Continuous spectrophotometric assay for aminoacyl-tRNA synthetases

A simple, continuous assay for aminoacyl-tRNA synthetases utilizing a commercially available pyrophosphate assay reagent kit was demonstrated. The method coupled aminoacyl-tRNA synthetase activity with pyrophosphate-dependent fructose-6-phosphate kinase, aldolase, triosephosphate isomerase, and glycerophosphate dehydrogenase. PPi formation was correlated with the oxidation of NADH, and was monitored continuously by the decrease of absorbance at 340 nm.     

Continuous spectrophotometric assay of glucosyltransferase and beta-fructofuranosidase activity

Sucrose 6-glucosyltransferase [(1→6)-α-D-glucan:D-fructose 2-glucosyltransferase, E.C. 2.4.1.5] converts sucrose into D-glucan and β-fructofuranosidase [β-D-fructofuranoside fructohydrolase, invertase, E.C. 3.2.1.26) hydrolyses sucrose, both releasing stoichiometric amounts of D-fructose. This study reports on a direct spectrophotometric method for measuring the kinetics of these enzymes. D-Fructose (in aqueous solution at 37°) has 2 absorption peaks at 188.5 and 278 nm (ε 133.6 and 1.12 respectively); D-glucose, which is also released by β-fructofuranosidase, has a λ_max at 186.0 nm (ε 123.0). The methods developed utilize the absorption peaks in the far u.v. range. Buffers exert a bathochromic shift and have a hypochromic effect. Glucosyltransferase was assayed in phosphate buffer (0.1mM, pH 6.8). From a plot of Δ(ε_Dfructose-ε_sucrosevs. λ, 195 nm was selected as the optimal wavelength since, at this wavelength, the contribution of sucrose was least (? 10%) and ΔA₁₉₅/min was linearly proportional to glucosyltransferase concentration. Determination of K_m by this method gave values comparable to those obtained by chemical assay of released reducing groups (2.05 vs. 2.00mM sucrose, respectively). β-Fructofuranosidase assayed at 207 nm in acetate buffer (0.5mM, pH 4.6) at 37° gave a K_m value of 12.3mM sucrose. This is in agreement with the results obtained by polarimetry and chemical assay of released reducing groups (14.6 and 12.3mM sucrose, respectively). The advantages of this method are simplicity, ability to measure initial reaction rates, and a continuous following of the course of the reaction.     

Continuous spectrophotometric assay for retroviral proteases of HIV-1 and AMV

Ac-Lys-Ala-Ser-Gln-Asn-Phe(NO2)-Pro-Val-Val-NH2 (peptide I) and Thr-Phe-Gln-Ala-Phe(NO2)-Pro-Leu-Arg-Glu-Ala (peptide II) undergo hydrolysis between the p-nitrophenylalanyl and prolyl residues catalyzed by the proteases of HIV-1 and AMV, respectively. The specific hydrolyses of peptides I and II are accompanied by a decrease in their uv absorption at 269 nm (delta epsilon = 1000) and an increase at 316 nm (delta epsilon = 600). The use of microspectrophotometric cells allows continuous uv measurements on a volume (60 to 120 microliters) comparable to that required for the HPLC point assay currently used. At the highest substrate concentration possible under the assay conditions, good first-order kinetics were observed with both proteases, and the values of Vmax/Km were obtained.     

Continuous spectrophotometric assay of protein tyrosine phosphatase using phosphotyrosine.

A continuous activity assay for protein tyrosine phosphatases (PTPs), employing phosphotyrosine (P-Tyr) as a substrate, has been developed and applied to measure the activities of two purified enzymes, namely, the full length T-cell protein tyrosine phosphatase (TC PTP) and its truncated form (TC delta C11 PTP). The reaction was followed by changes in ultraviolet absorption and fluorescence resulting from the dephosphorylation of P-Tyr. Both enzymes obey Michaelis-Menten kinetics, with Km = 304 microM, Vmax = 62,000 units/mg for TC PTP and Km = 194 microM, Vmax = 73,000 units/mg for TC delta C11 PTP. The D- and L-forms of P-Tyr are equally effective as substrates. The optimum pH for both enzymes is 4.75. The known effectors of PTPs have the predicted effects on catalytic activity.     

