Lactate dehydrogenase catalyzes the following reaction: pyruvate + NADH ---> lac
ID: 63486 • Letter: L
Question
Lactate dehydrogenase catalyzes the following reaction:
pyruvate + NADH ---> lactate + NAD+
The assay method described is used very frequently in biochemistry labs. In a spectrophotometer, NADH absorbs light at 340 nm, NAD+ does not.
The Beer-Lambert Law relates the absorbance of a solution to its concentration, and states that: A = .c.L Where A is absorbance, is the molar extinction coefficient, c is concentration, and L is the path length of the solution in the spectrophotometer. In all the calculations that follow assume L = 1 (ie all measurements were made in a cuvette with a 1 cm path length), so L can effectively be disregarded (although not its unit). The molar extinction coefficient for NADH is 6,300 litres/mol cm-1 [or (mol/litre)-1 cm-1 or M-1 cm-1 ].
The molar extinction coefficient is the absorbance of a 1 molar solution in a 1 cm path length cuvette. The molecular weight of NADH is 663.4. In a 1 ml cuvette, you mix 0.1 ml of 2.5 mM NADH, 0.1 ml of 0.2 M pyruvate, 0.7 ml of a suitable buffer, and 0.1 ml of a solution of purified lactate dehydrogenase.
You mix, place the cuvette in a spectrophotometer (with a chart recorder attached) and follow the absorbance at 340 nm. You measure a rate of decrease of absorbance (A) of 0.51 min-1 . You are told that the lactate dehydrogenase solution you used in the assay contained 8.17 mg of protein per ml.
show calculations.
(a) How much NADH should you weigh out in order to prepare 0.5 ml of a 2.5 mM solution?
(b) Calculate the rate of consumption of NADH in this assay, in units of micromoles/min [Tip: you need to consider the reaction volume]
(c) Calculate the specific activity of lactate dehydrogenase in this assay, in units of micromoles NADH oxidized/minute/mg protein.
(d) What is the specific activity of lactate dehydrogenase in this assay, in units of micromoles pyruvate reduced/minute/mg protein.
Explanation / Answer
Based on the given data,
(a)
So,
(0.0005 L × 2.5 mol/L× 663.43 g/mol) = 0.82 grams of NADH
Thus, 0.82 grams of NADH is required to prepare 0.5 ml of a 2.5 mM solution.
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