 47, 654-660]. After incubation of the sample containing hydrazine and hydrazide with trinitrobenzenesulfonate at pH 8.5 at room temperature for 40 min, the reaction mixture was diluted with a Na2CO3-NaHCO3 buffer (0.1 M, pH 10.8) rather than with 0.5 M HCl. Different chromogens were produced from the reaction of hydrazine (lambda max = 570 nm) and hydrazides (lambda max = 385 and 500 nm) with trinitrobenzenesulfonic acid. The method allowed simultaneous determination of hydrazine (5 to 60 nmol) with hydrazide (10 to 120 nmol) in a mixture with a standard deviation of less than 5%. The presence of amino compounds (except for amino sugars) did not interfere with the measurement of hydrazine or hydrazides. Interference by amino sugars in the determination of hydrazine or hydrazides was eliminated by pretreatment of the sample with NaBH4 to reduce the amino sugars to 2-amino-2-deoxy-hexitols.     

A spectrophotometric method for the determination of free fatty acid in serum using acyl-coenzyme A synthetase and acyl-coenzyme A oxidase

A mixture of Ti(IV) and 4-(2-pyridylazo)resorcinol was found to be useful in the spectrophotometric determination of trace amounts of hydrogen peroxide. The absorbance at 508 nm was proportional to the concentration of hydrogen peroxide added. The reagent was successfully applied to the assay of free fatty acid in serum through the combined use of acyl-CoA synthetase and acyl-CoA oxidase. The latter enzyme produces H₂O₂. As a result, hydrogen peroxide was produced through the enzymatic oxidation of free fatty acid. It was possible to determine free fatty acid in 50 μl of serum at concentrations ranging from 0.02 to 1.5 mm. The coefficient of variation was less than 3% at concentrations ranging from 0.1 to 1.5 mm. In the present method, there is the advantage of minimal influence from reducible substances as well as greater simplicity and accuracy.