Abstract: A transparent and stable aqueous solution of a substrate for a determination of lipase, comprising triglyceride uniformly solubilized therein, a method for determination of lipase activity in a sample, employing the aqueous solution, and a process for the manufacture of a lyophilized product capable of forming the aqueous solution are disclosed. A process for the manufacture of a transparent and stable aqueous solution of triglyceride is also disclosed.

Abstract: A method for assaying lipoprotein (a) in a liquid sample containing other lipoproteins, and a assay device for use in the method are disclosed. In the method, the liquid is contacted with a solid-support reagent containing lectin attached to a solid support, under conditions effective to bind of lipoprotein (a) to the support-bound lectin. After removing unbound lipoproteins, the amount of lipoprotein (a) bound to the support is assayed. In one embodiment, the method and assay device are designed for assaying cholesterol associated with lipoprotein (a).

Abstract: A method of measuring adenylate cyclase (AC) in a sample of physiological material which does not employ radioactive reagents is provided, comprising:(a) providing a physiological sample of physiological material comprising cAMP produced by endogenous AC, and other endogenous adenine nucleotides selected from the group consisting of ATP, AMP, ADP and mixtures thereof;(b) combining the sample with effective amounts of apyrase, 5'-nucleotidase and adenosine deaminase, so as to enzymatically eliminate the other endogenous adenine nucleotides in the sample;(c) enzymatically converting the cAMP into AMP; and(d) measuring the amount of AMP, the amount providing a measure of the amount of cAMP and AC in the sample.

Abstract: For the preparation of cholesterol esterases with variable substrate specificity by cloning of a cholesterol esterase gene, the active center of which has the sequence -Gly-His-Ser-X-Gly-, wherein X signifies an amino acid, into a vector, transformation of a micro-organism with this vector and expression of the cholesterol esterase gene, one exchanges the amino acid X of the active center for another amino acid by mutagenesis.

Abstract: A method of detecting hydrolase activity using as substrates the compounds of the formula ##STR1## wherein each of R.sup.1, R.sup.2 and R.sup.3 is C.sub.1 -C.sub.4 alkyl, or phenyl optionally substituted in the meta- or para- position by C.sub.1 -C.sub.6 alkyl, C.sub.1 -C.sub.6 alkoxy or mono- or di-(C.sub.1 -C.sub.6)-alkylamino, or optionally substituted by an O-X group in which X is a glycosyl, phosphate or acyl moiety of a natural substrate of the corresponding glycosidase, phosphatase or esterase.

Abstract: Low density lipoprotein are directly determined in body fluids by selectively precipitating very low density lipoprotein, forming clusters with low density lipoproteins, selectively consuming the high density lipoproteins, and resolubilizing the low density lipoproteins for direct determination thereof. The clusters are formed by treating the fluid sample with a mixture of a polyanionic compound, a divalent metal, and a nucleating agent.

Abstract: A method of monitoring cell culture which comprises digitizing an image of cultured cells inputted to an image processing equipment through a TV camera connected with an observing means and measuring the number and cell proliferation of cultured cells by a processing with using a spatial filter, can treat a large amount of cells in a short time and give a precise number of the cells.

Abstract: The present invention provides a diagnostic agent for the enzymatic determination of cholesterol, wherein, besides the necessary enzymes, it contains a scleroprotein or a scleroprotein hydrolysate; said invention also provides a process for the production of a reagent film for the detection of cholesterol, as well as a test strip for the detection of cholesterol; and said invention is also concerned with the use of scleroproteins and scleroprotein hydrolysates for increasing the enzyme stability in diagnostic agents for the detection of cholesterol and for increasing the temperature-independence of the detection of cholesterol with diagnostic agents.

Abstract: The present invention discloses the Pichia pastoris acid phospbatase gene, which includes the 5' regulatory region, signal sequence, structural gene, and 3' transcription termination sequence. Also disclosed are methods of using these fragments, which include but are not limited to the secretion of proteins from cells and the regulation of the transcription of DNA. DNA vectors containing the acid phospbatase gene or fragments thereof and hosts transformed with these vectors are also disclosed. Additionally, integrative vectors which direct integration at the Pichia pastoris PHO1 locus and a method of identifying these disruptants is disclosed.

