Direct Spectrophotometric Assay Research Articles

Comparison of the optimized direct spectrophotometric serum prolidase enzyme activity assay method with the currently used spectrophotometric assay methods and liver fibrosis indexes to distinguish the early stages of liver fibrosis in patients with chronic hepatitis B infection.

The aim of this study was to optimize the currently used direct spectrophotometric serum prolidase enzyme activity (SPEA) assay method and compare its diagnostic accuracy with current precipitation and direct spectrophotometric assay methods, AST-to-ALT ratio, age platelet index, AST-to-platelet ratio index, cirrhosis discriminate score, Doha score, FIB-4, FibroQ, fibrosis index, Goteborg University Cirrhosis Index , King's score, and Pohl score for distinguishing Ishak F0 from F1-F3 in patients with chronic hepatitis B (CHB) infection. Liver biopsy results from 112 patients were included in this study. The SPEA values were 529(292-794) U/L, 671(486-927) U/L, and 1077(867-1399) U/L with the precipitation, current, and optimized direct spectrophotometric assay methods, respectively. According to multivariate logistic regression analysis optimized direct spectrophotometric SPEA was the only statistically significant parameter to predict the early stages of liver fibrosis. Optimized direct spectrophotometric SPEA assay method could be used to distinguish early stages of liver fibrosis in patients with CHB infection instead of the currentlyused spectrophotometric SPEA assay methods and other evaluated liver fibrosis indexes.

Insights into the catalytic mechanism of a bacterial hydrolytic dehalogenase that degrades the fungicide chlorothalonil

Chlorothalonil (2,4,5,6-tetrachloroisophtalonitrile; TPN) is one of the most commonly used fungicides in the United States. Given TPN's widespread use, general toxicity, and potential carcinogenicity, its biodegradation has garnered significant attention. Here, we developed a direct spectrophotometric assay for the Zn(II)-dependent, chlorothalonil-hydrolyzing dehalogenase from Pseudomonas sp. CTN-3 (Chd), enabling determination of its metal-binding properties; pH dependence of the kinetic parameters kcat, Km , and kcat/Km ; and solvent isotope effects. We found that a single Zn(II) ion binds a Chd monomer with a Kd of 0.17 μm, consistent with inductively coupled plasma MS data for the as-isolated Chd dimer. We observed that Chd was maximally active toward chlorothalonil in the pH range 7.0-9.0, and fits of these data yielded a pKES1 of 5.4 ± 0.2, a pKES2 of 9.9 ± 0.1 (k'cat = 24 ± 2 s-1), a pKE1 of 5.4 ± 0.3, and a pKE2 of 9.5 ± 0.1 (k'cat/k' m = 220 ± 10 s-1 mm-1). Proton inventory studies indicated that one proton is transferred in the rate-limiting step of the reaction at pD 7.0. Fits of UV-visible stopped-flow data suggested a three-step model and provided apparent rate constants for intermediate formation (i.e. a k'2 of 35.2 ± 0.1 s-1) and product release (i.e. a k'3 of 1.1 ± 0.2 s-1), indicating that product release is the slow step in catalysis. On the basis of these results, along with those previously reported, we propose a mechanism for Chd catalysis.

