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.     

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.     

Synthesis of chromophoric, spin label enzyme substrates useful for cryoenzymology.

The spin label nitroxide derivative 3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oic acid has been synthesized and characterized by chemical methods. It is a useful intermediate in the preparation of a new class of chromophoric spin label substrates for enzyme studies, as shown by the synthesis of O-3-(2,2,5,5-tetramethylpyrroline-1-oxyl)-propen-2-oyl-L-beta-phenyllactic acid, a specific ester substrate of bovine pancreatic carboxypeptidase A (peptidyl-L-amino acid hydrolase; EC 3.4.12.2). Kinetic parameters of the esterolytic reaction are conveniently determined by UV spectrophotometric methods, and a reaction intermediate can be stabilized in fluid cryosolvent mixtures at subzero temperatures. Results are presented of preliminary electron spin resonance studies to demonstrate that structural relationships of the spin label substrate in a catalytically active configuration to active site residues can be determined for this low temperature-stabilized reaction intermediate. This substrate thus demonstrates the utility of this new class of spin label derivatives for characterization of enzyme reaction intermediates stabilized by cryoenzymologic techniques.     

A new spectrophotometric method of assay for chitosanase based on calcofluor white dye binding

A new spectrophotometric method for the assay of chitosanase based on complex formation of the substrate chitosan with Calcofluor white dye is described. The absorption maximum for the chitosan-Calcofluor complex is determined to be 406 nm. The apparent minimum size of chitosan for complex formation is 5–7 kDa. Therefore, those enzymes that do not generate glucosamine or reducing groups as products of hydrolysis at levels not measurable by the available methods of assay can be assayed by the present method. In the standardized procedure 200 μg of chitosan in acetate buffer pH 4.5 with the enzyme in a reaction volume of 1.5ml is incubated at 45°C for 1 h, after which 1.5 ml of Calcofluor white (0.05%) is added, kept for 1h and absorbance at 406 nm measured by a spectrophotometer. The chitosanase unit is arbitrarily defined as the reduction in absorbance by 0.01/min.     

Comparison of methods for following alkaline phosphatase catalysis: spectrophotometric versus amperometric detection

An amperometric method for alkaline phosphatase is described and compared to the most widely used spectrophotometric method. Catalytic hydrogenation of 4-nitrophenylphosphate (the substrate in the spectrophotometric method) gives 4-aminophenylphosphate (the substrate in the amperometric method). The latter substrate has the formula C6H6NO4PNa2.5H2O and a Mr of 323. The Michaelis constant for 4-aminophenylphosphate in 0.10 M, pH 9.0. Tris buffer is 56 microM, while it is 82 microM for 4-nitrophenyl phosphate. The amperometric method has a detection limit of 7 nM for the product of the enzyme reaction, which is almost 20 times better than the spectrophotometric method. Similarly, with a 15-min reaction at room temperature and in a reaction volume of 1.1 ml, 0.05 microgram/l alkaline phosphatase can be detected by electrochemistry, almost an order of magnitude better than by absorption spectrophotometry. Amperometric detection is ideally suited for small-volume and trace immunoassay.     

The use of diaminobenzidine for spectrophotometric and acrylamide gel detection of sulfite oxidase and its applicability to hydrogen peroxide-generating enzymes

DAB, in the presence of HRP, sulfite oxidase and FBS, polymerizes to a product with an absorption difference maximum at 352 nm. This reaction has been used as a sensitive and specific spectrophotometric assay for sulfite oxidase. Incubation of acrylamide gels containing sulfite oxidase with assay mixtures produces an insoluble product which precipitates where the enzyme is localized. This stain is at least as sensitive as the amido black protein stain and its specificity is such that no band is seen in the absence of substrate, and that only one band, migrating identically to purified enzyme is seen in rat liver homogenates. This polymerization reaction has been applied to other H₂O₂-generating enzymes and can be used to demonstrate the presence of these enzymes in the midst of other oxidases. DAB polymerization provides a sensitive spectrophotometric assay which can be used with either ultraviolet or visible optics. The application of this procedure to modified enzymes is discussed.     

