Use of Total Reflection X-ray Fluorescence (TRXF) for the Quantification of DNA Binding to Lipid Monolayers at the Air−Water Interface

Abstract

To use the TRXF technique for the quantification of DNA binding to monolayers at the air-water interface, DNA from salmon testes was labeled by covalently bound bromine. For this purpose, an analytical procedure for the quantification of bromine in labeled DNA with a detection limit of 10-20 μg was developed. It was found that the pH of the solution has a strong influence on the yield of brominated DNA (BrDNA) when Br(2) is used as a reagent. Much higher degrees of bromination can be achieved at pH 5 than at pH 7. A degree of bromination above a threshold of 2 to 3% (bromine per base) leads to the cross linking of BrDNA with the formation of an insoluble gel during the precipitation procedure. Finally, a reaction scheme with N-bromosuccinimide (NBS) that avoids precipitation has been established. Succinimide and some bromide ions remain in the solution as byproducts. However, these bromide ions are not competitive with BrDNA for binding at positively charged monolayers. Therefore, a new method for binding studies of model DNA to Langmuir monolayers at the air-water interface has been established. An important result of these studies is the finding that higher salt concentrations (representing physiological conditions) lead to an increased amount of adsorbed DNA. This can be explained by the decrease in the effective charge of the DNA molecules with decreasing Debye screening length.