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At least 271 records · Page 15Linked to original sources
PubMed1983

Chromophoric peptide substrates for activity determination of animal aspartic proteinases in the presence of their zymogens: a novel assay.

Solid-phase synthesis was used for the preparation of pyroglutamyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (I) and glycyl-glycyl-histidyl-p-nitrophenylalanyl-phenylalanyl-alanyl-leucine amide (II), two water-soluble and sensitive chromophoric substrates of chicken pepsin, hog pepsin A, and bovine spleen cathepsin D. The kinetic constants of hydrolysis of the p-nitrophenylalanyl-phenylalanyl bond of the substrates were measured by difference spectrophotometry at 308 nm (delta epsilon = 860 M-1 cm-1) and by ninhydrin colorimetry (substrate I, epsilon 570 = 2.31 X 10(4) M-1 cm-1). The pH optimum of cleavage is 5 for the pepsins and 3.7 for cathepsin D. Since all three proteinases still have a significant activity at pH 5.5-6 a new, simple assay was designed for submicrogram quantities of pepsins in the presence of pepsinogens without interference of the latter. The method is particularly suitable for the analyses of the zymogen activation mixtures.

Animals↗
PubMed1983

An improved method excluding hemoglobin interferences for lysosomal hydrolase assays using colorimetric synthetic substrates, 2-(N-hexadecanoylamino)-4-nitrophenol derivatives.

Artificial chromogenic substrates, derived from 2-(N-hexadecanoylamino)-4-nitrophenol, can be used to assay sphingomyelin phosphodiesterase, glucosylceramidase, or galactosylceramidase. Nevertheless, these enzymatic spectrophotometric assays cannot be realized on tissue preparations containing hemoglobin which interferes in the measurement. We present a selective extraction method of 2-(N-hexadecanoylamino)-4-nitrophenol which allows to avoid hemoglobin interference in this spectrophotometric assay of 2-(N-hexadecanoylamino)-4-nitrophenol. The solvents used have been tested to obtain on the one hand maximal absorbance and organic extraction of 2-(N-hexadecanoylamino)-4-nitrophenol, and on the other hand the minimal hemoglobin interference. None of the pure solvents studied being suitable, two solvent mixtures were selected: ethyl acetate/2-propanol (5/1) and 2-ethyl-1-hexanol/4-methyl-2-pentanone (1/1). These methods were tested to determine sphingomyelinase activity in enzymatic preparations and prove that they are available for lysosomal hydrolase assays using these colorimetric substrates.

Chromogenic Compounds↗
PubMed1984

Lowry method of protein quantification: evidence for photosensitivity.

Despite reports of its susceptibility to various interfering factors, the Folin Phenol protein quantification method of O. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951, J. Biol. Chem. 193, 265-275) remains the most convenient and accurate method for routine protein determinations. Our findings indicate that the Lowry assay is also photosensitive which can result in a discrepancy of up to 10% in estimated protein concentrations, unless appropriate precautions are taken.

Chromogenic Compounds↗
PubMed1984

Visible labeling of proteins for polyacrylamide gel electrophoresis with dabsyl chloride.

Several proteins, which are used as molecular weight markers in polyacrylamide gel electrophoresis, were reacted with dabsyl chloride. This labeled them deep orange and the chromophore attachment was stable throughout the electrophoretic procedure and fixation. Small amounts (10-50 micrograms) of the labeled proteins could be loaded onto gels and seen with the unaided eye so that the separation during electrophoresis could be followed. Dabsylation did not affect the mobility of the proteins. The location of the orange band gave a good indication of the position of the protein in the gel so that molecular weight estimations could be made during and immediately following electrophoresis.

Chromogenic Compounds↗
PubMed1984

L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide--a chromogenic substrate for thiol proteinase assay.

L-Pyroglutamyl-L-phenylalanyl-L-leucine-p-nitroanilide (PFLNA)--a convenient chromogenic substrate for assay of thiol proteinases papain, ficin, and bromelain--was prepared by enzymatic synthesis with chymotrypsin as a catalyst. The thiol proteinases hydrolyze PFLNA with the liberation of p-nitroaniline, estimated spectrophotometrically by its absorbance at 410 nm. The phenylalanine residue in the P2 position of PFLNA meets the specificity demands of thiol proteinases. The following values of Km were found for PFLNA hydrolysis: by papain, 0.34 mM; by ficin, 0.43 mM; by bromelain, 0.30 mM. This substrate was successfully applied to monitor thiol proteinase affinity chromatography on bacitracin-Sepharose, which resulted in a 2- to 4-fold purification from commercial preparations.