Abstract: A room temperature stable restriction enzyme in a glassified carbohydrate stabilizer is disclosed. A restriction enzyme reaction buffer containing Mg.sup.+2 is dispersed in the stabilizer. One cleaves DNA merely by adding water and DNA to the composition. The composition can reduce glycerol-caused star activity by avoiding the need for glycerol. The composition also reduces star activity caused by high concentrations of enzymes. Another method is provided of comparing cleaved DNA samples in connection with forensic and paternity testing applications.

Abstract: Novel assay methods employing compounds which are chemically or enzymatically cleavable and which give rise to an intermediate which further decomposes by an intramolecular anchimeric displacement reaction which releases a signal producing species are disclosed. Also disclosed are probe hybridization assays employing the compounds of the invention employing thermostable enzymes which are not denatured by the hybridization conditions. Such signal producing species may include chemiluminescent dioxetanes and other colored products.

Abstract: A single system reagent for the determination of lipase, formulated as an emulsion comprising a lipase-active fatty acid source; a first lipase activator comprising colipase; a second lipase activator; a lipoprotein lipase inhibitor; a first emulsion stabilizer comprising Triton X100; a second emulsion stabilizer; a buffer; and an anti-precipitant.

Abstract: The present invention provides a process for the determination of phospholipase A in body fluids, wherein the body fluid is incubated with a mixture containing a substrate having a fatty acid residue for the phospholipase and at least two emulsifiers of different chemical classes, namely, at least one neutral emulsifier and a bile acid salt, and the amount of the free fatty acids formed by the cleavage of the substrate is determined in known manner.

Abstract: Phosphoric acid derivatives represented by formula (I): ##STR1## wherein X is a halogen and R is --(CH.sub.2).sub.n CH.sub.3 (n=0 to 3), or salts thereof are stable to non-enzymatic hydrolysis and are capable of specifically reacting with acid phosphatase. Therefore, the activity of acid phosphatase in the sample can be determined extremely accurately by reacting said compound with a sample containing acid phosphatase and quantitatively determining the reaction product by colorimetry.

Abstract: Methods and compositions for membrane flow-through assays using substrate and enzyme conjugate is described. A preferred method of the present invention comprises providing E. coli alkaline phosphatase (ECAP) conjugate, with additional color enhancement provided by combining lithium salt with the substrate, calcium and/or magnesium salts with the conjugate, and polyalcohol polymers with the conjugate. Color development is further enhanced by spotting tetrazolium dyes, such as the Nitro Blue tetrazolium series, onto the membrane.

Abstract: A method of detecting, identifying, and enumerating microbes in biological and non-biological samples includes the steps of combining the sample with a triggerable chemiluminescent compound specifically susceptible to the initiation of chemiluminescent decomposition by at least one microbial enzyme in the sample and detecting and integrating light emission over an extended period of time as an indication of the presence, identification, or enumeration of the microbes in the sample.

Abstract: A unitary sterility indicator and a method for its use, the indicator comprising an outer container having liquid impermeable and gas non-absorptive walls, and having a gas-transmissive, bacteria impermeable opening therein; contained within the outer container, a detectable amount of a source of active enzyme and/or another microorganism commonly used to monitor sterilization; a sealed, openable gas and liquid impermeable inner container containing an aqueous medium and/or a nutrient growth medium, disposed in the outer container; an enzyme substrate system capable of reacting with active enzyme to produce a detectable enzyme-modified product and/or a detector material sensitive to microorganism growth, contained in one of the containers; and means contained within the outer container for restricting the area in which enzyme-modified product and/or growing microorganisms are contained, after the inner container is opened, to an area which is less than the volume of aqueous solution contained in the inner con

Abstract: A stable triglyceride assay solution for use with a chromogen system is comprised of a solution buffered with a Zwitterionic buffering agent and/or TRIS and stabilized with sortibol, gelatin, and ammonium sulfate. Solution pH is in the range of from 6.8 to 8.0. The functional enzymes are lipase, glyercol kinase, and glycerol phosphate oxidase. The preferred chromogen system is based on the inclusion of 4-aminoantipyrine for use in combination with oxidase to yield a detectable chromogen compound.

