A rapid and sensitive colorimetric assay of amine oxidase

A sensitive colorimetric assay for amine oxidase with Δ¹-pyrroline as the product is described based on the formation of a red-colored complex with ninhydrin reagent in acidic medium.     

A quantitative method for the measurement of membrane affinity by polydiacetylene-based colorimetric assay

The measurement of membrane affinity is an important early screening step during drug discovery. However, classical methods for membrane affinity measurement are tedious and difficult to implement in high-throughput screening. This article describes a quantitative method for the measurement of membrane affinity by colorimetric assay based on polydiacetylene (PDA) sensors. Prepared lipid/PDA chromatic vesicles were used to model cell membranes. By measuring the colorimetric response of the chromatic vesicles when drug-membrane interactions occurred, membrane affinity constant K(b) could be calculated using a simple quantitative model. Under optimized preparation conditions, the calculated log(K(b)) values exhibited an in-batch relative standard deviation (RSD) of less than 4% and a between-batch RSD of less than 8% for all three reference compounds. The logarithm of K(b) of the six β-blockers exhibited excellent linear correlation with the logarithm of the liposome/water partition coefficient (K(m)) with R(2)=0.9793. For neutral compounds, the log(K(b)) of n-fatty alcohols correlated with the logarithm of the n-octanol/water partition coefficient (K(oct)) with a linear correlation coefficient R(2)=0.9833. This work provides a simple, convenient, and reproducible method for the rapid measurement of membrane affinity and presents important implications for high-throughput screening.     

Human brain monoamine oxidase type B: mechanism of deamination as probed by steady-state methods

Recently, evidence has been published which suggests that [Husain, M., Edmondson, D. E., & Singer, T.P. (1982) Biochemistry 21, 595-600] monoamine oxidase [amine:oxygen oxidoreductase (MAO), EC 1.4.3.4] deaminates phenylethylamine and benzylamine via two distinct kinetic pathways which involve either binary or ternary complex formation, respectively. These conclusions were drawn largely from stopped-flow kinetic analysis performed on purified enzyme removed from its native membrane and in the presence of the inhibitory detergent Triton X-100. In this study, d-amphetamine and alternative substrates were used as steady-state probes of the kinetics of deamination by the B form of human brain MAO using native membrane-bound enzyme. Initial velocity studies showed mixed-type patterns for amphetamine inhibition of phenylethylamine, tryptamine, and tyramine when either amine or oxygen was the varied substrate. Slope and intercept vs. amphetamine concentration replots were linear in all cases except for phenylethylamine (hyperbolic); Ki values obtained from linear replots of slope or intercept values were comparable. In contrast, amphetamine was a competitive inhibitor of benzylamine deamination when amine concentration was varied and uncompetitive when oxygen concentration was varied; slope and intercept replots were linear for both. When benzylamine was the alternative substrate inhibitor and tyramine and tryptamine deamination was measured, mixed-type inhibition patterns were obtained when either amine or oxygen concentration was varied; replots of slope and intercept were linear in all cases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanism of inhibition by chlorgyline and deprenyl of tyramine deamination by mitochondrial monoamine oxidase of rat liver

The inhibition by chlorgyline and deprenyl of deamination of tyramine, i. e. substrate of two forms of monoamine oxidase (MAO) A and B, by fragments of rat liver mitochondrial membrane and the effects of competitive reversible inhibitors of the MAO activity, e. g. 4-ethylpyridine, benzyl alcohol, O-benzyl-hydroxylamine and 2-oxyquinoline, on this process were studied. It was shown that all the inhibitors used sharply increase the inhibiting effect of chlorgyline on tyramine deamination, the degree of the stimulating effect being the same irrespective of whether the inhibitors are added to the samples before or after a 30-min preincubation of chlorgyline with the enzyme at 23 degrees, i. e. after the onset of irreversible inhibition. The stimulating effect is due to the independent action of two inhibitors on the two different sites of the MAO active center: chlorgyline--on the isoalloxazine ring of FAD, that of 4-ethylpyridine, benzyl alcohol, O-benzylhydroxylamine, 2-oxyquinoline, respectively, on the hydrophobic region involved in tyramine binding. In similar experiments with deprenyl all the competitive inhibitors used, when added to the samples after a 30-min incubation of the inhibitor with the enzyme at 23 degrees, remove the inhibiting effect of deprenyl on tyramine deamination. The decrease of the inhibiting effect of deprenyl is indicative of an existence of competitive interactions between deprenyl and the above-mentioned compounds and of the reversible inhibition by deprenyl of tyramine deamination under the given experimental conditions. The data obtained revealed the differences in the type and mechanism of action of chlorgyline and deprenyl on tyramine deamination and showed that these inhibitors act on different sites of the MAO active center, responsible for tyramine oxidation. Chlorgyline blocks primarily the "flavin moiety" of the MAO molecule, essential for the catalytic act, while the effect of deprenyl is directed to the hydrophobic part of the enzyme active center essential for the enzyme binding to tyramine. In this case the irreversible inhibiting effect is achieved at a slower rate and the reversibility of tyramine oxidation by deprenyl is maintained for a longer period of time than the chlorgyline inhibition of deamination of this amine.     

A rapid and sensitive potentiometric assay for monoamine oxidase using an ammonia-selective electrode

A convenient method to store polyacrylamide gel slabs is described. The method involves sealing the wet gels between transparent plastic sheets using a kitchen-type bag sealer. The sealed gels are suitable for long-term storage, photography, and densitometry and can be used in autoradiography.     

A sensitive colorimetric high-throughput screening method for lipase synthetic activity assay

A sensitive and practical high-throughput screening method for assaying lipase synthetic activity is described. Lipase-catalyzed transesterification between vinyl acetate and n-butanol in n-hexane was chosen as a model reaction. The released acetaldehyde was determined by the colorimetric method using 3-methyl-2-benzothialinone (MBTH) derivatization. In comparison with other methods, the major advantages of this process include high sensitivity, simple detection, inexpensive reagents, and low requirements for instruments.     

A colorimetric assay for S-adenosylhomocysteine hydrolase

A colorimetric method for S-adenosyl-L-homocysteine hydrolase (SAHase) which uses S-adenosyl-L-homocysteine (SAH) as substrate is described. This method involves the hydrolytic conversion of SAH into adenosine (ADO) and L-homocysteine (HCY). The formation of HCY is quantified using Ellman's reagent and spectrophotometrical measured at 412 nm. Under these assay conditions, the product was followed continuously in a facile and quantitative manner until substrate conversion was complete. This method is an easy, cheap and shorter alternative to more complex methods and it is applicable to routine clinical analysis and in the assay and development of new S-nucleosidylhomocysteines to be used as therapeutic compounds.     

A colorimetric assay for immobilized chloroperoxidase

A rapid and sensitive colorimetric assay was developed for the estimation of chloroperoxidase activity. N,N,N',N'-Tetramethyl-p-phenylenediamine was chosen from four potential chromogenic substrates because the blue product resulting from chloroperoxidase conversion gave the highest molar absorption. This product exhibited two absorbance maxima, at 563 and 610 nm. Activity was monitored at 563 nm, and the product absorbance was stable for at least 1 h at 10 degrees C after treatment with an equal volume of a mixture (40:1) of methanol and phosphoric acid (85% w/v), pH 2. The linear range of the assay with respect to enzyme amount was determined. The assay was developed using soluble chloroperoxidase but worked well with the enzyme immobilized on glass beads.