Bacitracin↗
PubMed1984

The synthesis, purification, and evaluation of a chromophoric substrate for pepsin and other aspartyl proteases: design of a substrate based on subsite preferences.

A convenient chromophoric assay for porcine pepsin has been developed using a new synthetic substrate. The sequence of this substrate was chosen based on the known subsite preferences for this enzyme. The peptide contains a phenylalanyl-p-nitrophenylalanine sequence at the reactive site. Cleavage of this bond yields a change in absorbance at 310 nm of between 1700 and 2000 per mole. This allows kinetic data to be obtained readily and accurately. The products of cleavage have been identified by isolation of a peptide fragment by high-performance liquid chromatography. Values of kcat, Km, and kcat/Km of 94 +/- 6 s-1, 0.13 +/- .04 mM, and 815 +/- 210 s-1/mM-1 were obtained at pH 3.0 and 37 degrees C. The peptide is soluble over the pH range from 2 to 7, thus facilitating determination of the pH dependence of the kinetic parameters. The substrate is also valuable in studying the inhibition of pepsin.

Amino Acid Sequence↗
PubMed1984

Colorimetric assay for monoamine oxidase in tissues using peroxidase and 2,2'-azinodi(3-ethylbenzthiazoline-6-sulfonic acid) as chromogen.

Monoamine oxidase is assayed in tissue by a colorimetric reaction using horse radish peroxidase and 2,2'-azinodi(ethylbenzthiazoline-6-sulfonic acid to measure H2O2 formed during oxidation of amines. The method has a coefficient of variation of approximately 2.5% and provides results comparable with those of radiometric assay. Monoamine oxidase activities in rat liver mitochondria and crude mitochondrial fraction from brain and with tyramine as a substrate were 18.9 +/- 0.4 and 4.61 +/- 0.15 nmol/min/mg of protein, respectively, using this method. Kinetic parameters of liver and brain monoamine oxidase with various substrates and inhibitors appeared to be the same when determined by either colorimetric or radiometric methods.

Animals↗
PubMed1985

Chromogenic labeling of monosaccharides using 4'-N,N-dimethylamino-4-aminoazobenzene.

Twenty-three monosaccharides, e.g., D- or L-pentoses, D- or L-hexoses, heptose, 2- or 6-deoxyhexoses, 2-deoxy-2-aminohexoses, hexuronic acids, and N-acetylmuramic acid, were coupled to the azo dye 4'-N,N-dimethylamino-4-aminoazobenzene by reductive amination using sodium cyanoborohydride as reducing agent and in the presence of pentaerythritol. The structure of the colored glycamines was established by mass spectrometry. The average yield of the reaction was more than 80%. The sugar derivatives were separated either by silica-gel thin-layer chromatography or by high-performance liquid chromatography. Spectrophotometric quantitation was performed in the visible range at the picomole level. The method was applied to the determination of the sugar composition of the glycosphingolipid globotetraosyl ceramide and the human milk oligosaccharide lacto-N-fucopentaose I.

Amination↗
PubMed1985

Chromogenic substrate autography: a method for detection, characterization, and quantitative measurement of serine proteases after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing in polyacrylamide gels.

A chromogenic substrate autography is described which allows characterization and quantification of serine proteases in crude systems after sodium dodecyl sulfate-polyacrylamide gel electrophoresis or isoelectric focusing. Separation of samples containing proteases by either method is followed by an overlay of the gels on an indicator film prepared by incorporation of a suitable paranitroanilide substrate into agarose. Positions of the proteases are revealed by the formation of yellow-colored zones which can be quantified by densitometry at 405 nm. The technique proved suitable for determination of molecular weights, isoelectric points, and quantitative measurements of amidolytic activities of urokinase, tissue plasminogen activator, tissue and plasma kallikrein, and thrombin in biological fluids and purified preparations.

Animals↗