Enzymatic Assay of L-PHENYLALANINE DEHYDROGENASE (EC 1… 1.4.1.20L... · (EC 1.4.1.20)...

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Page 1 of 4 Enzymatic Assay of L-PHENYLALANINE DEHYDROGENASE (EC 1.4.1.20) PRINCIPLE: L-Phenylalanine + H 2 O + ß-NAD > Phenylpyruvate + NH 3 + ß-NADH Abbreviations used: ß-NAD = ß-Nicotinamide Adenine Dinucleotide, Oxidized Form ß-NADH = ß-Nicotinamide Adenine Dinucleotide, Reduced Form CONDITIONS: T = 30 ° C, pH = 10.5, A 340nm , Light path = 1 cm METHOD: Continuous Spectrophotometric Rate Determination REAGENTS: A. 250 mM Glycine Buffer, pH 10.5 at 30 ° C (Gly Buff) (Prepare 100 ml in deionized water using Glycine, Free Base. Adjust to pH 10.5 at 30C with 5 M NaOH.) B. 50 mM Potassium Phosphate, Monobasic Solution (Prepare 100 ml in deionized water using Potassium Phosphate, Monobasic, Anhydrous.) C. 50 mM Potassium Phosphate Buffer, pH 7.5 at 30 ° C (Enz Dil) (Prepare 50 ml in deionized water using Potassium Phosphate, Dibasic, Trihydrate. Adjust to pH 7.5 at 30 ° C with Reagent B.) D. 100 mM L-Phenylalanine Substrate Solution (Phe) (Prepare 10 ml in deionized water using L- Phenylalanine.) E. 30 mM ß-Nicotinamide Adenine Dinucleotide, Oxidized Form Solution (ß-NAD) (Dissolve the contents of one 20 mg vial of ß-Nicotinamide Adenine Dinucleotide in the appropriate volume of deionized ater or prepare 10 mlw in deionized water using ß-Nicotinamide Adenine Dinucleotide. 45-1 Ramsey Road, Shirley, NY 11967, USA Email: [email protected] Tel: 1-631-562-8517 1-631-448-7888 Fax: 1-631-938-8127

Transcript of Enzymatic Assay of L-PHENYLALANINE DEHYDROGENASE (EC 1… 1.4.1.20L... · (EC 1.4.1.20)...

Page 1: Enzymatic Assay of L-PHENYLALANINE DEHYDROGENASE (EC 1… 1.4.1.20L... · (EC 1.4.1.20) CALCULATIONS: (continued) units/ml enzyme Units/mg protein = mg protein/ml enzyme UNIT DEFINITION:

Page 1 of 4

Enzymatic Assay of L-PHENYLALANINE DEHYDROGENASE (EC 1.4.1.20) PRINCIPLE: L-Phenylalanine + H2O + ß-NAD > Phenylpyruvate + NH3 + ß-NADH Abbreviations used: ß-NAD = ß-Nicotinamide Adenine Dinucleotide, Oxidized Form ß-NADH = ß-Nicotinamide Adenine Dinucleotide, Reduced Form CONDITIONS: T = 30°C, pH = 10.5, A340nm, Light path = 1 cm METHOD: Continuous Spectrophotometric Rate Determination REAGENTS: A. 250 mM Glycine Buffer, pH 10.5 at 30°C (Gly Buff) (Prepare 100 ml in deionized water using Glycine, Free

Base. Adjust to pH 10.5 at 30C with 5 M NaOH.)

B. 50 mM Potassium Phosphate, Monobasic Solution (Prepare 100 ml in deionized water using Potassium

Phosphate, Monobasic, Anhydrous.) C. 50 mM Potassium Phosphate Buffer, pH 7.5 at 30°C

(Enz Dil) (Prepare 50 ml in deionized water using Potassium

Phosphate, Dibasic, Trihydrate.Adjust to pH 7.5 at 30°C with Reagent B.)

D. 100 mM L-Phenylalanine Substrate Solution (Phe) (Prepare 10 ml in deionized water using L-

Phenylalanine.) E. 30 mM ß-Nicotinamide Adenine Dinucleotide, Oxidized

Form Solution (ß-NAD) (Dissolve the contents of one 20 mg vial of

ß-Nicotinamide Adenine Dinucleotide in the appropriate volume of deionized ater or prepare 10 mlw in deionized

water using ß-Nicotinamide Adenine Dinucleotide.

