Wo 2012/058621 A2

(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International Publication Date 3 May 2012 (03.05.2012) WO 2012/058621 A2

(51) International Patent Classification: (81) Designated States (unless otherwise indicated, for every C12Q 1/527 (2006.01) kind of national protection available): AE, AG, AL, AM, AO, AT, AU, AZ, BA, BB, BG, BH, BR, BW, BY, BZ, (21) International Application Number: CA, CH, CL, CN, CO, CR, CU, CZ, DE, DK, DM, DO, PCT/US201 1/058432 DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT, (22) International Filing Date: HN, HR, HU, ID, IL, IN, IS, JP, KE, KG, KM, KN, KP, 28 October 201 1 (28.10.201 1) KR, KZ, LA, LC, LK, LR, LS, LT, LU, LY, MA, MD, ME, MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, (25) Filing Language: English NO, NZ, OM, PE, PG, PH, PL, PT, QA, RO, RS, RU, (26) Publication Language: English RW, SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM, TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, (30) Priority Data: ZM, ZW. 61/408,4 18 29 October 2010 (29.10.2010) U S (84) Designated States (unless otherwise indicated, for every (71) Applicant (for all designated States except US): SHIRE kind of regional protection available): ARIPO (BW, GH, HUMAN GENETIC THERAPIES INC. [US/US]; 300 GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, SZ, TZ, Shire Wau, Lexington, MA 02421 (US). UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, (72) Inventor; and DE, DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, (75) Inventor/Applicant (for US only): WIEDERSCHAIN, LT, LU, LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, Gherman [US/US]; 63 Bradwood Street, Roslindale, MA SE, SI, SK, SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, 0213 1 (US). GA, GN, GQ, GW, ML, MR, NE, SN, TD, TG). (74) Agent: TRE ANNIE, Lisa, M.; Morse, Barnes-brown & Published: Pendleton, P.C., Reservoir Place, 1601 Trapelo Road, Suite 205, Waltham, MA 0245 1 (US). — without international search report and to be republished upon receipt of that report (Rule 48.2(g))

(54) Title: ASSAYS AND KITS TO DETERMINE UREA CYCLE ENZYME ACTIVITY ON SOLID SUPPORT

- (57) Abstract: The present invention discloses compositions, methods, kits and assays which facilitate the rapid, high-throughput and sensitive detection of at least one enzyme in a test sample. Also disclosed are assays performed on a solid support which are o useful for the detection of urea cycle enzymatic activity. Inventor(s): Gherman Wiederschain

Attorney's Docket No.: SHIR-002-WO1

ASSAYS AND KITS TO DETERMINE UREA CYCL E ENZYME ACTIVITY ON

SOL ID SUPPORT

REL ATED APPL ICATIONS This application claims the benefit of U.S. Provisional Application No. 61/408,418, filed October 29, 2010. The entire teachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION The urea cycle represents a series of metabolic processes which occur in mammals that produce urea from ammonia. A deficiency in any of these metabolic processes, or more particularly the enzymes involved in these processes, may result in the accumulation of excess ammonia which may be toxic. Specifically, the urea cycle consists of a series of five biochemical reactions and serves two primary functions: the elimination of nitrogen as urea and the synthesis of arginine. Defects in the urea cycle result in the accumulation of ammonia and its precursor amino acids (glutamine, glutamic acid, aspartic acid, and glycine). The resulting high levels of ammonia are neurotoxic, and the triad of hyperammonemia, encephalopathy, and respiratory alkalosis frequently characterize the urea cycle metabolic disorders. As a result, such deficiencies may result in extensive ammonia accumulation, which in turn may lead to extensive liver damage and death if not properly treated. Advances in molecular biology have supported the development of recombinant biological agents {e.g., recombinant proteins and/or enzymes) which are useful for modulating protein or enzyme activity or nucleic acid expression. For example, such recombinantly prepared proteins and/or enzymes may be therapeutically administered to humans who demonstrate an endogenous deficiency of such protein and/or enzyme. The diagnosis of such endogenous deficiencies, as well as the in vitro production of recombinant biological agents both require means of rapidly assessing the enzymatic activity of the recombinantly prepared enzyme. Generally, assays used to assess enzymatic activity involve contacting a biological sample or a recombinantly prepared enzyme with a substrate with which the enzyme of interest is known to predictably react. Enzymatic activity may be subsequently measured by either evaluating the depletion of the substrate and/or the yield of an enzymatic product over time. Different methods of measuring the concentrations of substrates and/or products exist and many enzymes can be assayed in several different ways, many of which are time-consuming. Assays available to analyze enzymatic activity are frequently performed in a test tube which may prove especially cumbersome and time consuming, for example due to the handling of hazardous waste and/or the immobilization of substrates onto solid supports. Such analyses may be further complicated by the performance of multiple steps or reactions, each often performed on a different solid support. A number of time consuming assays for determining the enzymatic activity of the enzyme L-arginine amidinohydrolase (ARG) have been previously described. (See,

Chinard, FP., J . Biol. Chem. 199: 91-95 (1952); Konarska, L ., et al Clin. Chim.

Acta, 154: 7-17 (1986); Mellerup, B., Clin. Chim. 13 (10): 900-908 (1967); Jung, D., et al., Clin. Chim. 2 1 (8): 1136-1 140 (1975); and Levinson, S Clin. Chim. 24 (12):2199-2202 (1978)). Most of these assays are based on quantification of either ornithine or urea as the products of the ARG enzymatic reaction and are performed in a test tube. There is therefore a need for improved methods and high-throughput assays for the routine analysis of enzymatic activity, preferably which are performed in a quick and accurate manner, in a single solid support. The methods, assays and kits of the present invention are useful in carrying out such methods.

