A preliminary study on interaction of H4 histone with DNA in the unirradiated and gamma irradiated states

摘要

H4 histone (100 μg/ml) in 0.9% sodium chloride showed minimum conductance and absorbance values at 230 nm, 280 nm and A_(λmax) at pH 7.02. So exposition of chromophores was minimum and charge neutralization was maximum at this pH. DNA : H4 complex (50 : 50 μg/ml) in 0.9% sodium chloride was found to be stable in the pH range of 6.68 to 8.86. Minor variations in binding of H4 histone with DNA (pH range 6.68 to 7.15) for forming the DNA : H4 complex (25 : 25 μg/ml) have occurred when 0.9% sodium chloride, 0.15 M and 0.1 M phosphate buffers have been used as solvents. This is evidenced by variation in position of absorption maximum and intensity of absorption maximum. Nature of binding in last two solvents are alike but different for 0.9% sodium chloride. This is due to variations in (i) counter-ion concentration in solution (i.e. PO_4~(3-), HPO_4~(2-), H2PO_4~(1-), Na~+ and Cl~-), (ii) exposed functional groups and chromophores (both in far and near UV) of H4 histone (i.e. imidazole group of histidine, positively charged arginine and lysine) and that of DNA (i.e. -C = C-C = O and -C = C-C = N-) and (iii) charge interactions between amino-acids and peptides of H4 histone with phosphate groups of DNA. The value of the binding constant for DNA-H4 complex varied from 3.45 LM~(-1) (for 0.15 M phosphate buffer) to 3.66 LM~(-1) (0.1 M phosphate buffer) and to 3.77 LM~(-1) (for 0.9% sodium chloride). The complex formation of DNA with H4 histone has also been evidenced by increase in melting temperature by 9 °C more for DNA : H4 complex than that of DNA. H4 histone (25 μg/ml, pH 6.80, 0.15 M phosphate buffer) can be used as in vitro biological dosimeter by measuring A_(660nm) with Folin-Ciocalteau reagent in the dose range 30-80 Gy at 1.12 Gy/s. Similarly depletion of H4 histone from DNA-H4 complex (25 : 25 μg/ml) as detected by Folin-Ciocalteau reagent (A_(660nm)) can also be used as in vitro biological dosimeter in the dose range of 20 to 70 Gy at a dose rate of 1.12 Gy/s. In 0.9% sodium chloride the response for H4 histone (i.e A_(280nm)) did not change with dose. But in 0.01 N hydrochloric acid, pH range 3.92-4.44, the response for H4 histone (i.e A_(210nm) , A_(220nm) and A_(280nm)) decreased with dose up to 50 Gy for a dose rate of 0.0103 Gy/s. Here it can be used as a dosimeter for intestinal tract. These biological in vitro dosimeters may find use in radiation-induced nucleosomal aberration.