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.     

Characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors

A novel spectrophotometric method to study the kinetics of the guanine nucleotide exchange factors-catalyzed reactions is presented. The method incorporates two coupling enzyme systems: (a). GTPase-activating protein which stimulates the intrinsic GTP hydrolysis reaction of small GTPases and (b). purine nucleotide phosphorylase and its chromophoric substrate, 7-methyl-6-thioguanosine, for quantitation of the resultant inorganic phosphate. The continuous coupled enzyme system was used for characterization of the interactions between the small GTPase RhoA and its guanine nucleotide exchange factors, Lbc and Dbl. Kinetic parameters obtained here show that there is no significant difference in kinetic mechanism of these GEFs in interaction with RhoA. The Michaelis-Menten constants were determined to be around 1micro M, and the rate constants k(cat) were around 0.1s(-1).     

Continuous spectrophotometric assays for dopamine beta-monooxygenase based on two novel electron donors: N,N-dimethyl-1,4-phenylenediamine and 2-aminoascorbic acid.

Based on the novel chromophoric electron donors, N,N-dimethyl-1,4-phenylenediamine (DMPD) and 2-amino-2-deoxy-L-ascorbic acid (2-aminoascorbic acid), two sensitive, convenient, and continuous spectrophotometric assays for dopamine beta-monooxygenase (EC 1.14.17.1) are described. Both, DMPD and 2-aminoascorbic acid are kinetically and stoichiometrically well-behaved electron donors for dopamine beta-monooxygenase with kinetic parameters comparable to the most efficient physiological electron donor, ascorbic acid. During dopamine beta-monooxygenase turnover, DMPD is converted to its chromophoric cation radical which is stable under the standard assay conditions. The rate of the enzyme-dependent formation of DMPD cation radical under standard assay conditions could easily be followed at 515 nm with high accuracy and reproducibility. Similarly, dopamine beta-monooxygenase-mediated oxidation of 2-aminoascorbic acid results in the formation of the known, stable chromophoric product, 2,2'-nitrilodi-2(2')-deoxy-L-ascorbic acid (red pigment), which has a very strong absorption maximum at 385 nm. Both the above assays are superior to the existing assays in their convenience, reproducibility, and sensitivity for routine kinetic analysis of dopamine beta-monooxygenase and may be adopted as a simple color test for the enzyme. We propose that the above assays could also be adopted to design continuous and sensitive spectrophotometric assays for ascorbate oxidase, peptidyl alpha-amidating monooxygenase, and the chromaffin granule electron transport protein, cytochrome b561, due to their remarkable similarity to dopamine beta-monooxygenase in the chemistry of catalysis with regard to the electron donor.     

Binding of m-nitrobenzeneboronic acid to the active site of subtilisin BPN.

m-Nitrobenzeneboronic acid as a possible transition-state analog for serine proteases was found to cause absorption spectral change from 250 nm 350 nm upon binding with subtilisin BPN' (EC 3.4.21.14) at pH 6.5. Similar difference spectral changes of m-nitrobenzeneboronic acid were also observed at alkaline pH or upon addition of N-methylimidazole at pH 6.5. A characteristic circular dichroism spectrum of m-nitrobenezeneboronic acid was induced upon binding with subtilisin BPN' not only at pH 6.5, but also at alkaline pH. Circular dichroism spectral titration confirmed the stoichiometry of 1 : 1 for the m-nitrobenzeneboronic acid - subtilisin complex. m-Nitrobenzeneboronic acid was shown to be useful as a reversible chromophoric probe for the catalytic site of serine proteases.     