SUMMARY OF THE INVENTION The present invention relates to methods, assays and kits which are useful for the high-throughput determination of enzymatic activity of a test sample. Generally, such methods, assays and kits comprise the steps of contacting a test sample with a substrate and using routine means (e.g., absorption or fluorescence spectroscopy techniques) to quantify the product of an enzymatic reaction, or alternatively the depletion of a substrate. The methods, assays and kits of the present invention contemplate the use of substrates which are known to predictably react with an enzyme whose presence is suspected in a test sample. In some embodiments the methods, assays and kits of the present invention provide useful tools to measure the presence of urea cycle enzymes (i.e., carbamoyl phosphate synthetase, ornithine transcarbamylase, argininosuccinate synthetase, argininosuccinate lyase and L- arginine amidinohydrolase) in a test sample, or alternatively the principles presented herein can be applied generally to determine the presence of any particular enzyme in a test sample. The methods, assays and kits of the present invention provide tools which are useful to visually, colorimetrically, fluorometrically and/or chemically distinguish the presence or absence of a predicted enzymatic reaction, and are preferably capable of quantitatively determining the presence of an enzyme in a test sample using routine means (e.g., absorption or fluorescence spectroscopic techniques). For example, in one embodiment of the present invention, enzymatic activity, such as the hydrolysis of a substrate by an enzyme, may yield a fluorescently-detectable signal which can be measured using fluorescence spectroscopy and thus provide means of quantitatively assessing the presence of such enzyme. The methods, assays and kits may optionally be useful for the detection of an enzyme as a diagnostic marker, predictor or identifier of disease (e.g., diagnosis of particular urea cycle enzyme deficiencies by evaluating a test sample obtained from a human). One embodiment of the present invention relates to methods and assays which are useful for determining and/or measuring the presence and/or activity of the enzyme ornithine transcarbamylase (OTC) in a solid support. OTC is the second enzyme in the mammalian urea cycle and is responsible for catalyzing the transfer of a carbamoyl group of carbamyl phosphate to ornithine, thus producing citrulline. Such methods and assays comprise the steps of determining OTC activity by reacting a test sample with ornithine and a suitable substrate (e.g., carbamyl phosphate). The reaction is subsequently halted by contacting the reactants with an anti-catalyst or stop buffer (e.g., phosphoric acid and/or sulfuric acid) to enable quantification of the applicable reactants. The present invention contemplates determining and/or quantifying the presence of OTC in the test sample by, for example, quantifying the production of citrulline during the preceding reaction. n a preferred embodiment, citrulline production is determined by contacting the reactants with a chromogenic reagent (e.g., 2, 3 butanedione monoxime) under suitable conditions (e.g., in acidic media at about 95-100° C) such that a detectable signal is produced which enables a colorimetric quantification of the citrulline present in the reaction media, if any. OTC activity may accordingly be measured as a function of citrulline production in the solid support, for example using known spectroscopic techniques. In a preferred embodiment, the methods, assays and kits of the present invention are performed in a single solid support (e.g., a 96-well microplate). Also contemplated are kits for determining the OTC activity in a test sample. Such kits may comprise at least one solid support, carbamyl phosphate, at least one substrate, an anti-catalyst and a chromogenic reagent. The kits of the present invention may further comprise a buffer (e.g., sodium carbonate, sodium bicarbonate, potassium phosphate, tris(hydroxymethyl)aminomethane, DPBS and combinations thereof). Another embodiment of the present invention relates to methods and assays which are useful for determining and/or measuring the presence and/or activity of the enzyme argininosuccinate synthetase (AS) in a solid support. AS is the third enzyme in the mammalian urea cycle and is responsible for catalyzing the ATP-dependant condensation of citrulline with L-Aspartic acid to produce argininosuccinic acid, which is the immediate precursor of arginine and urea. Such methods and assays comprise the steps of determining AS activity by reacting a test sample with citrulline, a substrate (e.g., L-Aspartic acid) and adenosine triphosphate (ATP). The reaction is subsequently halted by contacting the reactants with an anti-catalyst or stop buffer (e.g., phosphoric acid and/or sulfuric acid) to enable quantification of the applicable reactants. The present invention contemplates determining and/or quantifying the presence of AS in the test sample by, for example, quantifying the depletion of citrulline during the preceding reaction. In a preferred embodiment, citrulline depletion is determined by contacting the reactants with a chromogenic reagent (e.g., 2, 3 butanedione monoxime) under suitable conditions (e.g., in acidic media at about

95-100° C) such that a detectable signal is produced which enables a colorimetric quantification of the citrulline present in the reaction media, if any. AS activity may accordingly be measured as a function of citrulline depletion in the solid support, for example using known spectroscopic techniques. In a preferred embodiment, the assays and methods of the present invention are performed in a single solid support (e.g., a 96-well microplate). Also contemplated are kits for determining AS activity in a test sample. Such kits may comprise at least one solid support, citrulline, at least one substrate, ATP, an anti-catalyst and a chromogenic reagent. The kits of the present invention may further comprise a buffer (e.g., sodium carbonate, sodium bicarbonate, potassium phosphate, tris(hydroxymethyl)aminomethane, DPBS and combinations thereof). Another embodiment of the present invention relates to methods and assays which are useful for determining and/or measuring the presence and/or activity of the enzyme argininosuccinate lyase (ASL ) in a solid support. ASL is the fourth enzyme in the mammalian urea cycle and is responsible for catalyzing the cleavage of L- argininosuccinate, thus producing the amino acid L-arginine and fumaric acid. Such methods and assays comprise the steps of determining ASL activity by reacting a test sample with a substrate (e.g., L-argininosuccinic acid). The present invention contemplates determining and/or quantifying ASL activity in the test sample by, for example, quantification of fumaric acid produced during the preceding reaction. In the context of the present invention, the product of the ASL activity (e.g., fumaric acid) may itself function as a detectable signal which can be colorimetrically quantified. In a preferred embodiment, fumaric acid production is measured, for example using known spectroscopic techniques. In accordance with the present invention, the production of fumaric acid is a function of ASL activity in the test sample and its presence in the reaction media may signal ASL activity. In a preferred embodiment, the assays and methods of the present invention are performed in a single solid support (e.g., a 96-well microplate). Also contemplated are kits for determining ASL activity in a test sample. Such kits may comprise at least one solid support and at least one substrate. The kits of the present invention may further comprise a buffer (e.g. , sodium carbonate, sodium bicarbonate, potassium phosphate, tris(hydroxymethyl)aminomethane, DPBS and combinations thereof). The present invention also contemplates the detection of fumaric acid by comparison to one or more colorimetric standards. One embodiment of the present invention relates to methods and assays which are useful for determining and/or measuring the presence and/or activity of the enzyme L-arginine amidinohydrolase (ARG) in a solid support. ARG is the fifth enzyme in the mammalian urea cycle and is responsible for the detoxification of ammonia by catalyzing the hydrolysis of L-arginine to produce L-ornithine and urea. Such methods and assays comprise the steps of determining ARG activity by reacting a test sample with a substrate (e.g., L-arginine). In a preferred embodiment, the test

sample is pre-treated with a solution comprising manganese chloride (MnCl2). The reaction is subsequently halted by contacting the reactants with an anti-catalyst or stop buffer (e.g., acetic acid) to enable quantification of the applicable reactants. The present invention contemplates determining and/or quantifying ARG activity of the test sample by, for example, quantification of L-ornithine production during the preceding reaction. In a preferred embodiment, L-ornithine production is determined by contacting the reactants with a chromogenic reagent (e.g., ninhydrin) under suitable conditions (e.g., in acidic media at about 95-100° C) such that a detectable signal is produced which enables a colorimetric quantification of the L-ornithine present in the reaction media, if any. ARG activity may accordingly be measured as a function of the production of L-ornithine in the solid support, for example using a spectrophotometer to determine the presence of the L-ornithine/ninhydrin complex. In a preferred embodiment, the assays and methods of the present invention are performed in a single solid support (e.g., a 96-well microplate). Also contemplated are kits for determining ARG activity in a test sample. Such kits may comprise at least one solid support, at least one substrate, an anti- catalyst and a chromogenic reagent. Such kits may optionally comprise manganese. The kits of the present invention may further comprise a buffer (e.g., sodium carbonate, sodium bicarbonate, potassium phosphate, tris(hydroxymethyl)aminomethane, DPBS and combinations thereof). The above discussed and many other features and attendant advantages of the present invention will become better understood by reference to the following detailed description of the invention when taken in conjunction with the accompanying examples. The various embodiments described herein are complimentary and can be combined or used together in a manner understood by the skilled person in view of the teachings contained herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the calibration curve for citrulline standards using the microplate format assay described in Example 1. FIG. 2 represents the calibration curve for citrulline standards using the microplate format assay described in Example 2 . FIG. 3 represents the calibration curve for fumaric acid standards using the microplate format assay described in Example 3. FIG. 4 represents the calibration curve for ornithine standards using the microplate format assays described in Example 4.

