Question

1. Why should someone measure absorbance at 340nm? What are the advantages/disadvantages of using 355nm vs 340nm? NADH absorbance is highest at 250nm, what are 2 distinct reasons we are not looking at that wavelength?

2. One of the known inhibitors of hexokinase is 2-deoxy-D-glucose. Predict the type of inhibitor that 2-deoxy-D-glucose might be and explain your answer.

Answer 1

Nicotinamide adenine dinucleotide,normally exists in two forms. Oxidosed and reduced forms.That is NAD+ and NADH and it is a coenzyme found in all living cell.The NAD+ which accepts an electrons from other molecules and become reduced.This forms NADH which is used to reduciing agent to donate electrons.Both NAD+ and NADH absorbs UV stongly because of the adinine base in it.NAD+ absorbs at 259nm and NADH at 339.This difference in the UV absorption spectra of the coenzymes at higher wavelengths makes it simple to measure the conversion of one to another in enzyme assays by measuring the amount of UV absorption at 340 nm using a spectrophotometer but only when protonated.

Since both the oxidized and reduced forms of nicotinamide adenine dinucleotide are used in these linked sets of reactions, the cell maintains significant concentrations of both NAD⁺ and NADH, with the high NAD⁺/NADH ratio allowing this coenzyme to act as both an oxidizing and a reducing agent.

NADH absorbance is highest at 250nm, it is may be because of its aromatic structure.

2. 2-deoxy-D-glucose is an glycolytic inhibitor and block cancer cell growth(glycolysis plays an important role in tumorigenesis and is a valid target for cancer therapy).It has two hydroxyl group which is replaced by Hydrogen.Also it will inhibit the production of gluco-6-phosphate from glucose at the phosphoglucoisomerase.2-deoxy-D-glucose is a good marker for the tissue glucose uptake and hexokinase activity.