question 1. work through the calculations to determine the amount of LDH enzyme present in one of your reactions. Obtain the initial rate (OD/sec) from your 20 l volume assay. Calculate how many Units of LDH were present in this enzymatic reaction.

Initial rate: 3.68E-03 OD/sec

Calculating LDH enzyme units (U) based on initial rate (OD/sec) measurements

Here is the step-by-step process that is used to convert your experimental observation (OD/sec) into a calculated quantity (enzyme units, U):

1. First, recall that U = mol/min by definition (see introduction section of this protocol)

2. Initial rate is obtained from the slope (m) of the trendline: y = mx + b

units of OD/sec

3. Convert time to minutes

[OD/sec]*( 60 sec/min) = OD/min

4. Convert OD to concentration units with Beers Law: c = OD/(e*l)

e is the extinction coefficient for NADH (6220 cm-1M-1)

l is the path-length of the cuvette = 1 cm,

c has units of molar concentration

5. At this stage, your value has units = M/min (concentration/time), but you want mol/min

6. Convert molar concentration to mol by including volume in calculation. Cuvette volume is 2.0 ml or 2 x 10-3 liter

[M/min]*[2x10-3 l] = mol/min

(mol/min)*106 mol/mol = final answer in mol/min = U

If you put all of these steps together into one equation, here is the way to convert OD/sec to U:

The above equation is valid only converting from OD/sec to LDH enzyme units with 2 ml assay volume and a 1-cm pathlength cuvette. The equation can be simplified to:

The conversion factor, 19.29, has units of (mol/min)/(OD/sec).