Time-course of human cholinesterases-catalyzed competing substrate kinetics

Competing substrate kinetic analysis of human butyrylcholinesterase (BChE) and acetylcholinesterase (AChE) from the time-course of enzyme-catalyzed substrate hydrolysis, using spectrophotometric assays is described. This study is based on the use of a chromogenic reporter “visible” substrate (substrate A), whose complete hydrolysis time course is retarded by a competing “invisible” substrate (substrate B). For BChE, four visible substrates were used, two thiocholine esters, benzoylthiocholine and butyrylthiocholine, and two aryl-acylamides, o-nitro trifluoro acetaminide and 3-(acetamido)-N,N,N-trimethylanilinium. Three different competing invisible substrates were used, phenyl acetate, acetylcholine and butyrylcholine. For AChE, two visible substrates were used, acetylthiocholine and 3-(acetamido)-N,N,N-trimethylanilinium. For AChE, acetylcholine was competing with visible substrates.The ratio (R) of bimolecular rate constants, kcat/Km, for all couples of substrates, invisible/visible (B/A) covered all possible limit situations, R ≪ 1, R ≈ 1 and R ≫ 1.The kinetic approach, based on the method developed by Golicnik and Masson allowed determination of binding and catalytic parameters of cholinesterases for both visible and invisible substrates. This analysis was applied to michaelian and non-michaelian catalytic behaviors (activation and inhibition by excess substrate). Reevaluation of catalytic parameters obtained for acetylcholine and butyrylcholine more than 50 years ago was made.The method is fast, reliable, and particularly suitable for poorly soluble substrates and for substrates B when no direct spectrophotometric assays exist. Moreover, replacing substrate B by a reversible inhibitor, mechanism of cholinesterase inhibition was possible to study. It is therefore, useful for screening libraries of new substrates and inhibitors, and/or screening of new cholinesterase mutants. This method can be applied to any other enzymes.

Characterization of dimethyl sulphoxide reductase from brewing yeast

Dimethyl sulphoxide (DMSO) reductase is present in all brewing yeast strains tested, but is more readily detectable using activity stains on polyacrylamide gels than through direct spectrophotometric assay of crude extracts. The enzyme is detectable at higher activities in yeast cultivated at lower temperatures and this is because of increased expression of the primary gene that codes for the enzyme in yeast, MXR1. DMSO somewhat increases enzyme expression, notably in lager yeast by up to 70%, whereas methionine suppresses it. Both ale and lager strains produce enzymes of ~100,000 in molecular weight, but ion exchange chromatography divides the activity for both ale and lager yeasts into two fractions with distinct properties (heat tolerance, pH optimum, kinetic parameters). Copyright © 2017 The Institute of Brewing & Distilling

Effects of genetic polymorphisms of apolipoprotein A1 on serum HDL cholesterol level in postmenopausal Korean women

Apolipoprotein A1 (ApoA1) is the major protein component of high density lipoprotein (HDL) cholesterol in blood, and ApoA1 genetic polymorphisms modulate the blood lipid profiles. This study was conducted in order to investigate the association between three genetic polymorphisms (rs670, rs5069, and rs5070) of ApoA1 and blood lipid profiles in postmenopausal Korean women. A total of 130 postmenopausal women who visited a hospital in order to undergo screening tests were subjects of this study. Genetic polymporphisms and blood lipid profiles were determined using a direct sequencing and spectrophotometric assay, respectively. A significant linkage disequilibrium was observed between all tested single nucleotide polymorphisms. ApoA1 rs5070 genetic polymorphism showed a marginally significant association with HDL cholesterol levels (p=0.066). After adjusting for age, body mass index, smoking, alcohol drinking, medication, hypertension, and diabetes mellitus, we found that the ApoA1 rs5070 genetic polymorphism is a significant determinant of HDL cholesterol levels (β=4.421, p=0.037). According to the results of this study, ApoA1 rs5070 genetic polymorphism may be an important genetic marker associated with HDL cholesterol in postmenopausal Korean women.

Generalized linear model describes determinants of total antioxidant capacity of refined vegetable oils