DETAIL ED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Provided herein are novel methods, assays and kits which are useful for the quick and accurate analysis of the enzymatic activity of a test sample. The present invention relies upon the intrinsic properties of an enzyme whose presence is suspected in a test sample as the means of detecting and/or quantifying such enzyme. The invention contemplates contacting the enzyme whose presence is suspected in the test sample with substrates and/or additional reactants, such that if the enzyme is present in the test sample, such enzyme will catalyze a predicted reaction, the result of which is the production or destruction of a detectable signal. In a preferred embodiment, the methods and assays of the present invention are based on the quantification of a detectable signal (e.g., specified chromogenic or fluoro genie reagents) where the all steps (e.g., incubation, color developing, heating, absorbance reading and the determination of enzymatic activity) are performed in a solid support (e.g., a 96-well microplate). The present invention contemplates the detection and/or quantification of a particular enzyme in a test sample based upon such enzyme's ability to catalyze a particular reaction. As used herein, the phrase "enzymatic activity" refers to an enzyme's ability to catalyze a repeatable biochemical reaction, for example, when contacted with a substrate with which such enzyme is known to react. In a preferred embodiment of the present invention, the enzymatic activity of an enzyme may be exploited to confirm the presence or absence of such enzyme in a particular test sample. For example, many enzymes have known and repeatable catalytic activity which may be enhanced under certain conditions (e.g., in the presence of a substrate) and the present inventions exploit such catalytic activity as a means of detecting the enzyme. As used herein, the term "catalyzes" means to accelerate the rate of a reaction by a substance which remains chemically unchanged by that reaction. The methods, assays and kits of the present invention are particularly useful for determining the enzymatic activity of the enzymes involved in the urea cycle. The urea cycle is comprised of five unique enzymes, the primary function of which are to convert toxic ammonia released from amino acid metabolism into urea for excretion in the urine. In inherited disorders of the urea cycle, this conversion is impaired, leading to high levels of blood ammonia which can cause severe illness and even death. The five enzymes which catalyze the urea cycle are carbamoyl phosphate synthetase, ornithine transcarbamoylase (OTC), argininosuccinate synthetase (AS), argininosuccinate lyase (ASL ) and L-arginine amidinohydrolase (ARG). The first two enzymes of the urea cycle are located within the mitochondrial matrix (Gamble, et al, J . Biol. Chem. 248: 610-618 (1973); Clark, J. Biol. Chem. 251:950-161 (1976)) and the remaining three enzymes are cytosolic (Pierson, et a , J. Biol. Chem. 252: 6464-6469 (1977)). OTC is the second enzyme in the mammalian urea cycle which catalyzes the formation of citrulline from carbamoyl phosphate. (Mori, et al, Mol. Cell Biochem. 49:97-1 11 (1982); Sheffield, et al Biochem. Biophys. Res. Commun. 134:21-28 (1986)). OTC is located in the mitochondria and catalyzes the transfer of a carbamoyl group of carbamyl phosphate to ornithine, producing citrulline and inorganic phosphate. OTC deficiency is a severe human metabolic disease for which there is no effective treatment. A number of methods for OTC analysis have been described based on colorimetric quantification of citrulline as the by-product of the OTC enzymatic reaction. (Archibald, RM., J . Biol. Chem. 156:121-142 (1944); Ratner, S., Methods in Enzymology, (Corowick S. P., Kaplan N.D., Eds.), vol. II, pp. 356-367, Academic Press, New York, 1955; Jones, ME., In: Methods in Enzymology, (Colowick S. P., Kaplan N. D., Eds,), vol. 5,. pp. 910-913, Academic Press, New York, 1962; Farkas, DH, et al., Anal. Biochem. 160:421-428 (1987)). AS is the third enzyme in the mammalian urea cycle and catalyzes the ATP- dependent condensation of citrulline with aspartate to form argininosuccinic acid. AS deficiency is a severe human autosomal recessive metabolic disease, for which there is no effective treatment. (Snodgrass, Pediatrics, 68: 273-283 (1981); Brusilow, et al, Urea cycle enzymes. In: The Metabolic and Molecular Bases of Inherited Diseases, 8th Edition, Scriver CR (Eds.), McGraw-Hill, Inc., New York, pp. 1909-19063 (2001)). ASL is the fourth enzyme in the mammalian urea cycle and catalyzes the cleavage of L-argininosuccinate into the amino acid L-arginine and fumaric acid. Deficiency of ASL is a severe human autosomal recessive metabolic disease for which there is no effective treatment. (Brusilow, et al, (2001); Snodgrass, (1981)). ARG is the fifth enzyme in the mammalian urea cycle, which is responsible for catalyzing the last reaction in the urea cycle and plays a key role in the detoxification of ammonia. ARG catalyzes the hydrolysis of arginine to ornithine and urea. Two distinct ARG isozymes, named ARG-I and ARG-II, have been identified. ARG-I is found primarily in the liver, and also in erythrocytes. Deficiency of ARG-I is a severe, rare human autosomal recessive metabolic disease for which there is no effective treatment. (Brusilow, et al, (2001); Snodgrass, (1981); Crombez, et al, Mel. Genet. Metab. 84 (3):243-251 (2005)). Although the methods, assays and kits of the present invention are useful for determining the activity of all of the urea cycle enzymes (e.g., carbamoyl phosphate synthetase), the inventions and concepts described herein are generally applicable to the detection of other enzymes. Enzymatic activity may be measured by routine means known to one of ordinary skill in the art (e.g., colorimetric, spectrophotometric, fluorometric or chromatographic detection assays) by determining, for example the consumption or depletion of substrate and/or the production of a product over time. In accordance with the present invention, substrate depletion of about 5%, 10%, 20%, 30%, 40%, 50% or more, or preferably about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more relative to the amount of substrate introduced may be indicative of enzymatic activity. Alternatively, following contacting an enzyme with a substrate, a relative increase in the formation of a product, or the conversion of that substrate to a product, in each case of about 5%, 10%, 20%, 30%, 40% , 50%o or more, or preferably about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more preferably 100% or more, may be indicative of enzymatic activity. The methods, assays and kits of the present invention contemplate the selection of appropriate enzyme substrates and/or reactants to determine enzymatic activity. As such, an understanding of enzyme kinetics and in particular the catalytic properties of the enzyme(s) being evaluated are required to practice the methods described herein. Knowledge of the substrate specificity of the enzyme whose presence is suspected in a test sample can enable the identification of such enzyme.

