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.     

An improved spectrophotometric assay for superoxide dismutase based on epinephrine autoxidation

Superoxide dismutase activity was assayed in terms of its ability to inhibit the radical-mediated chain-propagating autoxidation of epinephrine. The enzyme assay based on adrenochrome absorption at 480 nm has been improved by measuring the absorption change at 320 nm. This alternative procedure was found to be 6 to 10 times more sensitive and more consistent than that measured at 480 nm.     

Thiolysis of O-2,4-dinitrophenyltyrosines. Spectrophotometric monitoring of the reaction and its use in peptide synthesis.

The thiolytic cleavage of O-2,4-dinitrophenyl (Dnp) derivatives of phenols was applied to the synthesis of tyrosine-containing peptides. This paper describes the preparation and properties of starting materials for such syntheses and illustrates their use in the synthesis of some peptides containing tyrosine at either the C- or N-terminus. A spectrophotometric method for following the thiolytic removal of Dnp groups from O-Dnp-tyrosines was developed and used to establish optimal conditions for quantitative deblockage in aqueous and nonaqueous solvents. The method is based on the fact that upon thiolysis, the colorless solution of O-Dnp-tyrosine (λ_max at 298 nm, pH 8.5) becomes yellow due to the formation of a dinitrophenylated thiol (for S-Dnp-2-mercaptoethanol, λmax at 340 nm, pH 8.5). This gives rise to a difference spectrum with a maximum at 354 nm (Δ?_M = + 8680 M^?1 cm^?1), a minimum at 298 nm (Δ?_M = ?5900 M^?1 cm^?1) and a crossover point at 318 nm, which is different (in the 290–320 nm range) from the difference spectrum obtained upon thiolysis of N^Im-Dnp-histidine. This method provides a useful analytical tool in peptide and polypeptide synthesis as well as in protein chemistry.     

Rapid and direct spectrophotometric method for kinetics studies and routine assay of peroxidase based on aniline diazo substrates

Peroxidases are ubiquitous enzymes that play an important role in living organisms. Current spectrophotometrically based peroxidase assay methods are based on the production of chromophoric substances at the end of the enzymatic reaction. The ambiguity regarding the formation and identity of the final chromophoric product and its possible reactions with other molecules have raised concerns about the accuracy of these methods. This can be of serious concern in inhibition studies. A novel spectrophotometric assay for peroxidase, based on direct measurement of a soluble aniline diazo substrate, is introduced. In addition to the routine assays, this method can be used in comprehensive kinetics studies. 4-[(4-Sulfophenyl)azo]aniline (λ_max?=?390?nm, ??=?32 880 M^?1 cm^?1 at pH 4.5 to 9) was introduced for routine assay of peroxidase. This compound is commercially available and is indexed as a food dye. Using this method, a detection limit of 0.05?nmol mL^?1 was achieved for peroxidase.     

The chromophore of pholasin: A highly luminescent protein

Pholasin is the photoprotein extracted from the marine bivalve Pholas dactylus. It undergoes an oxidative chemiluminescent reaction to oxypholasin with superoxide anion, hypochlorite, peroxidases and other oxidants. Since the observed absorbance and chemiluminescent emission spectra of pholasin solutions cannot be brought about solely by the amino acids composing the protein, there has to be a chemiluminescent chromophore. However, little is known about the chemical nature of this molecule. This work seeks to identify the chemical structure of the luminescent prosthetic group of pholasin. Pholasin could not be reactivated using chromophores from the hydroid Obelia geniculata (coelenterazine) and from the ostracod shrimp Vargula (formerly Cypridina) hilgendorfi. Furthermore, the reaction product of the Vargula chromophore could not be detected in solutions containing oxypholasin. Fluorescence analysis of such a solution revealed a compound with an emission spectrum (γ_max 480 nm; excitation at 320 nm), resembling the emission spectrum of the chemiluminescent reaction. This fluorescent substance was separated by gel filtration. It exhibited an apparent molecular mass of < 2000. Fluorescence masurements of extracts of partially purified pholasin suggested that a flavin moiety may be involved in pholasin luminescence.     