Continuous spectrophotometric assay amenable to 96-well plate format for prostaglandin E synthase activity

The measurement of prostaglandin E synthase (PGES) activity is cumbersome because the product of the reaction, PGE(2), is not readily quantitated by spectral means. The activity of isolated PGES is typically determined by PGE(2) immunoassay or by high-performance liquid chromatography using radiolabeled substrate. A relatively rapid continuous spectrophotometric assay which uses 15-hydroxyprostaglandin dehydrogenase (PGDH) to couple the oxidation of the 15-hydroxy group of PGE(2) to the formation of NADH was developed. PGDH is relatively specific for PGE(2) over the substrate for the PGES reaction, PGH(2), allowing a highly reproducible assay of PGES activity to be obtained.     

A continuous spectrophotometric assay for catechol-O-methyltransferase

Catechol-O-methyl transferase (COMT) activity can be monitored continuously using a coupled enzyme assay in which the inhibitory product S-adenosylhomocysteine (SAH) is converted to S-inosylhomocysteine (SIH). A simple spectrophotometric assay for COMT is described based on the difference in the ultraviolet absorption spectra between SAH and SIH.     

A spectrophotometric assay for feruloyl esterases

We have developed a spectrophotometric assay for the quantitative determination of feruloyl esterase activity based on release of 4-nitrophenol from a novel substrate, 4-nitrophenyl ferulate in an emulsion of Triton X-100 in aqueous buffer solution. The release of 4-nitrophenol was linear with reaction time at an early stage of the reaction with various esterase preparations. The method proposed here is accurate, rapid, and easy to perform.     

A spectrophotometric method for calmodulin assay

The activity of calmodulin as an activator of cAMP phosphodiesterase was assayed. AMP was hydrolyzed by 5'-nucleotidase, and the adenosine formed was measured by both liquid scintillation counting and spectrophotometry at 265 nm. Calmodulin activities measured by the two methods were equivalent, indicating that spectrophotometric assay of calmodulin can be used in place of the isotopic method.     

A spectrophotometric assay for hypoxanthine-guanine phosphoribosyltransferase

The present paper describes a new spectrophotometric assay for HGPRTase activity which is more rapid than and as sensitive as the isotopic assays for this enzyme and which avoids the use of high-voltage electrophoresis and liquid scintillation counting.     

A spectrophotometric assay for dehydroascorbate reductase

A simple spectrophotometric assay for dehydroascorbate reductase based on the change in absorbance associated with the formation of ascorbic acid is described. Using a partially purified preparation from spinach leaves, the reaction was found to be linear with time and enzyme concentration. The reaction rate determined by this assay correlated well with that obtained by a high-performance liquid chromatography method. Possible advantages over currently available assays as well as potential applications are discussed.     

A rapid spectrophotometric assay for catechol-O-methyltransferase

A new assay technique for catechol-O-methyltransferase is described. 3,4-Dihydroxyacetophenone is used as the substrate for the assay and the products, 3-hydroxy-4-methoxyacetophenone and 4-hydroxy-3-methoxyacetophenone are detected spectrophotometrically at 344 nm in borate buffer, pH 10.0. This spectrophotometric procedure is simple, rapid, and inexpensive while retaining reasonably high sensitivity and precision.     

A spectrophotometric assay for strictosidine synthase

A spectrophotometric assay for strictosidine synthase is described. Strictosidine is extracted with ethyl acetate and, where high substrate concentrations are used, the organic extract is washed with dilute ammonia to remove coextracted secologanin; after evaporation of the solvent, the residue is heated with 5 M H2SO4 for 45 min and the A348 value is measured. Strictosidine production is calculated from the response of similarly treated standards. A minimum production of 10-25 nmol of strictosidine may be determined. The assay is demonstrated using extracts of cultured Cinchona ledgeriana cells.     

An o-phthalaldehyde spectrophotometric assay for proteinases

A rapid and convenient spectrophotometric assay has been devised to measure proteolysis. The assay is based on the reaction of o-phthalaldehyde (OPA) and 2-mercaptoethanol with amino groups released during proteolysis of a protein substrate. The reaction is specific for primary amines in amino acids, peptides, and proteins, approaches completion within 1 to 2 min at 25 degrees C (half-times of approx 10-15 s), and requires no preliminary heating or separation of the hydrolyzed products from the undegraded protein substrate prior to performing the assay. The OPA assay was relatively as successful as a 2,4,6-trinitrobenzenesulfonic acid (TNBS) procedure in predicting the extent of hydrolysis of a protein substrate. The utility of the OPA method was demonstrated by measuring the degree of proteolytic degradation caused by trypsin, subtilisin, Pronase, and chymotrypsin of various soluble protein substrates. Ethanethiol (instead of 2-mercaptoethanol) or 50% of dimethyl sulfoxide can be included in the assay solution to stabilize certain OPA-amine products. The present method approaches the sensitivity of ninhydrin and TNBS procedures, is more convenient and rapid, and could substitute for these reagents in most assay systems.     