The properties of an enzyme, such as for example, Michaelis-Menton constants (Km) and/or turnover numbers (Kc ) as they relate to a particular substrate, provide the basis for evaluating the sensitivity of an enzyme for one or more substrates and provide information regarding the reproducibility of the methods and assays contemplated by the present inventions. As used herein, the terms "react" and "reaction" are used in their broadest senses and refer to enzymatic, chemical, physical and/or biological processes (e.g., hydrolysis) which alter or transform one or more of the participating reactants. For example, test samples with known enzymatic activity (e.g., OTC) may be expected to react with a particular substrate (e.g., ornithine) which, under certain conditions will transform one or more of the reactants (e.g., OTC catalyzes the transformation of ornithine to citrulline). In an enzymatic reaction, a reactant may e expended i the reaction to yield a product. In accordance with the present invention, a determination of the depletion of one or more reactants and/or the yield of one or more products will signal an enzymatic reaction, and accordingly the presence of the enzyme in the test sample. The term "contact" or "contacting" means bringing two or more moieties together, or within close proximity of one another such that the moieties may react. For example, in one embodiment of the present invention, contacting an enzyme with its corresponding substrate may be expected to cause a particular reaction. To evaluate and quantify enzymatic activity, the enzymatic reaction should preferably be halted. The present invention contemplates the addition of an anti- catalyst or stop-buffer to the enzymatic reaction to slow, stop or terminate such enzymatic, chemical, or biological reaction. For example, the addition of an anti- catalyst to an ongoing enzymatic reaction would be expected to slow or stop the catalytic activity of the enzyme. Suitable anti-catalysts contemplated by the invention, include, for example, glacial acetic acid, phosphoric acid, sulfuric acid and combinations thereof. As used herein, the term "substrate" refers to a molecule, complex, material, substance or reactant upon which an enzyme acts (e.g., chemically or biologically). Generally, the substrate participates in a biochemical enzymatic reaction due to the enzymatic activity of the enzyme. Preferably, the substrates contemplated by the present inventions are specific for an enzyme present in a test sample. For example, L-argininosuccinic acid is a substrate which is specific for ASL . When contacted with L-argininosuccinic acid, the enzyme ASL would be expected to catalyze a reaction whereby L-argininosuccinic acid is transformed to the products L-arginine and fumaric acid. Substrates useful in the methods of the invention can be native or modified. Modified substrates useful in the invention retain the ability to be acted upon by the corresponding enzyme. Exemplary modifications suitable for substrates include, for example, labeling to confirm the presence r absence of enzymatic activity (e.g., fluorogenic substrates). As used herein, the phrase "test sample" is used in its broadest sense and means any solid or liquid preparation suspected of having enzymatic activity. Test samples are preferably obtained from biological media or materials, including biologically or recombinantly derived media which may contain, among other things, naturally occurring or recombinantly prepared peptides, polypeptides or proteins, enzymes, lipid or carbohydrate molecules, or glycosylated proteins or enzymes, or other samples obtained from an recombinant media, including any fractions thereof. The test samples contemplated by the present invention may be obtained from in- process or "dirty" biological systems, for example, those obtained during the preparation of a recombinant enzyme. In a preferred embodiment, test samples include biological samples (e.g., samples obtained for diagnostic purposes) such as, for example samples comprising tissue, whole blood, serum, plasma, cell lysates, lymphatic fluid, saliva, cerebrospinal fluid, synovial fluid, urine, nasal secretion, and other bodily fluids. The methods, assays and kits of the present invention advantageously require a limited amount of protein or enzymes in the test sample. In a preferred embodiment of the present invention, the methods and assays may be performed using a low volume or amount of a test sample or biological sample (e.g., 100ug, 75µg, 5 µg, µg , 3 0µg , 5µg, µg , 15 µg, 10µ g, 5µg, 1µg or less of tissue homogenate or pure enzyme or protein). In preferred embodiments, the test sample is obtained from a biological source, such as cells in culture or a tissue sample from an animal or microorganisms, most preferably, a human. A suitable test sample may be obtained from lysates of selected microorganisms and prepared in accordance with the present invention. To determine the enzymatic activity of a test sample, such test sample is contacted with selected substrates and/or reactants such that if the enzyme of interest is present in the test sample the presence of a detectable signal will indicate enzymatic activity. Detection and/or quantification of enzymatic activity are preferably determined by measuring the presence of one or more chemically, fluorometrically or colorimetrically detectable signals. As used herein, the term "detectable signal" is used in its broadest sense to refer to any indicator of enzymatic activity. Preferably, detectable signals are measurable using routine means. The presence of the detectable signal, and where appropriate its measurement, facilitate the determination and/or quantification of enzymatic activity when used in accordance with the present invention, Preferably, the presence or absence of a detectable signal correlates to the presence or absence, respectively, of enzymatic activity in the test sample. In one embodiment of the present invention, a detectable signal is produced by the addition of one or more chromogenic reagents to an enzymatic reaction under appropriate conditions such that the chromogenic reagent will conjugate to one or more of the participating reactants, or alternatively to the product of a reaction. The conjugation of the chromogenic reagent to the reactant or the product thus provides a detectable signal which will enable detection and/or quantification of the reactant or product to which it conjugates. In a particular embodiment of the present invention, the addition of a chromogenic reagent to the enzymatic reaction may conjugate with a reactant to form a detectable signal, that will in-turn enable quantification of such reactant in the reaction. For example, the addition of a chromogenic reagent which is known to predictably bind citrulline in strong acidic conditions (e.g., 2, 3-butanedione monoxime) will conjugate to citrulline present in an enzymatic reaction and produce a detectable signal. The detectable signal will then enable the detection of citrulline, the resulting optical density of which may be determined, for example in a spectrophotometer, read at 490nm. (Pearson WR., Biochem. J. 33:902-907 (1939); Gornall, AG., Biochem. J. 35:650-658 (1941)). A reduction in the amount, or the absence of such a detectable signal may be indicative of enzymatic activity. Alternatively, in another embodiment the chromogenic reagent may be capable of facilitating the detection of an enzymatic product (e.g., by conjugating to the product of an enzymatic reaction). For example, the chromogenic reagent ninhydrin is known to conjugate with ornithine under acidic conditions and produce a detectable signal which may be quantified based on its strong absorption at 512nm. In this particular embodiment, the presence of the detectable signal may be indicative of enzymatic activity. In another embodiment of the present invention, the use of a fluorogenic substrate may liberate a detectable signal when contacted with an enzyme with which it is known to react. The exposure of such a fluorogenic substrate to an enzyme, for example an enzyme known to predictably hydrolyze it under appropriate conditions, will liberate a detectable signal which is indicative of enzymatic activity. As used herein, the term "fluorogenic" refers to a state or condition of having the capability to be fluorescent. As used herein, the term "fluorogenic substrate" refers to a non- fluorescent or weakly-fluorescent enzyme substrate that becomes more fluorescent (e.g., at least about 2, 4, 6, 10, 20, 50 or 100 times more fluorescent) upon the occurrence of an enzymatic, chemical, biochemical, physical and/or other similar transformative event. In another embodiment, the products of the enzymatic reaction may themselves be capable of detection and/or quantification without the use of a chromogenic reagent. For example, the enzyme argininosuccinate lyase (ASL ) is responsible for catalyzing the cleavage of L-argininosuccinate into the amino acid L- arginine and fumaric acid. The formation of fumaric acid may be measured, for example spectrophotometrically from the increase in absorbance at 240 nm. (Ratner, S., Methods Enzymol. 17: 304-309 (1970)). Accordingly, based on its strong signals at 240nm, fumaric acid may be a detectable signal whose presence may be quantified as an indicator of enzymatic activity. The methods, assays and kits of the present invention contemplate the determination of enzymatic activity, including the steps of incubation and final absorbance reading, in a single solid support (e.g., a 96-well microplate), thus avoiding issues relating to, for example, multiple transfers of reactants or the reaction media. The methods, assays and kits of the invention permit real-time analysis of enzymatic activity while providing enhanced convenience and maintaining sensitivity. By determining enzymatic activity in a single solid support, the present invention provides a quick and accurate means of assessing enzymatic activity. The assays, methods and kits of the present invention are particularly distinguishable from traditional test tube-based assays, which often require the performance of multiple steps and multiple transfers between solid supports. As the term is used herein, "solid support" refers to an inert solid or semi-solid material in which, or on which the enzymatic activity of a test sample may be assessed in accordance with the assays and methods of the present invention. Typical solid supports include, for example, beads, tubes, chips, resins, plates, microplates, wells, films, and sticks. The solid supports may comprise various materials, for example, plastic, glass, ceramic, silicone, metal, cellulose, gels, polystyrene, polyester, and dextran. In a preferred embodiment, the solid support contemplated by the present invention is a standard multiple-well microplate (e.g., a standard polystyrene 96-well microplate, flat bottom with low evaporation lid and well volume.) The present invention also relates to kits which are useful for determining enzymatic activity of a test sample. Such kits preferably comprise reagents necessary to initiate an enzymatic reaction and facilitate the determination of enzymatic activity. For example, one embodiment of the present invention contemplates kits for determining the presence of AS in a test sample, wherein such kits may comprise a solid support, citrulline, L-aspartic acid, ATP, an anti-catalyst and a chromogenic reagent. In this particular embodiment, citrulline depletion may be measured by detection of the chromogenic reagent, which is indicative of AS activity. In one embodiment, the components of such kits are integrated into a single solid support such that the determination of enzyme activity is performed in that solid support. Preferably, the kits of the present invention further comprise suitable colorimetric standards which are useful for measuring enzymatic activity in solid support. The identification and quantification of the detectable signals described herein {e.g., the formation of colored products or the detection of color or the absorption of light) may be performed by any suitable means known. The simplest is visual observation of color development or color change. Alternatively, embodiments of the present invention requiring quantitative measurement will best be performed by spectrophotometry. Choice of the detection device will be governed by the intended application and considerations of cost, convenience, and whether creation of a permanent record is required. Detection of molecules by fluorescence has several advantages compared to alternative detection methods. Fluorescence provides an unmatched sensitivity of detection, as demonstrated by the detection of single molecules using fluorescence. (Weiss, S., Science 283: 1676-1683 (1999)). Detection of fluorescence, changes in fluorescence intensity or changes in emission spectra can be easily achieved by the selection of specific wavelengths of excitation and emission. Fluorescence provides a real-time signal, allowing real-time monitoring of processes and real-time cellular imaging by microscopy. (L akowicz, J. R. Principles of Fluorescence Spectroscopy, luwer Academic Plenum Press, New York, 1999, which is herein incorporated by reference). Additionally, well-established methods and instrumentation for high- throughput detection of fluorescence signals exist in the art. (Hill J., et al., Methods in Enzymol. 278: 390-416 (1997)). The articles "a" and "an" as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to include the plural referents. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Where elements are presented as lists, e.g., in Markush group or similar format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not in every case been specifically set forth in so many words herein. It should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. EXAMPL ES

