9. Suppose you have just purified an enzyme (lactase dehydrogenase - LDH) and wa
ID: 37769 • Letter: 9
Question
9. Suppose you have just purified an enzyme (lactase dehydrogenase - LDH) and want to check its activity. You know that in a pH 9 Iris butler that LDH catalyzes the following reaction: You are not aware of a simple colorimetric assay to monitor this reaction. You performed two wavelength scans, one on purified NAD and one on purified NADH: You decide to use the spectrophotometer to monitor the LDH activity. (3pts) List all the components that should be in the blank cuvette for this assay. (1pt) Prior to adding LDH, how much pyruvate should he present in the assay tube. (2pts) What is the best wavelength used to measure the LDH activity? (4pts) After adding LDH to the assay tube, you begin monitoring the reaction. What would you expect to happen to the absorbance while measuring at the best wavelength? (2pts) What is the second wavelength that could be used to measure the LDH activity? (3pts) Explain why this wavelength should not be the first choice used?Explanation / Answer
LDH ;Lactate dehydrogenase (LDH) is an oxidoreductase, present in a wide variety of organisms. It catalyses the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NAD?. When disease or injury or toxic material damages tissues, cells release LDH into the bloodstream.I t is a stable enzyme, LDH has been widely used to evaluate the presence of damage and toxicity of tissue and cells. Quantification of LDH has broad range of applications
Nicotinamide adenine dinucleotide (NAD) is a coenzyme found in all living cells.In metabolism , nicotinamide adenine dinucleotide is involved in redox reactions, , : NAD+ is an oxidizing agent it accepts electrons from other molecules and becomes reduced. This reaction forms NADH, which can then be used as a reducing agent to donate electrons. These electron transfer reactions are the main function of NAD.
what is the best wavelenth used to measure the ldh activity , what would you expect to happen to the absorbance while measuring at the bset wavelenth ?what is the first best and second best wavelength for ldh actvity?
Spectral scans from 200 nm to 600 nm of NAD+ and NADH solutions (1 mg/ml) demonstrate the differences between the oxidized and reduced forms of the molecule.NAD + and NADH strongly absorb uv ight because of the adenine. NAD+ is at 259 nm, NADH also absorbs at higher wavelengths, at 339 nm .This difference in the ultravioletabsorption spectra between the oxidized and reduced forms of the coenzymes at higher wavelengths makes it simple to measure the conversion of one to another in enzyme assay
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