Abstract: The claimed invention relates to a substrate for evaluating glycosidic enzymes comprising a fluorescein derivative of the general formula: ##STR1## wherein GlyX is a carbohydrate bonded to fluorescein by a glycosidic linkage;Y, which may be the same as GlyX or different, is an alkyl ether, an ester, or a glycosidically linked carbohydrate;R is a lipophilic residue containing from 1 to 21 carbon atoms; andL links the R residue to fluorescein.A preferred embodiment of the invention is a non-fluorescent substrate specifically hydrolyzable by a glycosidase inside a cell to yield, after greater than about 2 minutes, a fluorescent detection product excitable at between about 460 nm and 550 nm and with fluorescence observable at an emission wavelength longer than the excitation wavelength, which fluorescent detection product is retained inside a viable cell more than about 2 hours at greater than about 15.degree. C. and which is non-toxic to the cell.

Abstract: Chromogenic merocyanine enzyme substrate compounds of the general formula: ##STR1## where Y is an enzymatically-cleavable group such as a radical of a sugar, carboxylic acid, amino acid, peptide, phosphoric acid, or sulfuric acid; A and B represent residues that complete 5- or 6-membered ring systems; R.sup.1 is substituted or unsubstituted alkyl; R.sup.2 and R.sup.3, independently, are hydrogen or lower alkyl; m, n, and p, which can be different, are integers from 0 through 3 provided that m+n+p must be at least 2; and X is an appropriate counterion (anion).

Abstract: The present invention provides compounds of the general formula: ##STR1## wherein R.sup.1 is an amino acid residue or a residue of an oligopeptide in which the amino groups are optionally substituted by protective groups, R.sup.2 and R.sup.3, which are the same or different, are hydrogen atoms, a lower alkyl, lower alkoxy, carboxyl or lower alkoxycarbonyl radicals or carboxamido groups optionally substituted by lower alkyl or, if R.sup.2 and R.sup.3 are adjacent, can represent a --CH.dbd.CH--CH.dbd.CH-- radical, Z is conjugated-through positively charged, bicyclic heterocyclic system and X is an anion of an organic or inorganic acid. The present invention also provides processes for the preparation of these compounds and diagnostic agents containing them for the determination of peptide bond-cleaving enzymes.

Abstract: The present invention provides a new methodology and test composition for determining the presence of theophylline in test samples. The methodology employs enzymes that utilize or recognize theophylline as a substrate to measure the concentration thereof in samples, including body fluids. This new approach utilizes enzymes as opposed to traditional methods which use antibodies for the recognition of theophylline. The enzymatic approach to theophylline determination is quick, simple and convenient and allows test systems to be made in liquid as well as in dry-chemistry formats. Various protocols, systems or methodologies may be used for assaying and relating the results to the amount of theophylline present. Methods for obtaining theophylline utilizing or recognizing enzymes are also described.

Abstract: An assay for Chlamydia includes contacting Chlamydia organisms in a liquid with a solid support having an antispecies Fe antibody immobilized thereon and an anti-Chlamydia capture antibody. After binding of Chlamydia antigen to the capture antibody and binding of the capture antibody to the antispecies antibody on the support, a tracer including a label conjugated to a signal antibody is added. After binding of the signal antibody to the antigen, the presence of Chlamydia organisms in the liquid is detected by a signal associated with the label thereby bound to the support. The invention includes a kit of materials for performing an assay according to the method of the invention.

Abstract: A method is provided for the quantitative study of cytotoxic T-lymphocyte activation by measuring secreted granule-associated BLT esterase activity after incubating the cytotoxic T-lymphocytes with activating stimuli. This method can be used to screen inhibiting (drugs) or activating agents (monoclonal antibodies) (at selected sub-optimal levels of activation of cytotoxic T-lymphocytes.

Abstract: The present invention is directed toward a thermostable lipoprotein lipase capable of hydrolyzing triglycerides in lipoproteins to glycerol and fatty acids, a process for producing the thermostable lipoprotein lipase, and a blood triglyceride determining reagent containing the thermostable lipoprotein lipase. The thermostable lipoprotein lipase exhibits about 100% retention of the hydrolyzing activity when treated in a buffer having a pH of from about 4 to 7 at about 60.degree. C. for about 15 minutes and a glycerol forming activity/fatty acid forming activity ratio of at least about 15%. The process comprises cultivating a thermophilic actinomycetes, particularly Streptomyces 7825 (FERM P-9983, FERM BP-2489 and recovering lipoprotein lipase from the culture.