AbstractThe total antioxidant capacity of edible oils corresponds to the integrated action of antioxidant compounds. The antioxidant capacity may be used as a proxy of the chemical antioxidant activities of components in oils, but this hypothesis has not been tested by multivariate statistical analysis. Tocols, fatty acids, artificial antioxidants, peroxide value, conjugated dienes, and total antioxidant capacity were determined in samples of refined oils of soybean, corn, canola, and sunflower. Generalized linear models and multiple regression models were adjusted to estimate the integrated association between the total antioxidant capacity and chemical components of oils. The generalized linear model of antioxidant capacity better adjusted to the data because the descriptive variable type of oil was a significant predictor. The type of oil, γ‐ and δ‐tocopherols, and peroxide value explained 95% of the variability of antioxidant capacity in the model (p = 0.0006). Besides, γ‐tocopherol was the most important predictor variable for the model considering the p‐value and the estimated relative weight for the model. Therefore, γ‐tocopherol was the major determinant of total antioxidant capacity of refined oils of soybean, corn, canola, and sunflower.Practical applications: Direct spectrophotometric assay of antioxidant activity can be used to determine the antioxidant quality of fresh refined oils. Furthermore, the statistical evaluation of chemical determinants of vegetable oils based on generalized linear models might contribute to our understanding of the antioxidant quality and the stability of oils.

Direct Spectrophotometric Assay for Benzaldehyde Lyase Activity

Benzaldehyde lyase from Pseudomonas fluorescens Biovar I. (BAL, EC 4.1.2.38) is a versatile catalyst for the organic synthesis of chiral α-hydroxy ketones. To allow fast assessment of enzyme activity, a direct spectrophotometric assay is desirable. Here, a new robust and easy-to-handle assay based on UV absorption is presented. The assay developed is based on the ligation of the α-hydroxy ketone (R)-2,2′-furoin from 2-furaldehyde. A robust assay with direct monitoring of the product is facilitated with a convenient concentration working range minimising experimental associated with low concentrations.

Direct Spectrophotometric Assay of Laccase Using Diazo Derivatives of Guaiacol

Laccase (EC 1.10.3.2) is a widespread cuproenzyme able to oxidize various types of phenols and similar aromatic compounds through a one-electron transfer mechanism. The enzyme has already found its way into the market as a biocatalyst. Because of its ability to be paired by electron mediators, the expectation for employing laccases in versatile processes is very high. There are a few spectrophotometric methods for assaying the laccase activity; however, all of them are based on the formation of product(s) resulting from the enzymatic and inevitable succeeding chemical reactions. Use of diazo derivatives of guaiacol (DdG) was developed as a new spectrophotometric method based on substrate depletion allowing direct assessment of enzyme activity has been introduced. This method allows accurate comprehensive kinetic studies of laccases and provides reliable information about the quality of docking of different substrates or one substrate to the active sites of different laccases. Using this method, the kinetic parameters of various DdG carrying different electron donating and withdrawing substituents were used to assay laccase from Neurospora crassa. 2-Methoxy-4-[(4-phenyl)azo]-phenol (K(m) = 93.5 μM and V = 1.98 μM/min) was identified as an appropriate substrate for the accurate and routine spectrophotometric based assay of laccases.

Development and kinetic validation of an assay for the quantitative determination of peroxidase: Application in the detection of activity in crude plant tissues

A simple, rapid, and sensitive direct spectrophotometric assay of peroxidase activity based on its reaction with a chromogenic reagent 2,4-dimethoxyaniline (DMA) in aqueous media of pH 3.7–7.8 is reported. This method is based on the intermolecular coupling of activated DMA in the presence of peroxidase and H2O2 to produce a violet-colored species having λmax 540nm. This method has been used in the assay of H2O2 in the range of 40–330μM. At a H2O2 concentration of 660μM, the peroxidase linearity is established in the range of 0.2022–1.13 and 0.0079–0.756nM by rate and one-time detection method, respectively. Two substrate kinetic ping-pong mechanism is established. A Hanes-Woolf plot is used in the evaluation of Michaelis-Menten constants of H2O2 and dimethoxyaniline. The catalytic efficiency and catalytic power of the proposed method are 2.67×105min−1M−1 and 2.05×10−4min−1, respectively. The apparent k3 at H2O2 concentration ranging between 40 and 1320μM is found to be 469min−1. This method has been applied in the crude plant extracts with medicinal value.

