Question

(a) Explain the physical principles of absorbance of light by a chromophore.Why is light of specific wavelengths absorbed? Give two examples of biological chromophores.[S] (b) Explain using the appropriate equations

what is meant by (i) the Transmittance (T) and (ii) the Absorbance (A) of a solution. What is the relationship between A and T? 3 ml of a solution of the oxidized copper (Cu²) protein pseudoazurin was prepared. The solution had an absorbance of 0.25 at 590 nm in a cuvette of path length 1 cm. (c) Calculate the mass of pseudoazurin required to make up this solution if the molar absorptivity of oxidized pseudoazurin is 1390 M' cm' at 590 nm and the molecular weight is 12,000. (d) Explain what would happen to the absorbance of the material in (c) if the spectrum was recorded on a spectrophotometer with a lamp of 10 times the intensity of that used in (c). If pseudoazurin is reduced so that the copper ion is in the Cu* oxidation state,the protein no longer has any absorbance in the visible region of the electromagnetic spectrum. Physiologically, the copper of reduced pseudoazurindonates one electron to the enzyme nitrite reductase (NiR), which catalyses the one-clectron reduction of nitrite to nitric oxide. (e) 5 µl of a 10 mg ml" solution of NİR was added to a 1 cm path length cuvette containing 2.5 ml of a solution of 10 mg ml" reduced pseudoazurin and nitrite. The absorbance at 590 nm increased by 0.3 unit sin 2 minutes. Calculate ket (in units of s'; i.e. mol NO produced per mol of NiR per sec) for NiR if its molecular mass is 120,000 and the enzyme reduces nitrite to nitric oxide at a constant rate in this experiment. (f) Explain why pseudoazurin containing Cu* absorbs in the visible region of the electromagnetic spectrum whereas the protein containing Cu' does not.

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