Abstract: A method for measuring diaphorase activity comprising mixing a sample comprising diaphorase with nitro blue tetrazolium, EDTA or a salt thereof, at least one of nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, and a surface active agent, to form an assay solution; and measuring an increase in absorbance, due to the formation of diformazan, in the assay solution and reagents used in a method for measuring diaphorase activity comprising a first solution, a second solution, EDTA, and a surface active agent, wherein the first solution comprises at least one of nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate and the second solution comprises nitro blue tetrazolium.

Abstract: Disclosed herein is a novel monoglygeride lipase at least capable of catalyzing an enzymatic reaction of the following equation (a) and as substrate specificity, capable of acting on monoglyceride but incapable of acting on diglyceride and triglyceride:Monoglyceride+H.sub.2 O.fwdarw.Glycerol+Fatty acid (a)The monoglyceride lipase is produced by culturing a specific monoglyceride lipase producing microorganism of Bacillus and then collecting the monoglyceride lipase from the resulting culture. A method is also disclosed for the analysis of a monoglyceride-containing sample solution. The monoglyceride lipase is caused to act on the sample solution upon measurement of the monoglyceride in the sample solution. Either one of glycerol and the fatty acid formed in accordance with the equation (a) is then measured.

Abstract: This invention relates to a process for the recovery of a substantially insoluble, radiation imageable polyacetylene dispersion in aqueous binder which comprises contacting the dispersion with a proteolylic enzyme in an amount sufficient to decompose the binder, at a pH of from about 3 to about 9 and a temperature of between about 20.degree. C. and about 70.degree. C. and separating polyacetylene solids from the resulting liquid mixture, and which solids can be solubilized and recrystallized for further use.

Abstract: Reagent for analysis of triglycerides contained in blood serum is provided, which comprises lipases and monoglyceride lipases capable of acting on monoglycerides having substrate specificity and capable of catalyzing the following enzymatic reaction: monoglyceride+H.sub.2 O.fwdarw.glycerol+fatty acids. The glycerol or fatty acids are measured to learn an amount of the triglycerides or fatty acid by any known analytical method.

Abstract: An assay with high sensitivity for activity of lecithin-cholesterol acyltransferase in blood for functional analysis of liver, by bringing the blood into contact with lecithin and free cholesterol until lysolecithin and cholesterol ester are produced, allowing the lysolecithin produced to react with lysophospholipase and glycerophosphocholine phosphodiesterase and assaying glycerol-3-phosphate produced simultaneously or successively in the reaction by means of an enzymatic cycling reaction in which glycerol-3-phosphate, dihydroxyacetone-3-phosphate, nicotinamide adenine dinucleotide (NAD), reduced NAD, O.sub.2, H.sub.2 O.sub.2, glycerophosphate oxidase and glycerophosphate dehydrogenase take part.

Abstract: Chromogenic merocyanine enzyme substrate compounds of the general formula: ##STR1## where Y is an enzymatically-cleavable group such as a radical of a sugar, carboxylic acid, amino acid, peptide, phosphoric acid, or sulfuric acid; A and B represent residues that complete 5- or 6-membered ring systems; R.sup.1 is substituted or unsubstituted alkyl; R.sup.2 and R.sup.3, independently, are hydrogen or lower alkyl; m, n, and p, which can be different, are integers from 0 through 3 provided that m+n+p must be at least 2; and X is an appropriate counterion (anion).

Abstract: Method and apparatus for use in determining the concentration of a selected analyte in a body-fluid sample. As the fluid sample is applied to a wettable, absorbent reaction pad, the extent of sample wetting of the pad is monitored by surface reflectance. This surface monitoring is used to control the volume of sample applied to the pad, to prevent overfilling the pad and to allow determination of the final sample volume applied. From the known sample volume and amount of analyte, the concentration of analyte in the sample can be accurately calculated in reaction components, trapping agents, matrix configuration, and analyte tested can be made.

Abstract: This invention relates to an enzyme-controlled release method for the release of a leaving group comprising:contacting a compound represented by the formula ##STR1## wherein R, R.sub.1, R.sub.2 and R.sub.3 each independently is hydrogen, a substituent affecting the mobility or reactivity of the compound or a substituent including a biologically active group;X is leaving group;Z is an enzyme substrate moiety;--CR.sub.2 R.sub.3 X is either ortho or para to the --O--Z moiety;with an active capable of cleaving said enzyme substrate moiety Z from said compound;whereby said leaving group X is released from said compound.

