The pyruvate dehydrogenase complex catalyzes the following reaction: pyruvate + coa + nad acetyl-coa + co + nadh we will discuss this enzyme in detail on class, but for now all you need to know is the overall reaction that it catalyzes. 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 = e. c.l where a is absorbance, & is the molar extinction coefficient, c is concentration, and lis 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" for (mol/litre)cm or m'cm'). the molar extinction coefficient is the absorbance of a 1 molar solution in a 1 cm path length cuvette. the molecular weight of nad is 663.4. in a 1 ml cuvette, you mix 0.1 ml of 2.5 mm nad", 0.1 ml of 0.2 m pyruvate, 0.1 ml of 0.1m coa, 0.6 ml of a suitable buffer, and 0.1 ml of a solution of purified pyruvate 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 increase of absorbance (aa) of 0.37 min1. you are told that the pyruvate dehydrogenase solution you used in the assay contained 7.03 mg of protein per ml. for all questions, show your calculations. a. how much nad should you weigh out in order to prepare 0.5 ml of a 2.5 mm solution? b. what is the absorbance of a 0.02 mm solution of nadh, measured in a cuvette with a 1 cm path length calculate the rate of production of nadh in this assay, in units of micromoles/min [tip: you need to consider the reaction volume] d. calculate the specific activity of pyruvate dehydrogenase in this assay, in units of micromoles nadh produced/minute/mg protein. focus