EXAMPL E 1

Determining Ornithine transcarbamylase (OTC) Activity in 96- ell Plate Format

The present experiments were designed to provide a specific, sensitive and high-throughput assay for determining the presence of ornithine transcarbamylase (OTC) activity of a test sample in a 96-well microplate and to provide a rapid, high- throughput, simple, and sensitive method for routine analysis and measurement of OTC activity. Generally, OTC activity was determined by citrulline quantification, which was performed by reacting the citrulline with the chromogenic reagent 2, 3- butanedione monoxime during heating at approximately 95-100°C in strongly acid solution. The detectable signal thus obtained was specific for citrulline and the resulting optical density was read in a spectrophotometer at 490 ran.

Materials and Equipment • Polystyrene 96-well plate, flat bottom with well volume of approximately 350µ1(Falcon, Becton Dickinson Labware, cat. # 353075 or equivalent); • Centrifuge with plate carrier (Beckman GS-6 Centrifuge); • Dry-bath with heating block/platform for plate; • Thermowell sealing aluminum foil tape for 96-well plates (Corning, Cat. # 6570); • Thermosetting plate shaker (The Jitterbug, Boekel Scientific); • Plate reader (SpectraMax2, Molecular Devices); and • Pipettes for various volumes.

Assay Protocol

Sample Preparation Citrulline assay standard solutions were prepared by diluting citrulline (Acros Organics, Cat. # 110470250) to ImM citrulline working solution with water, and then adding 0, 10, 20, 30, 50, 80 and 10ΟµΙ of the working solution to the separate wells in duplicate for each concentration. Accordingly, the volume of working solution was equivalent to 0, 10, 20, 30, 50, 80 and 100 nmoles of citrulline, respectively. Additional blank wells were set up with 100µ of water per well. The final volume of each well was adjusted to 100µ with water and the microplate was placed on ice, and covered with aluminum foil to prevent condensation on the bottom of the plate. To determine the OTC activity of the test samples, the test samples were prepared by adding 2-10µL of cell lysates from the test sample, containing approximately 10 µg of total protein, to the microplate wells in duplicate. A background control was also prepared by adding the same volume of cell lysates in duplicate as previously added for the OTC activity samples. An internal control was also prepared by adding approximately 2-10µΙ of mouse liver cell lysates with known OTC-activity to the duplicate wells. For all of the above samples the final volume was adjusted to 100µL/we ΙΙ with water.

Assay Procedure • 25µL of ornithine solution (50mM) was added to each of the wells containing standards, OTC activity samples, and internal control; • 25µΙ of triethanolamine solution (2700mM) (Acros Organics, Cat. # 42163 1000) was added to each of the wells containing standards, OTC activity samples, and internal control; and • 25µL of Carbamyl Phosphate substrate solution (150mM) (Sigma, Cat. #C4135-1G) was added to each of the wells containing standards, OTC activity samples, and internal controls;

• 75µL of water was added to each of the background control wells and the internal control background wells; • The microplate was covered using aluminum adhesive sealing tape and the microplate was placed on the plate shaker at setting one for 30 minutes at 37°C; • After incubation, the enzymatic reaction was stopped by adding an anti-

catalyst, which consisted of 80µL· of a phosphoric acid/sulfuric acid mixture (3:1), to each well, followed by the addition of a chromogenic reagent solution which consisted of 20µ of 3% 2, 3 butanedione monoxime (Acros Organics; cat. #150371000); • The plate was then covered with an aluminum adhesive plate sealer and again shaken for 30 seconds at setting one on the plate shaker and placed on a heat block set at 95°C for 30 min.; * The plate was then removed from the heat block, allowed to cool at room temperature for 5 minutes and centrifuged for approximately 10-15 seconds at approximately 2000 RPM using the plate carrier; and » The adhesive sealing tape was then removed and the detectable signal was measured by reading the plate using a spectrophotometer plate reader at 490nm.

Results Calibration curves for the citrulline standards were prepared by using previous test- tube formats and this microplate assay, and demonstrated a very close sensitivity.

Optical density values at 490nm for citrulline 1OOnmol were equal to approximately 1.0-1 .2. The statistical parameters for the calibration curve obtained from measuring various concentrations of citrulline standards gave in most cases R2 close to 1.0 with acceptable CV and SD. The data obtained in this microplate assay are represented in the following table and in FIG. 1, and demonstrated good sensitivity, and linearity of citrulline standards in the range from 5-10 to 80-1OOnmol. Based on this data it is expected that this efficient, high-throughput assay is capable of being employed as a replacement of traditional test-tube-based assays. EXAMPL E 2

Determining Argininosuccinate Synthetase (AS) Activity in 96-Well Plate Format

The present experiments were designed to provide a specific, sensitive and high- throughput assay for determining argininosuccinate synthetase (AS) activity in a test sample in a 96-well microplate and to provide a rapid, high-throughput, simple, and sensitive method for routine analysis and measurement of AS activity. Generally, AS activity was determined by citrulline quantification, which was performed by reacting citrulline with the chromogenic reagent 2, 3-butanedione monoxime during heating at approximately 95-100°C in strongly acid solution. The detectable signal obtained in this reaction was specific for citrulline and the resulting optical density was read in a spectrophotometer at 490nm. Assessment of the citrulline concentration of the incubation mixture is a critical component of the present assay because detection of AS activity was based on quantification of the depletion of citrulline during the enzymatic reaction.

Materials and Equipment • Polystyrene 96-well plate, flat bottom with well volume of approximately 350µ1(Falcon, Becton Dickinson Labware, cat. 353075 or equivalent); • Centrifuge with plate carrier (Beckman GS-6 Centrifuge); • Dry-bath with heating block/platform for plate; • Thermowell sealing aluminum foil tape for 96-well plates (Corning, Cat. # 6570); • Thermosetting plate shaker (The Jitterbug, Boekel Scientific); • Plate reader (SpectraMax2, Molecular Devices); and • Pipettes for various volumes.

