You perform the LDH enzyme assay and measure absorption at 340 nm 30 seconds after addition...

You perform the LDH enzyme assay and measure absorption at 340 nm 30 seconds after addition of the enzyme. Your absorbance reading is 0.97. What is the enzyme activity (in units of µmol NADH/min)? (show your work)

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Expert Solution

You need to know the the extinction coefficient (epsilon: e) of your product then you apply the Beer Lambert Abs= e c l (l is the pathlength if you use cuvette of 1 cm then you can calculate c (concentration of product that appeared or substrate that disappeared) by Abs/el . Be careful with the units of e, to determine the C (usually in mM). If you have c in mM for instance and you are working in 1 mL you will know that you have let say if c = 0.2 mM 0.2 µMol in 1 mL . If now you know that you have a delta Abs in 1 min then means you have 0.2 µmol (200 nmol) per 1 min and you have to know how much enzyme you put in your cuvette (let say 2 nM) then your kcat (catalytic constant) will be 100 min-1. You can also work out activity as nmol/min/mg (then you need to know how much you put in the cuvette let say 1 µg in the 1 mL then meaning that you got 200 nmol/min for 100 µg so you mutliply by 10 to get 2000 nmol/min/mg or 2 µmol/min/mg that is also the enzyme activity.

Value of extinction coefficient must be given to calculate the concentration?

queen_honey_blossom answered 1 year ago

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