5. Human serum albumin (MW 66.5 k Da ) is the most abundant protein in human blo

Question

5. Human serum albumin (MW 66.5 kDa) is the most abundant protein in human blood plasma. Considering that the behavior of HAS in aqueous media can be satisfactory described as that of a sphere:

(i) Calculate the rotational correlation time of HAS in water at 37 oC.  To this end, consider that the specific volume of HAS is 0.73 mL/g, and that its hydration is 0.23 g H2O per gram of protein. Given:  viscosity of water at 37 oC  = 0.691 centipoise; R = 8.31 x 107erg Mol-1K-1.

(ii) Calculate the anisotropy that can be expected from samples of HAS covalently tagged with the following two distinct fluorescent dyes. In the first sample HAS is tagged with a fluorescent dye that shows fundamental anisotropy equal to 0.37 and 18 ns lifetime. In the second sample HAS is tagged with a fluorescent dye that shows fundamental anisotropy equal to 0.40 and 1.0 ns lifetime.

(iii) Which one of the HAS-tagged samples considered above would be preferable for use is studies dealing with the binding of HAS to other large biomolecules (if any). In other words, in which case the dynamic range associated with the experimental measurements would be larger. Explain your rational.  Here assume that the lifetime of the tagging dye does not change upon binding of the respective HAS-tagged adduct to other biomolecules.

Explanation / Answer

(i)Rotational Correlation time = ??r3h3/?g2?21024 = 2667.51*0.46/3.14*0.53*0.477=1227.05/766.86

=1.600096

(ii)Anisotropy (r) = Iz-Iy/Ix+Iy+Iz = 6.66/0.4 = 16.65

(iii) The HAS Tagged sample that would be preferably be used in studies dealing with the binding of HAS to

other large molecules(if any) is the HAS MOLECULE that shows fundamental anisotropy equal to 0.37 and 18ns life time , as the life time is more for thismolecule.

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