A rapid spectrophotometric assay for ferrochelatase activity in preparations containing much endogenous hemoglobin and its application to soybean root-nodule preparations.

A simple and rapid spectrophotometric assay for ferrochelatase (EC 4.99.1.1) activity is described which is suitable for use with enzyme preparations containing large amounts of hemoglobin. In this method mesoporphyrin IX is used as substrate and the product, mesoheme IX, is measured by recording the reduced minus the oxidized pyridine hemochrome spectrum. Pyridine mesohemochrome has an α peak at 547 nm and a trough at 531 nm while the α peak and trough of pyridine protohemochrome (from hemoglobin) are at 557 and 541 mm, respectively. Thus the contribution of the protohemochrome to E_1cm^547-531nm, which can be allowed for, is small, and so the method gives very reliable determinations of the amounts of mesoheme IX formed.By means of this assay, it was shown that in excess of 90% of the ferrochelatase activity of soybean root-nodules is present in the bacteroid cells and less than 10% in the plant mitochondria: No ferrochelatase activity could be detected in the soluble plant fraction.     

A new colorimetric method for determination of creatine phosphokinase

A new colorimetric method has been developed for the determination of creatine phosphokinase (CPK). This method is based on the reaction of creatine, formed enzymatically from creatine phosphate and ADP, with p-nitrophenylglyoxal (PNPG) under alkaline conditions to produce a colored product which absorbs maximally at 480 nm. At 25 degrees C the reaction was complete after 10 min in 0.1 M sodium pyrophosphate, pH 12, containing 0.15 M sodium ascorbate. The colored product was stable for at least 24 h and obeyed Beer's law in the range 0.005-0.05 mM creatine. The color reaction was used to determine the activity of CPK in serum and tissue extracts. The results obtained by this method agreed well with the results obtained by other available methods for CPK determination. However, the PNPG method was more rapid and more sensitive than other colorimetric methods and required a single chromogenic reagent.     

A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay

In order to determine the activity of O-linked GlcNAc transferase (OGT), a modified coupled enzyme method was proposed. This method was based on the measurement of uridine 5′-(trihydrogen diphosphate) (UDP), a product generated in transglycosylation reaction. In the assay, UDP was coupled to the conversion of phosphoenolpyruvate to pyruvate using pyruvate kinase. Using a commercial pyruvate assay kit, the pyruvate was converted to a red terminal product, which could be photometrically measured at 570 nm or fluorometrically measured at 587 nm (E _m = 535 nm) on a microplate reader. Kinetic study of a truncated recombinant mOGT and quantitative analysis of OGT in two biological samples indicated that this method was practical and competitive for quantitative analysis of OGT.     

A spectrophotometric method for the direct detection and quantitation of nitric oxide, nitrite, and nitrate in cell culture media

A method for the spectrophotometric determination of nitric oxide, nitrite, and nitrate in tissue culture media is presented. The method is based on the nitric oxide-mediated nitrosative modification of sulfanilic acid that reacts with N-(1-naphthyl)ethylenediamine dihydrochloride forming an orange-colored product absorbing at 496 nm. Nitric oxide levels were determined in culture media from this absorbance measurement using chemiluminescence standardization. Extinction coefficients of 5400 and 6600 M(-1) cm(-1) were determined for the nitric oxide product in assay solutions containing 0.1 or 100 mM KPO4 buffer (pH 7.4), respectively, with a limit of detection of 1 microM. Acidification of these reactions (pH 2.4) generated a pink-colored product absorbing at 540 nm allowing for quantitation of total nitric oxide/nitrite levels using extinction coefficients of 38,000 and 36,900 M(-1) cm(-1), for the assay solutions described. The limit of detection of this assay was approximately 300 nM. Using the 100 mM KPO4 buffer system, nitrate levels were determined following reduction to nitrite using a copper-coated cadmium reagent with an extinction coefficient of 29,500 M(-1) cm(-1) and a detection limit of 0.5 microM. The utility of these assays was demonstrated in the standardization of nitric oxide-saturated cell culture media, and the release of nitric oxide by the NONOate compound DEA/NO.     