Metmyoglobin Oxidation during Electron Transport Reactions in Mitochondria

Studies of the intracellular role of myoglobin were carried out by recording spectrophotometric changes in acid metmyoglobin and oxymyoglobin during electron transport reactions with mitochondria prepared from pigeon heart muscle by the method of Chance and Hagihara. The absorption peak of metmyoglobin at 409 mµ disappeared when substrate was added to normal or antimycin-inhibited preparations, and was replaced by a new maximum at 423 to 424 mµ, identified as due to the oxidation to ferrylmyoglobin. Further investigation revealed that the oxidation of metmyoglobin took place with the simultaneous oxidation of reduced flavoprotein. Hydrogen peroxide, formed by the reaction of reduced flavoprotein with oxygen, was considered to be the probable intermediate for the oxidation of metmyoglobin in experiments in which catalase was added as a competitor for the oxidant. When DPNH was added to the reaction mixture, the reductant acted to resynthesize the ferri-derivative by reaction with ferrylmyoglobin. Oxymyoglobin could not be used in place of metmyoglobin in these systems. Under the experimental conditions, oxymyoglobin dissociated when dissolved oxygen was depleted from the medium by enzyme oxidations; the resultant ferromyoglobin underwent oxidation to metmyoglobin.     

Mechanistic investigation of medium-chain fatty acyl-CoA dehydrogenase utilizing 3-indolepropionyl/acryloyl-CoA as chromophoric substrate analogues.

The CoA derivative 3-indolepropionyl-CoA (IPCoA) serves as a competent pseudosubstrate for the medium-chain fatty acyl-CoA dehydrogenase (MCAD)-catalyzed reaction. The reaction product trans-3-indoleacryloyl-CoA (IACoA) exhibits a characteristic UV-vis absorption spectrum with lambda max = 367 nm and epsilon 367 = 26,500 M-1 cm-1. The chromophoric nature of IACoA allows us to measure the direct conversion of substrate to product (at 367 nm) without recourse to absorption signals for either the enzyme-bound flavin or the coupling electron acceptors, as well as probe the enzyme site environment. The interaction of IACoA with medium chain fatty acyl-CoA dehydrogenase (MCAD)-FAD is characterized by resultant (spectra of the mixture minus the individual components) absorption peaks at 490, 417, and 355 nm. These absorption peaks increase in magnitude as the pH of the buffer media decreases. Transient kinetic analysis for the interaction of MCAD-FAD with IACoA suggests that the formation of the enzyme-IACoA complex proceeds in two steps. The first (fast) step involves the formation of an E-IACoA collision complex, which [formula: see text] is isomerized (concomitant with changes in the protein structure) to an E*-IACoA complex in the second (slow) step. We have studied the effect of pH on Kc, k2, and k-2. While Kc shows practically no dependence on pH (within a 2-fold variation between pH 6.0 and 9.5), k2 and k-2 show a strong dependence on pH. Both k2 and k-2 exhibit a sigmoidal dependence on the pH of the buffer media, with pKa's of 7.53 and 8.30, respectively. In accordance with the model presented herein, the pKa of 7.53 represents an enzyme site group which is involved in the interaction with IACoA within the E-IACoA collision complex. This pKa is perturbed to 8.30 upon isomerization of the collision complex. The pH-dependent changes in k2 and k-2 are such that the equilibrium distribution between E-IACoA and E*-IACoA is favored to the latter complex (by about 20-fold) at lower pH than at higher pH. A cumulative account of the spectral, kinetic, and thermodynamic properties of the enzyme-IACoA complexes has allowed us delineate the microscopic pathway by which the E-IACoA isomerization (presumably via protein conformational changes) is coupled to the proton equilibration steps.     

A chromogenic oxidative coupling reaction of laccase: applications for laccase and angiotensin I converting enzyme assay

A sensitive chromogenic assay for p-hydroxybenzoic acid, laccase activity, and angiotensin I converting enzyme activity is described. The method relies on the oxidative coupling of 2,2'-azino-di(3-ethylbenzothiazoline-6-sulfonic acid) and p-hydroxybenzoic acid. Lacase catalyzes the formation of a deep-purple compound, which shows a broad absorption between 530 and 630 nm with a maximum at 593 nm (the molar absorption coefficient was calculated to be 26,900). By means of this chromogenic coupling reaction, a spectrophotometric method for the assay of laccase activity and estimation of the amount of p-hydroxybenzoic acid was developed; laccase activity in the range 1-10 pmol protein could be estimated with a 10-min incubation time. Angiotensin I converting enzyme was also assayed by the laccase-catalyzed indicator reaction, using p-hydroxybenzoyl-glycyl-histidyl-leucine as the substrate, and N alpha-carbobenzoxy amino acid urethane hydrolase as the coupling enzyme.     