Continuous spectrophotometric assay for restriction endonucleases using synthetic oligodeoxynucleotides and based on the hyperchromic effect.

A continuous spectrophotometric assay for the EcoRV restriction endonuclease has been developed. The synthetic self-complementary oligonucleotide d(GACGATATCGTC) (which is double stranded under the assay conditions) is used as the substrate. The EcoRV endonuclease recognizes d(GATATC) sequences cutting between the central T and dA bases. Thus d(GACGATATCGTC) is converted to d(GACGAT) and d(pATCGTC) during catalysis. Both of the hexameric products are single stranded under the assay conditions. The conversion of the dodecameric substrate to the two hexameric products and the concomitant change from double- to single-stranded DNA is associated with an increase in absorbance at 254 nm due to the hyperchromic effect. This change can be used to monitor column effluents for endonuclease activity and also for Km and kcat determination under steady-state kinetic conditions.     

Continuous spectrophotometric assay of phospholipase A2 activity hydrolyzing plasmalogens using coupling enzymes

We developed a continuous spectrophotometric assay of the phospholipase A2 activity specific for choline plasmalogen using rat liver lysoplasmalogenase and horse liver alcohol dehydrogenase as coupling enzymes and Naja naja venom phospholipase A2 as a source of the phospholipase A2 activity. In these coupling reactions, choline lysoplasmalogen is hydrolyzed by lysoplasmalogenase to glycerophosphocholine and free aldehyde. The free aldehyde is quantitatively converted to alcohol by alcohol dehydrogenase with the oxidation of NADH. The disappearance of NADH is measured spectrophotometrically at 340 nm. The assay is sensitive to about 0.2 nmol aldehyde produced/ml/min and also is rapid, convenient, and continuous.     

Continuous spectrophotometric assays for cytosolic epoxide hydrolase

Two convenient and sensitive continuous spectrophotometric assays for cytosolic epoxide hydrolase are described. The assays are based on the differences in the ultraviolet spectra of the epoxide substrates and their diol products. The hydrolysis of 1,2-epoxy-1-(p-nitrophenyl)pentane (ENP5) is accompanied by a decrease in absorbance at 302 nm, while the hydration of 1,2-epoxy-1-(2-quinolyl)pentane (EQU5) produces an increase in absorbance at 315.5 nm. The Km, Vmax values for ENP5 and EQU5 with purified mouse liver cytosolic epoxide hydrolase were 1.7 microM, 11,700 nmol/min/mg and 25 microM, 8300 nmol/min/mg, respectively. Both substrates are hydrolyzed significantly faster than trans-stilbene oxide, which is currently the most commonly used substrate for measuring cytosolic epoxide hydrolase activity. No spontaneous hydrolysis of the substrates is detectable under normal assay conditions. The assays are applicable to whole tissue homogenates as well as purified enzyme preparations. p-Nitrostyrene oxide and p-nitrophenyl glycidyl ether were also examined and found to be very poor substrates for cytosolic epoxide hydrolase from mouse liver.     

A general coupled spectrophotometric assay for decarboxylases

A rapid, continuous spectrophotometric method has been developed for the assay of decarboxylases. The assay uses a coupled enzyme system in which liberated CO2 is reacted with phosphoenolpyruvate and phosphoenolpyruvate carboxylase to form oxaloacetate, which in turn is reduced by malate dehydrogenase to L-malate concomitantly with the oxidation of NADH to NAD. The resultant decrease in absorbance at 340 nm accurately reflects the activity of the decarboxylase. The method is capable of detecting the liberation of as little as 1 nmol of CO2/min and was tested in assays of lysine decarboxylase, orotidine-5'-phosphate decarboxylase, and 4'-phosphopantothenoyl-L-cysteine decarboxylase.