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In which sense does the reaction take place? Calculate alanine, LDH, NADH and a-ketoglutarate concentrations in the reaction medium at t = 0 (in the same units as the initial concentrations). Draw the graph A340 = f(t). Comment the shape of the curve, particularly the portion that corresponds to a plateau.

In which sense does the reaction take place? Calculate alanine, LDH, NADH and a-ketoglutarate concentrations in the reaction medium at t = 0 (in the same units as the initial concentrations). Draw the graph A340 = f(t). Comment the shape of the curve, particularly the portion that corresponds to a plateau.

Biochemistry
Biochemistry
9th Edition
ISBN:9781319114671
Author:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Publisher:Lubert Stryer, Jeremy M. Berg, John L. Tymoczko, Gregory J. Gatto Jr.
Chapter1: Biochemistry: An Evolving Science
Section: Chapter Questions
Problem 1P
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We want to measure the activity of alanine aminotransferase (ALAT) present in a serum. The reaction catalyzed by the enzyme is: Reaction 1: +H3N- glutamate H C CH₂ CH₂ COO -COO + pyruvate CH3 C=0 0.1 M phosphate buffer pH 7.4 : 550 µL 1.2 M alanine : 100 μL CH3 time (min) A340 COO COO™ pyruvate lactate dehydrogenase* (LDH, 300 µg.mL-¹): 50 μL 1.5 mM NADH: 200 μL 0.04 M a-ketoglutarate: 500 μL serum containing ALAT: 600 μL The enzyme reaction is realized in the following conditions: In a 1 cm-cuvette are added: 0 0.915 ALAT NADH + H+ LDH a-cétoglutarate COO * Lactate dehydrogenase (LDH) reduces pyruvate into lactate, with the concomitant oxydation of NADH. This allows to indirectly measure the amount of product formed. Reaction 2: NAD+ 1 0.741 C=O H CH₂ CH₂ COO™ CH3 C-OH COO lactate The reaction is performed at 25 °C and the absorbance at 340 nm is monitored every minute, for 5 min. The absorbance values are given in the table below: Data: alanine ENADH at 340 nm = 6220 M¹.cm1. One international unit corresponds to the amount of enzyme that catalyzes the conversion of 1 µmole of substrate to product in one minute. *H3N C COO 3 2 0.572 0.402 CH3 4 0.233 5 0.159 1- In which sense does the reaction take place? 2- Calculate alanine, LDH, NADH and a-ketoglutarate concentrations in the reaction medium at t = 0 (in the same units as the initial concentrations). 3- Draw the graph A340 = f(t). Comment the shape of the curve, particularly the portion that corresponds to a plateau.
Transcribed Image Text:We want to measure the activity of alanine aminotransferase (ALAT) present in a serum. The reaction catalyzed by the enzyme is: Reaction 1: +H3N- glutamate H C CH₂ CH₂ COO -COO + pyruvate CH3 C=0 0.1 M phosphate buffer pH 7.4 : 550 µL 1.2 M alanine : 100 μL CH3 time (min) A340 COO COO™ pyruvate lactate dehydrogenase* (LDH, 300 µg.mL-¹): 50 μL 1.5 mM NADH: 200 μL 0.04 M a-ketoglutarate: 500 μL serum containing ALAT: 600 μL The enzyme reaction is realized in the following conditions: In a 1 cm-cuvette are added: 0 0.915 ALAT NADH + H+ LDH a-cétoglutarate COO * Lactate dehydrogenase (LDH) reduces pyruvate into lactate, with the concomitant oxydation of NADH. This allows to indirectly measure the amount of product formed. Reaction 2: NAD+ 1 0.741 C=O H CH₂ CH₂ COO™ CH3 C-OH COO lactate The reaction is performed at 25 °C and the absorbance at 340 nm is monitored every minute, for 5 min. The absorbance values are given in the table below: Data: alanine ENADH at 340 nm = 6220 M¹.cm1. One international unit corresponds to the amount of enzyme that catalyzes the conversion of 1 µmole of substrate to product in one minute. *H3N C COO 3 2 0.572 0.402 CH3 4 0.233 5 0.159 1- In which sense does the reaction take place? 2- Calculate alanine, LDH, NADH and a-ketoglutarate concentrations in the reaction medium at t = 0 (in the same units as the initial concentrations). 3- Draw the graph A340 = f(t). Comment the shape of the curve, particularly the portion that corresponds to a plateau.
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