Abstract: An assay device for assaying multiple analytes in a drop-size blood sample. The device includes a sample dispenser designed to distribute a small-volume blood sample to multiple transfer sites, by capillary flow of the blood sample through sieving and distributing matrices which separate blood cells from serum as the sample fluid migrates toward the transfer sites. A test plate in the device carries multiple absorbent test pads, each containing reagent components for use in detection of a selected analyte. The test plate is mounted on the dispenser for movement toward and away from a transfer position at which the exposed surface regions of the pads are in contact with associated sample-transfer sites, for simultaneous transfer of sample fluid from such sites to the pads in the support.

Abstract: The invention provides a novel bile acid sulfate sulfatase, a process for its preparation, and a method of assaying bile acid 3.alpha.-sulfates and total bile acids using the bile acid sulfatase.

Abstract: A method for the detection of a fungal infection within 1-2 hours comprises the steps of: (a) contacting a sample suspected of containing a peroxidase-containing fungus with a mixture which comprises (i) a peroxide from hydrogen peroxide and addition compounds thereof containing latent hydrogen peroxide; (ii) a scavenger to remove heavy metals which may potentially decompose said peroxide catalytically; (iii) an alkaline buffer solution having the capacity to maintain the pH at a value within the range of 9.5 to 14; and (iv) a colorless oxidizable substrate (such as tyrosine, .beta.-(3,4-dihydroxyphenyl)-.alpha.-alamine (DOPA) or caffeic acid), which on reaction with oxygen at said pH value gives a visibly colored oxidation product; and (b) observing whether a color develops indicating the presence of peroxidase-containing fungus in the sample. Also included is a test kit for carrying out the method of the invention.

Abstract: A multi-pH chemiluminescence-based assay method comprising first-stage light production by the activation by cleavage of a phosphatase cleavable dioxetane derivative within a pH range optimal for enzyme catalysis, followed by the second-stage generation of increased light energy from the products of enzyme-catalyzed cleavage of the dioxetane derivative by the adjustment of the pH to a strongly alkaline pH optimal for the generation of light energy. The assay method is suitable for both solution phase and solid state assay formats. Light generation enhancers can be used further to increase the light yield.

Abstract: The invention provides a novel bile acid sulfate sulfatase, a process for its preparation, and a method of assaying bile acid 3.alpha.-sulfates and total bile acids using the bile acid sulfatase.

Abstract: The present invention concerns a spectrometric technique to determine microorganism detection and identification by taking advantage of the inherent extracellular enzymes present in living organisms, as opposed to dead, non-enzyme producing organisms. These enzymes are harnessed in the in vivo reactions with a non-fluorescent dye containing a select organic functional group that is known to be cleaved or hydrolyzed by the certain enzyme. The dye is tailored such that one of the products fluoresces, so that by employing a conventional spectrofluorimeter, the rate of fluorescence can be determined. By subjecting a plurality of samples having different cellular concentrations of viable microorganisms to the same non-fluorescent dye, or by subjecting the same bacterial sample to a number of different non-fluorescent dyes, a pattern of fluorescent rates emerge.

Abstract: Fluorogenic substrates and compositions containing the same are useful for detecting enzymatic activity within intact cells. The fluorogenic substrates are lysosomotropic derivatives of 2,3-dicyano-hydroquinone (DCH). These derivatives may be employed for detecting lysosomal enzymatic activity within intact cells using fluorescent microscopy and cytometry.

Abstract: The present invention is concerned with the use of aniline derivatives of the general formula: ##STR1## wherein R.sub.1 is a hydrogen atom or a lower alkyl or aryl radical, R.sub.2 is a hydrogen atom or a lower alkyl radical which can be substituted by hydroxyl, amino, carboxyl, lower alkoxycarbonyl, lower alkanoylamido, aryl lower alkyl or aryl or by a group of the structure: ##STR2## in which R.sub.5 and R.sub.6 have the meanings given hereinafter, R.sub.3 is a hydrogen or halogen atom or a carboxyl group, R.sub.4 and R'.sub.4, which can be the same or different, are hydrogen or halogen atoms, carboxyl groups or lower alkoxy radicals or lower alkyl radicals which are preferably in the m-position and R.sub.1 and R.sub.4 or R.sub.2 and R.sub.4 or R.sub.4 and R'.sub.4, when the two substituents are ortho to one another, can together form a saturated or unsaturated hydrocarbon chain containing 2 to 6 carbon atoms which can be substituted by hydroxyl or oxo groups, R.sub.