Assay Protocol

Sample Preparation Citrulline assay standard solutions were prepared by diluting citrulline (Acros Organics, Cat. # 110470250) to ImM citrulline working solution with water, and then adding 0, 10, 20, 30, 50, 80 and 100µ of the working solution to the separate wells in duplicate for each concentration. Accordingly, the volume of working solution was equivalent to 0, 10, 20, 30, 50, 80 and 100 nmoles of citrulline. respectively. Additional blank wells were set up with 100µ of water per well. The final volume of each well was adjusted to 100µ , with water and the microplate was placed on ice, and covered with aluminum foil to prevent condensation on the bottom of the plate. To determine the AS activity of the test samples, the test samples were prepared by adding 2-10 µl of cell lysates from the test sample, containing approximately 10µg of total protein, to the microplate wells in duplicate. The same volume of enzyme source was added to control wells and used as a control for background at OD 490 nm (enzyme source background, samples without substrates). An internal control was prepared by adding approximately 2-10 µL of mouse liver cell lysates with known AS-activity to the duplicate wells. Separate wells for a negative AS-control (e.g., E.coli cell lysates without induction of AS gene) were also set up. Assay Procedure

• 10µ of citrulline solution (5mM) was added to each well containing test samples and blank control (all components of incubation mixture without enzyme); • 10µΙ of L-Aspartic acid substrate solution (50mM) (Sigma-Aldridge, Cat. # A9256) was added to each well containing test samples; µΙ • 10 of MgCl 2 solution (150mM) (Sigma-Aldridge, cat. #M1028) was added to each well containing test samples; • 10µl of ATP solution (50mM) (MP Biomedicals, Cat. # 150266) was added to each well containing test samples; • 10µΙ of test sample cell lysates were added to test sample wells only; • DPBS buffer, pH 7.2 was added to blank control wells; 50µ1of 1 Tris-HCl buffer, pH 7.4 was added to all wells making their final volume 100 µΙ; • The microplate was then covered using aluminum adhesive sealing tape and the microplate was placed on the plate shaker at setting one for approximately 15-30 minutes at 37°C; • After incubation, the enzymatic reaction was stopped by adding an anti- catalyst, which consisted of 80µ1of phosphoric acid/sulfuric acid mixture

(3: 1) to each well, followed by the addition of a chromogenic reagent which consisted of 20µ1of 3% 2, 3 butanedione monoxime; • The plate was covered with aluminum adhesive sealing tape, gently shaken in the plate shaker and placed into a dry-bath heater at 95°C in the dark for 30 min.; • After heating the plate was kept in the dark for another approximately 5 minutes to adjust the plate to room temperature and centrifuged at 2000 RPM for approximately 10-15 seconds using the plate carrier; • After centrifugation, the adhesive sealing tape was removed and the plate was read using a spectrophotometer at 490 nm and depletion of citrulline per mg of protein and time incubation was calculated. Results Calibration curves for citrulline standards using the microplate assays have shown the acceptable sensitivity in the nmoles range. Statistical parameters for the calibration curves obtained from measuring various concentrations of citrulline standards gave in the most cases R2 close to 1.0 with acceptable CV and SD. The data obtained in this microplate assay are represented in the following table and in FIG. 2, and demonstrated good sensitivity, and linearity of citrulline standards in a range from 5-10 to 80-100nmol. Based on this data it is expected that this efficient, high-throughput assay is capable of being employed as a replacement of traditional test-tube-based assays.

EXAMPL E 3

Determining Argininosuccinate Lyase (ASL ) Activity in 96-Well Plate Format

The present experiments were designed to provide a specific, sensitive and high-throughput assay for determining argininosuccinate lyase (ASL ) activity of a test sample in a 96-well microplate and to provide a rapid, high-throughput, simple and sensitive method for the routine analysis and measurement of ASL activity. Generally, ASL activity was determined by quantification of the fumaric acid.

Materials and Equipment • Standard 96-well UV Microplate, flat bottom with well volume about 370µ1 (Corning, Cat. # 3635);

. Adhesive Sealing Films (Excel Scientific, Cat. # 100-SEAL -PL T); • Thermosetting plate shaker, (The Jitterbug Boekel Scientific); · Plate reader, (SpectraMax2, Molecular Devices); and • Pipettes with various volumes.

Assay Protocol

Sample Preparation Fumaric acid assay standard solutions were prepared by diluting fumaric acid (Sigma, Cat. # F1506-100G) to ImM fumaric acid working solution with water, and then adding 0, 10, 20, 30, 50, 80, 90, 100, 120, 150, 200, 300 µΐ of working solution to the separate wells in duplicate for each concentration. Accordingly, the volume of working solution was equivalent to 0, 10, 20, 30, 50, 80 100, 120, 150, 200, and 300nmoles of fumaric acid, respectively. Additional blank wells were set up with 300 µ1of water per well. The final volume of each well was adjusted to 300 µ1with water, the microplate was placed on ice and covered with aluminum foil to prevent enzymatic reaction before incubation. To determine ASL activity of the test samples, the test samples were prepared by adding 2-1Οµΐ of cell lysates, containing approximately 10µg of total protein to DPBS buffer solution, pH 7.2, with proteases inhibitors (Roche), such that the test sample solution contained approximately l mg protein/ml. The test sample solution was then added to the microplate wells in duplicate (ASL -activity test samples). The same volume of enzyme source was added to the separate wells and used later for background at OD 240nm (enzyme source background, samples without substrates). An internal control was also prepared by adding approximately 2-1Οµΐ of mice liver cell lysates with known ASL activity. Separate wells for a negative ASL -control (e.g., E.coli cell lysates without induction of ASL gene) were also set up.

Assay Procedure • Incubation mixture wells (total volume of 300µ1 ν 1Ι) were prepared by adding 25µ1of argininosuccinic acid substrate solution (11.7mM) (Sigma, Cat. # A-5707), ΙΟµΙ ASL test sample solution, 200 µ1of Potassium Phosphate buffer solution (l OOmM), pH 7.4 and 65µ1of cold water; • Blank control wells were prepared by adding all of the previous components of the incubation mixture wells without the ASL enzyme solution; • The microplate was covered using adhesive sealing tape and the microplate was placed on the plate shaker at setting one in the dark at 37°C for 5-15 minutes; • After incubation, the microplate was placed on ice and after 5 min. of cooling the adhesive sealing tape was removed, the detectable signal (fumaric acid) was measured by reading the plate using a spectrophotometer plate reader at 240 nm, and using the obtained data the amount of fumaric acid per mg of protein and time incubation was calculated.

Results

Calibration curves for fumaric acid standards using microplate format assays have shown the acceptable sensitivity (nmoles range). Statistic parameters for calibration curves obtained from measuring various concentrations of fumaric acid standards gave in the most cases R2 close to 1.0 with acceptable CV and SD parameters. The data obtained in this microplate assay are represented in the following table and in FIG. 3, and demonstrate good sensitivity, and linearity of fumaric acid standards in range from 10 to 300 nmol. Based on these results, it is expected that this efficient, high-throughput assay is capable of being employed as a replacement of traditional test-tube-based assays. EXAMPL E 4 Determining L-arginine amidinohydrolase (ARG) Activity in 96-Well Plate Format

The present experiments were designed to provide a specific, sensitive and high-throughput assay for determining L-arginine amidinohydrolase (ARG) activity of a test sample in a 96-well microplate format and to provide for rapid, high- throughput, simple and sensitive spectrophotometric methods for routine analysis of ARG activity. ARG activity was based on spectrophotometric quantification of a detectable signal based on its strong absorption at 512nm during formation of a chromogen complex with the chromogenic reagent ninhydrin and ornithine in the presence of an acetic and phosphoric acid mixture. Materials and Equipment • Polystyrene 96-well plate, flat bottom with low evaporation lid and well volume of approximately 370µ 1(Falcon, Becton Dickinson, Cat. #353075); • Thermosetting plate shaker, (The Jitterbug, Boekel Scientific); • Isotemp Heat Plate Block (Fisher Scientific); • Plate reader, (SpectraMax2, Molecular Devices); and • Pipettes with various volumes.