Dual-wavelength β-correction spectrophotometric determination of trace concentrations of cyanide ions based on the nucleophilic addition of cyanide to imine group of the new reagent 4-hydroxy-3-(2-oxoindolin-3-ylideneamino)-2-thioxo-2H-1,3-thiazin-6(3H)-one

A simple, fast, low cost and sensitive direct β-correction spectrophotometric assay of cyanide ions based on its reaction with the reagent 4-hydroxy-3-(2-oxoindolin-3-ylideneamino)-2-thioxo-2H-1,3-thiazin-6(3H)-one, abbreviated as HOTT in aqueous media of pH 7–10 is described. The electronic spectrum of the produced brown-red colored species showed well defined and sharp peak at λ max = 466 nm. The effective molar absorptivity for the produced cyano compound was 2.5 × 10 4 L mol −1 cm −1. Beer's law and Ringbom's plots were obeyed in the concentration range 0.05–2.0 and 0.30–1.5 μg mL −1 cyanide ions, respectively. The proposed method offers 16.0 and 50.3 μg L −1 lower limits of detection (LOD) and quantification (LOQ) of the cyanide ion, respectively. The analytical utility of the method for the analysis of cyanide ions in tap and drinking water samples was demonstrated and the results were compared successfully with the conventional cyanide ion selective electrode. The short time response and the detection by the naked eye make the method available for the detection and quantitative determination of cyanide in a variety of samples e.g. fresh and drinking water. Moreover, the structure of the produced colored species was determined with the aid of spectroscopic measurements (UV–Vis, IR, 1H and 13C NMR) and elemental analysis.

A direct spectrophotometric γ-glutamyltransferase inhibitor screening assay targeting the hydrolysis-only mode

γ-Glutamyltransferase (GGT, E.C. 2.3.2.2) catalyzes the hydrolysis and transpeptidation of extracellular glutathione. Due to its central role in maintaining mammalian glutathione homeostasis, GGT is now believed to be a valuable drug target for a variety of life-threatening diseases, such as cancer. Unfortunately, however, effective tools for screening GGT inhibitors are still lacking. We report here the synthesis and evaluation of an α-phenylthio-containing glutathione peptide mimic that eliminates thiophenol upon GGT-catalyzed hydrolysis of the γ-glutamyl peptide bond. The concurrent, real-time spectrophotometric quantification of the released thiophenol using Ellman’s reagent creates a GGT assay format that is simple, robust, and highly sensitive. The versatility of the assay has been demonstrated by its application to the kinetic characterization of equine kidney GGT, and enzyme inhibition assays. The ability of the glutathione mimic to behave as an excellent donor substrate (exhibiting Michaelis–Menten kinetics with a K m of 11.3 ± 0.5 μM and a k cat of 90.1 ± 0.8 nmol mg −1 min −1), coupled to the assay’s ability to study the hydrolysis-only mode of the GGT-catalyzed reaction, make our approach amenable to high-throughput drug screening platforms.

Detection ofDNA damage: effect of thymidine glycol residues on the thermodynamic, substrate and interfacial acoustic properties of oligonucleotide duplexes

Thymidine glycol residues in DNA are biologically active oxidative molecular damage sites caused by ionizing radiation and other factors. One or two thymidine glycol residues were incorporated in 19- to 31-mer DNA fragments during automatic oligonucleotide synthesis. These oligonucleotide models were used to estimate the effect of oxidized thymidines on the thermodynamic, substrate and interfacial acoustic properties of DNA. UV-monitoring melting data revealed that modified residues in place of thymidines destabilize the DNA double helix by 8-22 degrees C, depending on the number of lesions, the length of oligonucleotide duplexes and their GC-content. The diminished hybridizing capacity of modified oligonucleotides is presumably due to the loss of aromaticity and elevated hydrophilicity of thymine glycol in comparison to the thymine base. According to circular dichroism (CD) data, the modified DNA duplexes retain B-form geometry, and the thymidine glycol residue introduces only local perturbations limited to the lesion site. The rate of DNA hydrolysis by restriction endonucleases R.MvaI, R.Bst2UI, R.MspR9I and R.Bme1390I is significantly decreased as the thymidine glycol is located in the central position of the double-stranded recognition sequences 5'-CC / WGG-3' (W = A, T) or 5'-CC / NGG-3' (N = A, T, G, C) adjacent to the cleavage site. On the other hand, the catalytic properties of enzymes R.Psp6I and R.BstSCI recognizing the similar sequence are not changed dramatically, since their cleavage site is separated from the point of modification by several base-pairs. Data obtained by gel-electrophoretic analysis of radioactive DNA substrates were confirmed by direct spectrophotometric assay developed by the authors. The effect of thymidine glycol was also observed on DNA hybridization at the surface of a thickness-shear mode acoustic wave device. A 1.9-fold decrease in the rate of duplex formation was noted for oligonucleotides carrying one or two thymidine glycol residues in relation to the unmodified analog.