Abstract: A method for the quantitative determination of polyamines, which comprises allowing a polyamine oxidizing enzyme, an .omega.-aminoalkylaldehyde dehydrogenase, an oxidized nicotinamide coenzyme and, as required, an acylpolyamine anidohydrolase to act upon a sample solution containing polyamines (for example, urine, blood and other kinds of body fluid), and measuring the reduced nicotinamide coenzyme thus formed by, for example, colorimetry, thereby determining the amount of said polyamines.

Abstract: A transparent and stable aqueous solution of a substrate for a determination of lipase, comprising triglyceride uniformly solubilized therein, a method for determination of lipase activity in a sample, employing the aqueous solution, and a process for the manufacture of a lyophilized product capable of forming the aqueous solution are disclosed. A process for the manufacture of a transparent and stable aqueous solution of triglyceride is also disclosed.

Abstract: Chromogenic substrates for the detection of hydrolyzing enzymes, processes for the preparation of these chromogenic substrates and the use of the chromogenic substrates.The quantification of hydrolyzing enzymes for diagnostic purposes has to date been carried out by determination of the amounts of a fluorescent or highly absorbent chromogenic dyestuff liberated in the hydrolytic reaction, detection of which has in part been possible exlusively by instruments or in the neutral to alkaline pH range. After enzymatic hydrolysis, the chromogenic substrates according to the invention lead to highly sensitive and specifically measurable signals regardless of the pH range.Azo dyestuff compounds with the general formulaA--N.dbd.N--B (OR)in which A) and B) have the meanings given in claim 1 andR) is a radical which can be liberated by enzymatic hydrolysis, excluding a carbonyl radical,are provided.After enzymatic hydrolysis, highly sensitively and specifically measurable signals are obtained regardless of the pH range.

Abstract: An assay for detecting the presence of Mycoplasma fermentans is disclosed, along with a diagnostic kit that uses the assay. The presence of Mycoplasma fermentans is indicated by the presence of the restriction endonuclease MfeI. This endonuclease recognizes the sequence CAATTG and cleaves between the C and the first A.

Abstract: A simple, rapid and reliable assay for measuring functional enzyme inhibitor levels in body fluids and tissues, especially functional .alpha..sub.1 -proteinase inhibitor levels in human plasma or serum. .alpha..sub.2 -Macroglobulin is first inactivated, then porcine pancreatic elastase incubated with the samples to form a complex between the elastase and the functional .alpha..sub.1 -PI. Deficient individuals are detected by the presence of a color change following addition of substrate. If desirable, residual enzyme activity can then be calculated and the .alpha..sub.1 -PI levels present in the original sample determined. The method provides a means for early screening of individuals with a genetic deficiency in circulating levels of .alpha..sub.1 -PI, thereby facilitating treatment and prevention of familial emphysema.

Abstract: A method for distinguishing an alcoholic person from non-alocholic persons which comprises measuring the potential of lymphocytes from that person to produce phosphatidylenthanol and comparing the results obtained with a standard obtained from the lymphocytes of persons which are known not to be at substantial risk of becoming alcohol dependent which lymphocytes were incubated under identical conditions.

Abstract: Novel xanthene dyes are disclosed which excite in the range of 450-650 nm and which will emit to the red of fluoroscein. These dyes may be coupled to tagging agents, such as monoclonal antibodies, and used to detect cells in a sample.

Abstract: A method of determining the susceptibility of microorganisms to an antimicrobial substance wherein the microorganisms are cultured in the presence of the antimicrobial substance, and growth or lack of growth is assessed by detecting a fluorescent product resulting from the enzymatic hydrolysis of a fluorogenic substrate. The fluorogenic substrates include 7-N-(aminoacyl)-7-amido-4-methylcoumarin, 7-N-(alanyl)-7-amido-4-methylcoumarin, or a combination of 7-N-(alanyl)-7-amido-4-methylcoumarin and 4-methyl umbelliferyl phosphate.

Abstract: A process for performing an enzyme-catalyzed reaction in a reaction mixture comprising two phases, a first aqueous phase containing an enzyme which is not accessible and a second substantially non-aqueous phase containing a non-water miscible solvent wherein a compound derived from fluorescein or a substituted fluorescein is present for the purpose of measuring aqueous pH by monitoring spectroscopic properties. Novel compounds derived from fluorescein or a substituted fluorescein which may be used in the process and a method for producing some of the novel compounds are also claimed.