Estimate the specific activity of the two samples.Why is it important/useful to measure specific activity?

Data Handling Task Document
Lactate dehydrogenase is an enzyme which can be found in most major tissues. Serum levels of LDH are elevated in a wide variety of pathologic conditions, most notably cardiac and hepatic disease. LDH catalyses the formation of lactate from pyruvate and at the same time oxidises.
NADH to NAD+. This is a reversible reaction as shown below.
The measurement of LDH in the serum can be used to diagnose whether tissue damage has occurred.
Students were tasked with the development of an assay for LDH activity in human serum samples from a control patient and a patient who had suffered cardiac damage. Two samples were provided, labelled C and D .
The students decided to follow the loss of absorbance of NADH in an appropriate assay buffer containing pyruvate as the substrate.
Q1 (a) By examining the absorbance spectra of both NAD+ and NADH below, comment why this
approach is suitable strategy.
Q1 (b). What would be the optimum wavelength at which they should measure
absorbance? Give a reason for your answer.
The students conducted the assay for LDH activity using the two serum samples. Each cuvette contained 3ml of a suitable assay buffer . 20 microlitres of either serum was added to the cuvette and the absorbance values immediately
recorded at the optimum wavelength for a period of 5 minutes .
Protein concentration of serum sample

Change in absorbance at opti
per minute
Control serum (C) 8 -0.04
Diseased serum (D) 7.8 -0.6
Q2. Using the molar absorption coefficient of NADH as 6220 M-1 cm-1, and by application of the
Beer-Lambert law, estimate the enzyme activity in the two samples . Express activity as
moles per second.
Q3 (a) Estimate the specific activity of the two samples
Q3 (b) Why is it important/useful to measure specific activity?
The students were given a compound X. They are told that it inhibits LDH activity. The students decided to check that by including it in the assay and testing the effect of the inhibitor using various concentrations of substrate , in the presence of NADH. They evaluated the rates of the LDH reaction with and without the inhibitor compound. Data obtained are shown below.
1 2.04 1.18
2 2.86 2.0
4 3.70 2.8
8 4.35 3.6
12 4.76 4.0
Q4. Estimate Vmax and Km for LDH with and without the inhibitor.
Q5 (a) Determine the type of inhibition of the enzymatic reaction from the data the students collected in the presence and absence of the inhibitor X.
Q5 (b) Students were told that compound X is N-propyl oxamate. The structures of pyruvate and N-propyl oxamate are shown below. Is the information shown below consistent with your answer in Q5 part (a) ? Explain your answer.