A new colorimetric assay for methionyl aminopeptidases: Examination of the binding of a new class of pseudopeptide analog inhibitors

A direct and convenient spectrophotometric assay has been developed for methionine aminopeptidases (MetAPs). The method employs the hydrolysis of a substrate that is a methionyl analogue of p-nitroaniline ( l-Met- p-NA), which releases the chromogenic product p-nitroaniline. This chromogenic product can be monitored continuously using a UV–Vis spectrophotometer set at 405 nm. The assay was tested with the type I MetAP from Escherichia coli ( EcMetAP-I) and the type II MetAP from Pyrococcus furiosus ( PfMetAP-II). Using l-Met- p-NA, the kinetic constants k cat and K m were determined for EcMetAP-I and PfMetAP-II and were compared with those obtained with a “standard” high-performance liquid chromatography (HPLC) discontinuous assay. The assay has also been used to determine the temperature dependence of the kinetic constant k cat for PfMetAP-II as well as to screen two novel pseudopeptide inhibitors of MetAPs. The results demonstrate that l-Met- p-NA provides a fast, convenient, and effective substrate for both type I and type II MetAPs and that this substrate can be used to quickly screen inhibitors of MetAPs.

Study of a toxin–alkaline phosphatase conjugate for the development of an immunosensor for tetrodotoxin determination

This paper describes a direct competitive immunoenzymatic spectrophotometric assay (ELISA) for tetrodotoxin (TTX) determination and the adaptation of this method for use in an electrochemical assay format. The novelty of this work involves the use of the antigen labelled with alkaline phosphatase (AP); this conjugate was prepared in our laboratory as there is no commercially available conjugate of any kind for TTX. The new conjugate was characterized in terms of its affinity for the specific antibody as well as the residual concentration and the residual activity of the enzyme (AP) incorporated as label. The proposed method based on the new conjugate showed satisfactory results for TTX determination: for the spectrophotometric method the dynamic range was 4-15 ng mL(-1) with a limit of detection (LOD) of 2 ng mL(-1) (R=0.9247), whereas for the electrochemical protocol the dynamic range was 2-50 ng mL(-1) and the LOD was 1 ng mL(-1).

Can the DCoHα isozyme compensate in patients with 4a-hydroxy-tetrahydrobiopterin dehydratase/DCoH deficiency?