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 spectrophotometric procedure for determining the activity of various rat tissue oxidases.

A continuous spectrophotometric method suitable for the determination of the activities of several peroxisomal oxidases in rat tissue homogenates is described. The assay involves the continuous spectrophotometric measurement of the reaction product, H₂O₂, by coupling it to the reduction of a chromogen, o-dianisidine, with horseradish peroxidase. Catalase interference was overcome using azide to inhibit its activity and a H₂O₂ standard curve used to quantitate oxidase activity in terms of microkatals per milliliter of enzyme.     

A continuous spectrophotometric assay for determination of the aureusidin synthase activity of tyrosinase

Introduction – Aurones (aureusidin glycosides) are plant flavonoids that provide yellow colour to the flowers of some ornamental plants. In this study we analyse the capacity of tyrosinase to catalyse the synthesis of aureusidin by tyrosinase from the chalcone THC (2′,4′,6′,4–tetrahydroxychalcone). Objective – To develop a simple continuous spectrophotometric assay for the analysis of the spectrophotometric and kinetic characteristics of THC oxidation by tyrosinase. Methodology – THC oxidation was routinely assayed by measuring the increase in absorbance at 415 nm vs. reaction time. Results – According to the mechanism proposed for tyrosinase, the enzymatic reaction involves the o‐hydroxylation of the monophenol THC to the o‐diphenol (PHC, 2′,4′,6′,3,4 – pentahydroxychalcone), which is then oxidised to the corresponding o‐quinone in a second enzymatic step. This product is highly unstable and thus undergoes a series of fast chemical reactions to produce aureusidin. In these experimental conditions, the optimum pH for THC oxidation is 4.5. The progress curves obtained for THC oxidation showed the appearance of a lag period. The following kinetic parameters were also determined: K_m = 0.12 mM, V_m = 13 μM/min, V_m/K_m = 0.11/min. Conclusion – This method has made it possible to analyse the spectrophotometric and kinetic characteristics of THC by tyrosinase. This procedure has the advantages of a short analysis time, straightforward measurement techniques and reproducibility. In addition, it also allows the study of tyrosinase inhibitors, such as tropolone. Copyright © 2009 John Wiley & Sons, Ltd.     

Continuous spectrophotometric assays for beta-glucosidases acting on the plant glucosides L-picein and prunasin.

The neutral pH optimum beta-glucosidases of mammalian liver and almonds are each capable of hydrolyzing a number of plant glucosides, including L-picein (p-hydroxyacetophenone-beta-D-glucoside) and prunasin (D-mandelonitrile-beta-D-glucoside). Taking advantage of the marked differences in the spectra of the substrate/product pairs of L-picein/p-hydroxyacetophenone and prunasin/mandelonitrile, we have devised spectrophotometric assays that permit the continuous monitoring at pH 7.0 of p-hydroxyacetophenone (piceol) release from L-picein by guinea pig hepatic cytosolic beta-glucosidase and mandelonitrile from prunasin by almond beta-glucosidase. When L-picein hydrolysis was monitored at 320 nm and prunasin at 282 nm, the molar absorption coefficients determined for their products, namely piceol and mandelonitrile, were 3200 and 1360 M-1 cm-1, respectively. The kinetic parameter Km and Vmax values obtained using these spectrophotometric procedures for the guinea pig liver cytosolic beta-glucosidase acting on L-picein were 0.88 mM and 5.29 x 10(5) units/mg protein and for the almond beta-glucosidase acting on prunasin, Km 1.1 mM and Vmax 5.24 x 10(6) units/mg protein. These values agreed well with previously reported values obtained using less convenient, discontinuous assay procedures.     

An o-toluidine method for detection of carbohydrates in protein hydrolysates

The o-toluidine high-performance thin-layer chromatography (HPTLC) method for detection of reducing sugars has been demonstrated to be a facile method for composition analysis of protein hydrolysates with a maximum sensitivity range of 50-100 pmol. The solution phase reaction of o-toluidine with reducing sugars has been previously used for spectrophotometric detection of glucose at 480-630 nm. In contrast, the heterogeneous reaction of o-toluidine with reducing sugars resolved by thin-layer chromatography produces chromophoric derivatives which have a broad absorbance at 295 nm. Detection of these chromophoric derivatives is achieved by uv diffuse reflectance scanning densitometry. It is demonstrated that detection limits of less than 10 ng can be achieved by using HPTLC plates and is therefore equal or more sensitive for some sugars than recently reported high-pressure liquid chromatography methods using amperometric or fluorescence detection.     