Assay Protocol

Sample Preparation Ornithine assay standard working solutions (WS) were prepared by diluting 1 mM ornithine with water, and then adding 0, 10, 20, 30, 40, 50, and 60µL of the working solution to the separate wells in duplicate for each concentration. Accordingly, the volume of working solution was equivalent to 0, 10, 20, 30, 40, 50, and 60nmoles of ornithine, respectively. Additional blank wells were set up with ΙΟΟµΙ of water per well. The final volume of each well was adjusted volume to 1ΟΟµΙ/well with water. ARG requires pre-incubation with Mn2+ for maximal activation. ARG activation was performed immediately prior to incubation by treating diluted test samples of ARG with l OmM solution of MnCl2 (Fisher BioReagents, Cat. #BP541- 100). E . coli lysates activation was performed during this assay by serial dilutions of ARG-1 lysates (30 and 60 fold) prepared preliminary using DPBS-buffer, pH 7.2.

Diluted ARG-1 samples containing MnCl2 were incubated for 20 min. at 55°C to activate enzyme activity. To determine ARG activity of the test samples, the test samples were prepared by adding 2-1Οµ ΐ of cell lysates containing approximately 10µ g of total protein to the ARG-activity test sample wells in duplicate. The protein concentration of enzyme source and, therefore, its volume in incubation mixture, might be varied and depends from ARG-activity which is extremely high compared to other urea cycle enzymes. The correct choice may need to be made after preliminary experiments. For example, to get correct data about ARG activity in E . coli cell lysates, samples with enzyme source were diluted in 120 and 240 fold using DPBS buffer, with pH 7.2. The protein concentration in these diluted samples was very low («1 µg/incubation mixture), but ARG-activity for 5 min. incubation was acceptable for quantification using the ornithine standards values.

Assay Procedure • Incubation mixture wells were prepared by adding 10µΙ of diluted and activated ARG-1, 20µ1of Arginine substrate solution (l OOmM) with pH 9.5, and 70µ1of carbonate-bicarbonate buffer with pH 10; • Blank control wells were prepared by adding all of the previous components of the incubation mixture wells without the ARG, but instead included 10µΙ of DPBS buffer; • After incubation at 37°C for 10-15 min. under gentle shaking, the enzymatic reaction was stopped by adding an anti-catalyst, which consisted of 180µ1of glacial acetic acid, followed by 40µ1of the chromogenic reagent ninhydrin solution (Acros Organics, cat. # 165870250); • The plate was then covered with a lid and placed on dry bath plate platform at 95-100°C for 1 hour in the dark; • The plate was kept in the dark for another 5 min. to adjust to the room temperature and centrifuged for several seconds at approximately 2000 RPM using the plate carrier; and • The lid was then removed and the detectable signal was measured by reading the plate using a spectrophotometer plate reader at 515nm and the amount of ornithine per mg of protein and time incubation was calculated.

Results Calibration curves for ornithine standards using microplate format assays have shown the acceptable sensitivity (nmoles range) as demonstrated in FIG. 4. Statistical parameters for calibration curve obtained from measuring various concentrations of ornithine standards gave in the most cases R2 close to 1.0 with acceptable CV and SD parameters as reflected in the table below. As was mentioned above, Mn is a strong activator for ARG-activity and it needed to be used for ARG-activation. In the present experiment when comparatively high active bacterial cell lysates were used Mn-concentration in incubation mixture did not exceed 2mM. ARG-activity in 240 fold diluted lysates was higher (54mmol/mg protein/min.) than that in sample with 120 fold dilution (22 mmol/mg protein/min.). These results may be a result of higher Mn + concentration in more diluted samples or the existence of some inhibitor(s) of ARG. The enzyme showed optimum pH at pH 10 in carbonate buffer. Glycine buffer, which is frequently used for ARG-assays, is unsuitable in this procedure since it significantly decreased the sensitivity of the method. Based on data obtained it is expected that this efficient, high-throughput assay is capable of being employed as a replacement of traditional test-tube-based assays. What is claimed is:

1. A method of measuring argininosuccinate synthetase activity of a test sample in solid support, comprising: (i) contacting said test sample with citrulline, a substrate and adenosine triphosphate; (ii) contacting the reactants of step (i) with an anti-catalyst; (iii) contacting the reactants of step (ii) with a chromogenic reagent; and (iv) measuring the depletion of citrulline in said solid support.

2. The method of claim 1, wherein said substrate comprises L-Aspartic acid.

3. The method of claim 1, wherein said anti-catalyst is selected from the group consisting of phosphoric acid, sulfuric acid and combinations thereof.

4. The method of claim 1, wherein said chromogenic reagent comprises 2, 3 butanedione monoxime.

5. The method of claim 1, wherein said chromogenic reagent produces a detectable signal upon contacting citrulline.

6. The method of claim 5, wherein argininosuccinate synthetase activity is measured by detection of said detectable signal using a spectrophotometer.

7. The method of claim 6, wherein argininosuccinate synthetase activity is directly proportional to the depletion of said detectable signal.

8. The method of claim 6, wherein the optical density of said detectable signal is measured using a spectrophotometer read at about 490nm.

9. The method of claim 1, wherein said solid support is selected from the group consisting of beads, tubes, chips, resins, plates, wells, microplates and films. 10. The method of claim 1, wherein said solid support is selected from the group consisting of plastic, glass, ceramic, silicone, metal, cellulose, gels, polystyrene, polyester, and dextran.

11. The method of claim 1, wherein said solid support is a 96-well microplate.

12. The method of claim 1, wherein steps (i), (ii), (iii), and (iv) are performed in the same solid support.

13. A kit for measuring argininosuccinate synthetase activity of a test sample, comprising: (i) at least one solid support; (ii) citrulline; (iii) at least one substrate; (iv) adenosine triphosphate; (v) an anti-catalyst; and (vi) a chromogenic reagent.

14. The kit of claim 13, wherein said substrate comprises L-Aspartic acid.

15. The kit of claim 13, wherein said anti-catalyst is selected from the group consisting of phosphoric acid, sulfuric acid and combinations thereof.

16. The kit of claim 13, wherein said chromogenic reagent comprises 2, 3 butanedione monoxime.

17. The kit of claim 13, wherein said chromogenic reagent produces a detectable signal upon contacting citrulline.

18. The kit of claim 17, wherein argininosuccinate synthetase activity is directly proportional to depletion of said detectable signal.

19. The kit of claim 17, wherein argininosuccinate synthetase activity is measured by detection of said detectable signal using a spectrophotometer. 20. The kit of claim 19, wherein the optical density of said detectable signal is measured using a spectrophotometer read at about 490nm.

21. The kit of claim 13, wherein said kit further comprises a buffer.

22. The kit of claim 2 1, wherein said buffer is selected from the group consisting of sodium carbonate, sodium bicarbonate, potassium phosphate, tris(hydroxymethyl)aminomethane, DPBS and combinations thereof.