4a-Hydroxy-tetrahydrobiopterin dehydratase/DCoH is a bifunctional protein. In the cytoplasm it is an enzyme required for the regeneration of tetrahydrobiopterin, an essential cofactor for phenylalanine hydroxylase. In the nucleus it functions as a transcriptional coactivator by forming a 2:2 heterotetramer with the hepatic nuclear factor HNF1α (HNF1). Patients with a deficiency of dehydratase activity have elevated levels of phenylalanine, and accumulate 7-pterins due to degradation of its substrate 4a-hydroxy-tetrahydrobiopterin. Curiously, the hyperphenylalaninemia is transient, and no defects in the transcriptional coactivator function have been reported. Recently, a human isozyme, dehydratase/DCoHα, has been detected which shares 60% identity with dehydratase/DCoH. This investigation was undertaken to ascertain if dehydratase/DCoHα has the pre-requisite properties to compensate in individuals lacking an active form of DCoH. DCoHα demonstrated the ability to quantitatively alter HNF1-dependent DNA-binding in vitro whereas DCoH was ineffective in vitro. This characteristic, due to the presence of dimeric DCoHα, demonstrates that DCoHα does not require any additional mammalian regulation process to alter DNA binding and therefore, may be more effective than DCoH at low concentrations. The dehydratase activity of each isoform was measured by a direct spectrophotometric assay. K m and V max for DCoHα were both 2–3 times higher than for DCoH, thus leaving the catalytic efficiency ( V max/ K m) the same for both enzymes. In conclusion, the properties of dehydratase/DCoHα are consistent with the hypothesis that the activity of this isozyme could account for the relatively mild symptoms reported for patients with a defect in dehydratase/DCoH.

Optimization and validation of a dissolution test for famotidine tablets using flow injection analysis

A dissolution test for famotidine tablets was optimized and validated using flow injection analysis (FIA). The effect of dissolution parameters such as pH, medium and stirring speed was studied, while the ruggedness of the procedure was validated. All measurements were performed using a simple direct spectrophotometric flow injection assay ( λ max = 265 nm) that has also been optimized and fully validated in terms of linearity, limit of detection, precision, selectivity and accuracy. Linearity was obeyed in the range 50–150% of famotidine (20–60 mg L −1), while the detection limit (0.1 mg L −1) and repeatability ( s r < 1.0%, n = 12) were satisfactory. The sampling rate was 30 h −1. The dissolution results during quality and stability control of two batches of famotidine tablets obtained by the flow injection method were in good agreement with high-performance liquid chromatography (HPLC).

Flow injection direct spectrophotometric assay for the speciation of trace chromium(III) and chromium(VI) using chromotropic acid as chromogenic reagent

A new rapid and sensitive FI assay is reported for the simultaneous direct spectrophotometric determination of trace Cr(VI) and Cr(III) in real samples. The method is based upon the reaction of Cr(VI) with chromotropic acid (CA) in highly acidic medium to form a water-soluble complex ( λ max = 370 nm). Cr(III) reacts with CA only after its on-line oxidation to Cr(VI) by alkaline KIO 4. The determination of each chromium species in the sample was achieved by absorbance differences. The calibration curves were linear over the range 3–4000 μg l −1 and 30–1200 μg l −1 for Cr(VI) and Cr(III), respectively, while the precision close to the quantitation limit was satisfactory in both cases ( s r = 3.0% for Cr(VI) and 4.0% for Cr(III) ( n = 10) at 10 and 50 μg l −1 level, respectively). The method developed proved to be adequately selective and sensitive ( c L = 1 and 10 μg l −1 for Cr(VI) and Cr(III), respectively). The application of the method to the analysis of water samples (tap and mineral water) gave accurate results based on recovery studies (93–106%). Analytical results of real sample analysis were in good agreement with certified values.

New chromogenic dipeptide substrate for continuous assay of the D-alanyl-D-alanine dipeptidase VanX required for high-level vancomycin resistance.