A novel method for the measurement of elemental selenium produced by bacterial reduction of selenite

The measurement of elemental selenium (Se⁰) is needed to assess the rate and magnitude of bacteria reduction of selenite or selenate. We have developed a spectrophotometric method for the measurement Se⁰ that is rapid and can be employed to measure the quantity of Se⁰ produced by bacterial cultures. This method employs the use of 1 M Na₂S to convert the insoluble elemental selenium to a red-brown solution and with this method there is a direct correlation between concentration of elemental selenium and the absorption at 500 nm. To demonstrate the utility of this assay, we have followed the reduction of selenite to Se⁰ by Moraxella bovis, and by bacterial consortia in soil and water samples.     

Determination of urinary glucuronide conjugates by high-performance liquid chromatography with pre-column fluorescence derivatization

A simple and highly sensitive high-performance liquid chromatographic method for the direct determination of urinary glucuronide conjugates is described. The method is based on the direct derivatization of the glucuronic acid moiety in glucuronide conjugates with 6,7-dimethoxy-1-methyl-2 (1 H)-quinoxalinone-3-propionylcarboxylic acid hydrazide. The derivatization reaction proceeds in aqueous solution in the presence of pyridine and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at 0–37°C. The resulting fluorescent derivatives are separated on a C₁₈ column using methanol—acetonitrile—0.5% triethylamine in water (1:1:2, v/v) as mobile phase, and are detected spectrofluorimetrically at 445 nm with excitation at 367 nm. The detection limits (signal-to-noise RATIO = 3) for the glucuronides are 13–48 fmol for an injection volume of 10 μl (130–480 fmol per 5 μl of human urine). The method was applied to the measurement of etiocholanorone-3-glucuronide and androsterone-3-glucuronide in human urine. The method is simple and rapid without conventional liquid—liquid extraction of the glucuronides from urine.     

A simple spectrophotometric assay for arogenate dehydratase

A simple spectrophotometric assay for arogenate dehydratase, the enzyme that catalyzes the formation of L-phenylalanine from L-arogenate, is presented. The method couples the arogenate dehydratase reaction with that of an aromatic aminotransferase partially purified from Acinetobacter calcoaceticus. In the presence of 2-ketoglutarate, phenylpyruvate formation is measured at 320 nm at basic pH. The method was compared with two other methods already in use in our laboratory for arogenate dehydratase. The new method is simple, quick, fairly sensitive, and especially suitable for the screening of a large number of samples.     

N-Acetyl-L-Tyrosine (NAT) as a Substrate for Mushroom Tyrosinase

N-acetyl tyrosine (NAT) is hydroxylated by mushroom tyrosinase and the N-acetyl dopa formed is oxidized by the enzyme to N-acetyl dopaquinone (λ_max= 390 ± 10 nm). H₂O₂ and NH₂OH each shortened the lag period of NAT hydroxylation by the enzyme. H₂O₂ had an effect on the changes with time in the spectrum of product(s) formed and on the spectrum of the final product(s) obtained when NAT was hydroxylated by mushroom tyrosinase, in a manner suggesting that H₂O₂ converts N-acetyl dopaquinone to a pink-violet product(s) (λ_max= 490 nm), whereas such a product(s) was not formed in the absence of H₂O₂. A pink-violet product(s) (λ_max 490 ± 20 nm) was also formed when NAT was hydroxylated by mushroom tyrosinase in the presence of NH₂OH or para amino benzoic acid (PABA), probably as a result of an interaction between N-acetyl dopaquinone and NH₂OH or PABA forming mono- or di-oximes. Kojic acid (5-hydroxy-2-hydroxymethyl)-4H-pyran-4-one) inhibited effectively the rate of NAT hydroxylation by mushroom tyrosinase in the absence or presence of H₂O₂. When NAT was oxidized by the enzyme in the absence of kojic acid, N-acetyl dopaquinone was formed at once and a shoulder at 490–530 nm appeared later. Under identical conditions but in the presence of kojic acid, a yellow product(s), characterized by a peak at 320±10 nm, was detected, suggesting that N-acetyl dopaquinone oxidizes kojic acid to the yellow product(s). Maltol(3-hydroxy-2-methyl-4H-pyran-4-one), a γ-pyrone derivative structurally related to kojic acid, also inhibited the rate of NAT hydroxylation by mushroom tyrosinase. The addition of maltol at the plateau phase of the reaction resulted in an immediate decline in absorbance at 400 nm, suggesting that maltol conjugates with N-acetyl dopaquinone, yielding a product(s) characterized by a lower extinction coefficient at 400 nm than that of N-acetyl dopaquinone alone. The final brown-red product(s) formed when NAT was hydroxylated by mushroom tyrosinase was bleached in the presence of ascorbic acid or H₂O₂.     