23. A method of measuring ornithine transcarbamylase activity of a test sample in solid support, comprising: (i) contacting said test sample with ornithine and a substrate; (ii) contacting the reactants of step (i) with an anti-catalyst; (iii) contacting the reactants of step (ii) with a chromogenic reagent; and (iv) measuring the production of citrulline in said solid support.

24. The method of claim 23, wherein said substrate comprises carbamyl phosphate.

25. The method of claim 23, wherein said anti-catalyst is selected from the group consisting of phosphoric acid, sulfuric acid and combinations thereof.

26. The method of claim 23, wherein said chromogenic reagent comprises 2, 3 butanedione monoxime.

27. The method of claim 23, wherein said chromogenic reagent produces a detectable signal upon contacting citrulline.

28. The method of claim 27, wherein ornithine transcarbamylase activity is directly proportional to production of said detectable signal.

29. The method of claim 27, wherein ornithine transcarbamylase activity is measured by detection of said detectable signal using a spectrophotometer. 30. The method of claim 29, wherein the optical density of said detectable signal is measured using a spectrophotometer read at about 490nm.

31. The method of claim 23, wherein said solid support is selected from the group consisting of beads, tubes, chips, resins, plates, wells, microplates, and films.

32. The method of claim 23, wherein said solid support is selected from the group consisting of plastic, glass, ceramic, silicone, metal, cellulose, gels, polystyrene, polyester, and dextran.

33. The method of claim 23, wherein said solid support is a 96-well microplate.

34. A kit for measuring the activity of ornithine transcarbamylase of a test sample, comprising: (i) at least one solid support; (ii) ornithine; (iii) a substrate; (iv) an anti-catalyst; and (v) a chromogenic reagent.

35. The kit of claim 34, wherein said substrate comprises carbamyl phosphate.

36. The kit of claim 34, wherein said anti-catalyst is selected from the group consisting of phosphoric acid, sulfuric acid and combinations thereof.

37. The kit of claim 34, wherein said chromogenic reagent comprises 2, 3 butanedione monoxime.

38. The kit of claim 34, wherein said chromogenic reagent produces a detectable signal upon contacting citrulline.

39. The kit of claim 38, wherein ornithine transcarbamylase activity is directly proportional to production of said detectable signal. 40. The kit of claim 38, wherein ornithine transcarbamylase activity is measured by detection of said detectable signal using a spectrophotometer.

4 1. The kit of claim 40, wherein the optical density of said detectable signal is measured using a spectrophotometer read at about 490nm.

42. The kit of claim 34, wherein said kit further comprises a buffer.

43. The kit of claim 42, wherein said buffer is selected from the group consisting of sodium carbonate, sodium bicarbonate, potassium phosphate, tris(hydroxymethyl)aminomethane, DPBS and combinations thereof.

44. A method measuring argininosuccinate lyase activity of a test sample in a solid support, comprising: (i) contacting said test sample with a substrate; and (ii) measuring the production of fumaric acid in said solid support.

45. The method of claim 44, wherein said substrate comprises L-argininosuccinic acid.

46. The method of claim 44, wherein argininosuccinate lyase activity is measured by detection of fumaric acid.

47. The method of claim 46, wherein argininosuccinate lyase activity is directly proportional to fumaric acid production.

48. The method of claim 46, wherein argininosuccinate lyase activity is measured by detection of fumaric acid using a spectrophotometer.

49. The method of claim 48, wherein the optical density of said fumaric acid is measured using a spectrophotometer read at about 240nm. 50. The method of claim 44, wherein said solid support is selected from the group consisting of beads, tubes, chips, resins, plates, wells, films, and microplates.

51. The method of claim 44, wherein said solid support is selected from the group consisting of plastic, glass, ceramic, silicone, metal, cellulose, gels, polystyrene, polyester, and dextran.

52. The method of claim 44, wherein said solid support is a 96-well microplate.

53. The method of claim 44, wherein steps (i) and (ii) are performed in one solid support.

54. A kit for measuring argininosuccinate lyase activity of a test sample, comprising: (i) at least one solid support; and (ii) a substrate.

55. The kit of claim 54, wherein said substrate comprises L-argininosuccinic acid.

56. The kit of claim 54, wherein argininosuccinate lyase activity is measured by detection of fumaric acid using a spectrophotometer.

57. The kit of claim 56, wherein the optical density of said fumaric acid is measured using a spectrophotometer read at about 240nm.

58. The kit of claim 54, wherein argininosuccinate lyase activity is directly proportional to fumaric acid production.

59. The kit of claim 54, wherein said kit further comprises a buffer.

60. The kit of claim 59, wherein said buffer is selected from the group consisting of sodium carbonate, sodium bicarbonate, potassium phosphate, tris(hydroxymethyl)aminomethane, DPBS and combinations thereof. 6 1. A method of measuring L-arginine amidinohydrolase activity of a test sample in a solid support, comprising: (i) contacting said test sample with a substrate; (ii) contacting the reactants from step (i) with an anti-catalyst; (iii) contacting the reactants from step (ii) with a chromogenic reagent; and (iv) measuring the production of ornithine in said solid support.

62. The method of claim 6 1, wherein said substrate comprises L-arginine.

63. The method of claim 61, wherein said method comprises the additional step of contacting said test sample with manganese chloride prior to step (i).

64. The method of claim 61, wherein said anti-catalyst comprises acetic acid.

65. The method of claim 6 1, wherein said chromogenic reagent comprises ninhydrin.

66. The method of claim 6 1, wherein said chromogenic reagent produces a detectable signal upon contacting ornithine.

67. The method of claim 66, wherein L-arginine amidinohydrolase activity is directly proportional to the production of said detectable signal.

68. The method of claim 66, wherein L-arginine amidinohydrolase activity is measured by detection of a detectable signal using a spectrophotometer.

69. The method of claim 68, wherein the optical density of said detectable signal is measured using a spectrophotometer read at about 515nm.

70. The method of claim 61, wherein said solid support is selected from the group consisting of beads, tubes, chips, resins, plates, wells, films, and microplates. 7 1. The method of claim 61, wherein said solid support is selected from the group consisting of plastic, glass, ceramic, silicone, metal, cellulose, gels, polystyrene, polyester, and dextran.

72. The method of claim 61, wherein said solid support is a 96-well microplate,

73. The method of claim 6 1, wherein steps (i), (ii), (iii), and (iv) are performed in one solid support.

74. A kit for measuring L-arginine amidinohydrolase activity in a test sample, comprising: (i) at least one solid support; (ii) a substrate; (iii) an anti-catalyst; and (iv) a chromogenic reagent.

75. The kit of claim 74, wherein said substrate comprises L-arginine.

76. The kit of claim 74, wherein said anti-catalyst comprises acetic acid.

77. The kit of claim 74, wherein said chromogenic reagent comprises ninhydrin.

78. The kit of claim 74, wherein said chromogenic reagent produces a detectable signal upon contacting ornithine.

79. The kit of claim 78, wherein L-arginine amidinohydrolase activity is directly proportional to ornithine production.

80. The kit of claim 78, wherein L-arginine amidinohydrolase activity is measured by detection of said detectable signal using a spectrophotometer.

81. The kit of claim 80, wherein the optical density of said detectable signal is measured using a spectrophotometer read at about 515nm.

82. The kit of claim 74, wherein said kit further comprises manganese chloride. 83. The kit of claim 74, wherein said kit further comprises a buffer.

84. The kit of claim 83 wherein said buffer is selected from the group consisting of sodium carbonate, sodium bicarbonate, potassium phosphate, tris(hydroxymethyl)aminomethane, DPBS and combinations thereof.