A direct continuous UV-Vis spectrophotometric assay has been developed for VanX, a D-alanyl-D-alanine aminodipeptidase necessary for vancomycin resistance. This method is based on the hydrolysis of the alternative substrate D-alanyl-alpha-(R)-phenylthio-glycine D-Ala-D-Gly(S-Ph)-OH (H-DAla-DPsg-OH, 5a). Spontaneous decomposition of the released phenylthioglycine generates thiophenol, which is quantified using Ellman's reagent. The dipeptide behaved as an excellent substrate of VanX, exhibiting Michaelis-Menten kinetics with a kcat of 76 +/- 5/s and a KM of 0.83 +/- 0.08 mm (kcat = 46 +/- 3/s and KM = 0.11 +/- 0.01 mm for D-Ala-D-Ala). Determination of the kinetic parameters of the previously reported mechanism-based inhibitor D-Ala-D-Gly(SPhip-CHF2)-OH (H-D-Ala-DPfg-OH, 5c) [Araoz, R., Anhalt, E., René, L., Badet-Denisot, M.-A., Courvalin, P. & Badet, B. (2000) Biochemistry 39, 15971-15979] using the substrate reported in the present study yielded values of Kirr of 22 +/- 1 microM and kinact of 9.3 +/- 0.4/min in good agreement with values previously obtained in our laboratory (Kirr = 30 +/- 1 mm; kinact = 7.3 +/- 0.3/min). In addition, inhibition by the competing substrate D-Ala-D-Ala resulted in determination of a Ki = 70 +/- 6 microM close to the previously reported KM value. These results demonstrate that the present assay is a convenient, rapid and sensitive tool in the search for VanX inhibitors.

Development of a solid-phase assay for measurement of sulfated glycosaminoglycan concentrations in equine synovial fluid.

To develop a new 1,9-dimethylmethylene blue (DMMB) assay for measurement of sulfated glycosaminoglycan (sGAG) concentrations in equine synovial fluid (SF) by use of membrane technology and to compare the assay's ability to measure sGAG concentrations with that of 2 other established DMMB assays. 25 samples of SF collected from affected joints of 14 horses and 13 samples of SF collected from nonaffected (control) joints of 4 horses. A solid-phase DMMB assay was developed to measure sGAG concentrations in SE Results for the assay were then compared with results obtained by use of the direct spectrophotometric method (ie, Famdale method) and microplate DMMB assay. The solid-phase assay and direct spectrophotometric assay measured the same sGAG concentrations in identical equine SF, but those concentrations differed significantly from results obtained by use of the microplate DMMB assay. All other aspects of the solid-phase DMMB assay were comparable to both the direct spectrophotometric and microplate DMMB assays. The new solid-phase assay can be used interchangeably with the direct spectrophotometric method to measure sGAG concentrations in equine SF samples, but it cannot be interchanged with the microplate DMMB assay. Results can be rapidly obtained with the solid-phase assay. Also, the solid-phase assay can detect nanogram quantities of sGAGs in SF, circumvent the problem of premature precipitation of sGAG-dye complexes, and provide quantitative or qualitative results. The solid-phase assay may replace other DMMB assays for measuring sGAG concentrations in SF obtained from horses.

Direct spectrophotometric assay of monooxygenase and oxidase activities of mushroom tyrosinase in the presence of synthetic and natural substrates

Alternative substrates were synthesized to allow direct and continuous spectrophotometric assay of both monooxygenase (cresolase) and oxidase (catecholase) activities of mushroom tyrosinase (MT). Using diazo derivatives of phenol, 4-[(4-methoxybenzo)azo]-phenol, 4-[(4-methylphenyl)azo]-phenol, 4-(phenylazo)-phenol, and 4-[(4-hydroxyphenyl)azo]-benzenesulfonamide, and diazo derivatives of catechol 4-[(4-methylbenzo)azo]-1,2-benzenediol, 4-(phenylazo)-1,2-benzenediol, and 4-[(4-sulfonamido)azo]-1,2 benzenediol (SACat), as substrates allows measurement of the rates of the corresponding enzymatic reactions through recording of the depletion rates of substrates at their λ max(s) with the least interference of the intermediates’ or products’ absorption. Parallel attempts using natural compounds, p-coumaric acid and caffeic acid, as substrates for assaying both activities of MT were comparable approaches. Based on the ensuing data, the electronic effect of the substituent on the substrate activity and the affinity of the enzyme for the substrate are reviewed. Kinetic parameters extracted from the corresponding Lineweaver–Burk plots and advantages of these substrates over the previously used substrates in similar assays of tyrosinases are also presented.