Spectrophotometric and fluorometric assay of superoxide ion using 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole

Two highly sensitive spectrophotometric methods are developed and described for the measurement of superoxide ion radical derived from KO2 as well as O2*- generated either from the xanthine-xanthine oxidase reaction or by the addition of nicotinamide adenine dinucleotide (NADH) to skeletal muscle sarcoplasmic reticulum (SR) vesicles. These methods allow quantification of superoxide ion concentration by monitoring its reaction with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), either by recording absorbance of the final reaction product at a wavelength of 470 nm or by measuring its fluorescence emission intensity at 550 nm using an excitation wavelength of 470 nm. The extinction coefficient of the active product was determined to be 4000 M(-1) cm(-1). A lower limit second-order bimolecular rate constant of 1.5+/-0.3x10(5) M(-1) s(-1) was estimated from kinetic stopped-flow analysis for the reaction between NBD-Cl and KO2. A plot of absorbance versus concentration of superoxide was linear over the range 2 to 200 microM KO2, whereas higher sensitivities were obtained from fluorometric measurements down into sub-micromolar concentrations with a limit of detection of 100 nM KO2. This new spectrophotometric assay showed higher specificity when compared with some other commonly used methods for detection of superoxide (e.g., nitroblue tetrazolium). Results presented showed good experimental agreement with rates obtained for the measurement of superoxide ion when compared with other well-known probes such as acetylated ferri cytochrome c and 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT). A detailed discussion of the advantages and limitations of this new superoxide ion probe is presented.     

Green spectrophotometric and spectrofluorimetric determination of biperiden hydrochloride using erythrosine B sensing probe

Erythrosine B (EB) is a food colorant antiviral xanthene dye that has many applications as a color additive in pharmaceuticals and cosmetics. Its use as a sensor for spectrofluorimetric and spectrophotometric analysis of amine-based pharmaceuticals renders many advantages because of its availability, low cost, rapid labeling, and high sensitivity. Herein, two fast and sensitive spectrofluorimetric and spectrophotometric methods were established for the estimation of the anti-Parkinson drug, biperiden (BIP) hydrochloride (HCl), in its raw material and tablet forms. The proposed methods depended on the interaction between the phenolic group of EB and the tertiary amino group of the studied analyte to form an ion-pair complex at pH 4 using the Britton Robinson buffer. The spectrofluorimetric method is based on the measurement of the quenching power of BIP HCl on the fluorescence intensity of EB at λ_ex/em = 527.0/550.9 nm. This method was rectilinear over the concentration range of 0.1–1.0 μg/mL with a limit of detection (LOD) = 0.017 μg/mL and a limit of quantification (LOQ) = 0.05 μg/mL. Meanwhile, the colorimetric method involved monitoring the absorbance of the formed ion-pair complex at 555 nm, showing a linearity range of 0.4–5.0 μg/mL with LOD = 0.106 μg/mL and LOQ = 0.322 μg/mL. The proposed methods were assessed for the greenness, indicating the greenness of the developed methods.