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 (healthy) and a patient who had suffered cardiac damage. Two samples were provided, labelled C and D (Control and Diseased serum). 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(in the image attached) 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 (path length 1 cm) contained 3ml of a suitable assay buffer (including pyruvate as substrate). 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 (absorbance readings taken every 30 seconds). Protein concentration of serum sample (mg/ml) Change in absorbance at optimum wavelength per minute Control serum (C) 8 -0.04 Diseased serum (D) 7.8 -0.6 1c. 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 (C and D). Express activity as moles per second. 1d. Estimate the specific activity of the two samples (moles per second per microgram).
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 (healthy) and a patient who had suffered cardiac damage. Two samples were
provided, labelled C and D (Control and Diseased serum).
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(in the image attached) 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 (path length 1 cm) contained 3ml of a suitable assay buffer (including pyruvate as substrate). 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 (absorbance readings taken every 30 seconds).
|
|
Protein concentration of serum sample (mg/ml) |
Change in absorbance at optimum wavelength per minute |
|
Control serum (C) |
8 |
-0.04 |
|
Diseased serum (D) |
7.8 |
-0.6 |
1c. 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 (C and D). Express activity as moles per second.
1d. Estimate the specific activity of the two samples (moles per second per microgram).
Trending now
This is a popular solution!
Step by step
Solved in 3 steps