45-1 Ramsey Road, Shirley, NY 11967, USAEmail: [email protected]

Tel: 1-631-562-8517 1-631-448-7888 Fax: 1-631-938-8127

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Page 2: Enzymatic Assay of L-PHENYLALANINE DEHYDROGENASE (EC 1… 1.4.1.20L... · (EC 1.4.1.20) CALCULATIONS: (continued) units/ml enzyme Units/mg protein = mg protein/ml enzyme UNIT DEFINITION:

Page 2 of 4 45-1 Ramsey Road, Shirley, NY 11967, USA

Email: [email protected]: 1-631-562-8517 1-631-448-7888

Fax: 1-631-938-8127

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Page 3: Enzymatic Assay of L-PHENYLALANINE DEHYDROGENASE (EC 1… 1.4.1.20L... · (EC 1.4.1.20) CALCULATIONS: (continued) units/ml enzyme Units/mg protein = mg protein/ml enzyme UNIT DEFINITION:

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Enzymatic Assay of L-PHENYLALANINE DEHYDROGENASE (EC 1.4.1.20) REAGENTS: (continued) F. L-Phenylalanine Dehydrogenase Enzyme Solution (Prepare a solution containing 3-10 units of

L-Phenylalanine Dehydrogenase in cold Reagent C. Store at 4°C. Immediately before use, dilute to

0.10 - 0.20 unit/ml in cold Reagent A.) PROCEDURE: Pipette (in milliliters) the following reagents into

suitable cuvettes: Test Blank Reagent A (Gly Buff) 2.30 2.30 Reagent D (Phe) 0.30 0.30 Reagent E (ß-NAD) 0.30 0.30 Mix by inversion and equilibrate to 30°C. Monitor the

A340nm until constant, using a suitably thermostatted spectrophotometer. Then add:

Reagent F (Enzyme Solution) 0.10 ------ Reagent C (Enz Dil) ------ 0.10 Immediately mix by inversion and record the increase in

A340nm for approximately 5 minutes. Obtain the r A340nm/min using the maximum linear rate for both the Test and Blank.

CALCULATIONS: (rA340nm/min Test - r A340nm/min Blank)(3)(df) Units/ml enzyme = (6.22)(0.1) 3 = Total volume (in milliliters) of assay df = Dilution factor 6.22 = Millimolar extinction coefficient of ß-NADH at 340

nm 0.1 = Volume (in milliliter) of enzyme used units/ml enzyme Units/mg solid = mg solid/ml enzyme

45-1 Ramsey Road, Shirley, NY 11967, USAEmail: [email protected]

Tel: 1-631-562-8517 1-631-448-7888 Fax: 1-631-938-8127

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Page 4: Enzymatic Assay of L-PHENYLALANINE DEHYDROGENASE (EC 1… 1.4.1.20L... · (EC 1.4.1.20) CALCULATIONS: (continued) units/ml enzyme Units/mg protein = mg protein/ml enzyme UNIT DEFINITION:

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Enzymatic Assay of L-PHENYLALANINE DEHYDROGENASE (EC 1.4.1.20) CALCULATIONS: (continued) units/ml enzyme Units/mg protein = mg protein/ml enzyme UNIT DEFINITION: One unit will oxidize 1.0 µmole of L-phenylalanine per

minute at pH 10.5 at 30°C in the presence of ß-NAD. FINAL ASSAY CONCENTRATION: In a 3.00 ml reaction mix, the final concentrations are 192 mM glycine, 10 mM L-phenylalanine, 3 mM ß-nicotinamide

adenine dinucleotide, 1.7 mM potassium phosphate and 0.01 - 0.02 unit L-phenylalanine dehydrogenase. REFERENCE: Asano, Y., Nakazawa, A. and Endo, K. (1987) Journal of

Biological Chemistry 262, 10346-10354 NOTES: 1. This assay is based on the cited reference. 2. Where our Product or Stock numbers are specified,

equivalent reagents may be substituted. This procedure is for informational purposes. For a current copy of our quality control procedure contact our Technical Service Department.

45-1 Ramsey Road, Shirley, NY 11967, USAEmail: [email protected]

Tel: 1-631-562-8517 1-631-448-7888 Fax: 1-631-938-8127

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Restriction Enzyme Analysis of Listeria monocytogenes ... · RESTRICTION ENZYME ANALYSIS OF L. MONOCYTOGENES added (130 RI at 50 mg/ml). After incubation (37°C, 1 h), 20 [lI ofproteinase

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Enzymatic Assay of CHONDROITINASE ABC (EC 4.2.2.4 ... 4.2.2.4...Page 2 of 4 Enzymatic Assay of CHONDROITINASE ABC (EC 4.2.2.4) REAGENTS: (continued) G. Chondroitinase ABC Enzyme Solution

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