Synthesis of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside and its use as a substrate to assess feruloyl esterase activity

A synthetic scheme was developed for the production of methyl 5-O-trans-feruloyl-alpha-L-arabinofuranoside (FA-Ara) in gram quantities. This molecule accurately models the chemical attachment of ferulic acid to polysaccharides found in cell walls of plants in the Gramineae family. It is therefore a realistic substrate that can be used to monitor feruloyl esterase activity. Ultraviolet spectral analysis indicated that FA-Ara has an absorption maximum distinct from the hydrolytic product, ferulic acid (FA), over a wide range of solution pH values. The log molar extinction coefficient ranges from 4.16 to 4.36 for FA-Ara and 4.16 to 4.33 for FA depending upon the pH of the buffered solution. Consequently a convenient spectrophotometric assay can be utilized to monitor esterase activity. Three different methods were developed for using this model substrate to assess esterase activity, including thin-layer chromatography, a spectrophotometric assay, and the use of high-performance liquid chromatography.     

The use of hemoglobin for determination of phospholipase A2 activity

A spectrophotometric method is proposed for determining phospholipase A2 activity, which is based on the conversion of hemoglobin into hemichrome under the fatty acid action. The spectral difference between hemoglobin and hemichrome was registered by the difference spectrum with a minimum at 405 nm and a maximum at 423 nm. The absorption value determined as the difference between the spectrum maximum and minimum was proportional to the amount of the fatty acid derived from hydrolysis of phospholipids. The method enables the enzyme activity to be determined directly in the spectrophotometric cell.     

Phosphotyrosine as a substrate of acid and alkaline phosphatases

A new spectrophotometric method for following dephosphorylation of phosphotyrosine has been described. The absorption spectra of phosphotyrosine and tyrosine were plotted over the pH range from 3 to 9. The change in absorbance accompanying the conversion of phosphotyrosine to tyrosine was the greatest at 286 nm. The difference absorption coefficients were calculated for several pH values. Dephosphorylation of phosphotyrosine by acid phosphatases from human prostate gland, from wheat germ and potatoes obeys the Michaelis-Menten equation, whereas alkaline phosphatases calf intestine and E. coli are inhibited by excess of substrate.     

A direct continuous spectrophotometric assay for glycosidases with 3-nitro-2-pyridyl glycosides by tautomerization of 2-hydroxy-3-nitropyridine

Two kinds of 3-nitro-2-pyridyl glycosides were synthesized and evaluated as substrates for continuous spectrophotometric assay for glycosidases. The liberated aglycon, 2-hydroxy-3-nitropyridine, immediately tautomerized to 3-nitro-2(1H)-pyridone, causing an absorption shift of ca. 60 nm even under acidic conditions (pH 3-6). Consequently, the enzymatic hydrolysis of these glycosides was monitored continuously in the acidic to neutral pH range (pH 4-7), the optimum pH for most glycosidases. The absorbance of liberated aglycon increased linearly at 390 nm until 10% consumption of the substrate to enable the initial rate to be determined at once without terminating the reaction. The kinetic parameters for the hydrolysis of 3-nitro-2-pyridyl glycosides were obtained from the slopes of the progress curves and were compared with those obtained from the conventional discontinuous assay using p- and o-nitrophenyl glycosides as substrates. The kinetic parameters indicated that 3-nitro-2-pyridyl glycosides were more activated and specific substrates, but with less affinity to the enzymes than the corresponding nitrophenyl glycosides. Moreover, the absorbance shift by tautomerization should promise further applications to continuous spectrophotometric assays for other enzymes acting under acidic conditions, such as acid proteases and acid phosphatases.     

