List all the compnents that should be in the \"Blank\" cuvette for this assay. P

Question

List all the compnents that should be in the "Blank" cuvette for this assay.

Prior to adding LDH how much pyruvate should be present in the assay tube

What is the best wavelength used to measure the LDH actvitity

After adding LDH to the assay tube, you begin monitoring the reaction. what would you expect to happen to the abosrabance while measuring at the best wavelength

What is the second best wavelength that could be used to measure the LDH activity

Explain why this wavelength should not be the first choice used?

9. Suppose you have just purified an enzyme (lactase dehydrogenase - LDH) and want to check its activity. You know that in a pH 9 Tris buffer that 1.1)11 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: List all the compnents that should be in the Blank cuvette for this assay. Prior to adding LDH how much pyruvate should be present in the assay tube What is the best wavelength used to measure the LDH actvitity After adding LDH to the assay tube, you begin monitoring the reaction. what would you expect to happen to the abosrabance while measuring at the best wavelength What is the second best wavelength that could be used to measure the LDH activity Explain why this wavelength should not be the first choice used?

Explanation / Answer

The components in the blank should be lactate, NAD and Tris buffer of pH 9, without the enzyme Pyruvate should not be present prior to addition of LDH in the assay tube. It is the product of the enzymatic reaction. The best wavelength to measure LDH activity would be 250 nm. The absorbance would reach highest peak in the curve when the enzymatic activity is optimum. The second best wavelength to measure LDH activity would be 350 nm. The 350 nm wavelength is favourable for measuring absorbance in presence of NADH. Since NADH is product of the enzymatic reaction and NAD (best wavelength is 250 nm) is substrate. This wavelength should not be the first choice used.

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