A simple device for automated spectrophotometric kinetics using a diode array spectrophotometer

In this report we describe an automated system that rapidly and automatically mixes reagents and records results, such as spectrophotometric changes. It employs a commercial diode array spectrophotometer and a novel dilution chamber in a flow stream that allows repetitive spectrophotometric rate measurements at accurately measured incremental substrate concentrations. When applied to enzyme kinetic studies, initial velocities at 15 different substrate or inhibitor concentrations, or pH values, can be recorded in a few minutes with high reproducibility, i.e., standard deviations less than 1%, and high sensitivity. Reactions occur in an 8-microliters flow cell and the reagent consumption is minimal. The concentration of incrementally diluted reagent in the cell is measured directly by means of an indicator dye added to the substrate. Michaelis-Menten parameters, inhibition constants, and pH profiles are determined for several enzymes including dehydrogenases producing NADH, a kinase requiring a coupled assay, and a hydrolase, carboxypeptidase A, in a reaction that produces a small decrease in absorbance.     

Active site-specifically reconstituted nickel(II) horse liver alcohol dehydrogenase: Optical spectra of binary and ternary complexes with coenzymes,coenzyme analogues,substrates, and inhibitors

Insertion of nickel ions into the empty catalytic site of horse liver alcohol dehydrogenase yields an active enzyme with 65% metal substitution and about 12% intrinsic activity. The electronic absorption spectrum is characterized by bands at 357 nm (2900 M^?1 cm^?1, 407 nm (3500 M^?1 cm^?1), 505 nm (300 M^?1 cm^?1), 570 nm (?130 M^?1 cm^?1), and 680 nm (?80 M^?1 cm^?1). The absorption and CD spectra are similar to those of nickel(II) azurin and nickel(II) aspartate transcarbamoylase and prove coordination of the nickel(II) ions to sulfur in a distorted tetrahedral coordination geometry. Changes of the spectra upon ligand binding at the metal or conformation changes of the protein induced by coenzyme, or both, indicate alterations of the metal geometry.The chromophoric substrate trans-4-(N, N-dimethylamino)-cinnamaldehyde forms a ternary complex with Ni(II) liver alcohol dehydrogenase and the coenzyme analogue 1,4,5,6-tetrahydronicotinamide-adenine-dinucleotide, stable between pH 6 and 10. The corresponding ternary complex with NADH is only stable at pH > 9.0. The spectral redshifts induced in the substrate are 11 nm larger than those found in the zinc enzyme. We suggest direct coordination of the substrate to the catalytic metal ion which acts as a Lewis acid in both substrate coordination and catalysis.     

Determination of absorption coefficients of purified proteins by conventional ultraviolet spectrophotometry and chromatography combined with multiwavelength detection

The direct, ultraviolet spectrophotometric determination of protein absorption coefficients was found to be more reproducible and accurate when diluting was replaced by chromatography and multiwavelength detection. Four different ultraviolet spectrophotometric methods, described in the literature, were compared by calculating A0.1%280 values from the spectra of 25 proteins, obtained by chromatography. Only two methods, i.e., one based on the absorbance at 210 nm and the other on the absorbance at 205 nm, corrected for the absorbance of aromatic amino acids at that wavelength, were sufficiently accurate to be of potential use for the determination of unknown proteins. It was found, however that with uncorrected A203 values even better results could be achieved. Using 7 well-defined proteins the equation A0.1%280 = 38.69 X A280/A203 - 0.01 was established by linear regression. A0.1%280 values for 14 pure proteins calculated with this equation showed a mean deviation of only 4% from literature values. Since similar deviations were seen with 5 chromophoric and 7 glycoproteins, 3 and 7% respectively, the method may have universal applicability. In the configuration used, only 40 micrograms of a protein is required for the chromatographic determination of its absorption coefficient.