Study of neutral red interaction with DNA by resolution of rank deficient multi-way fluorescence data

The interaction of neutral red (NR) as an efficient anticancer drug with DNA was studied under physiological pH condition. Three-way data array were recorded by measuring excitation-emission fluorescence during the titration of neutral red with DNA at constant pH. The acid-base equilibrium constant of protonated and deporonated forms of NR was determined by using rank annihilation factor analysis, as pKa = 7.210 ± 0.003. Multivariate curve resolution (MCR) with trilinearity constraint along the alternating least squares optimization process was applied to estimate pure profiles. Concentration profiles of the equilibrium state suggested that both NR and HNR could bind concurrently to DNA. For the first time, rank annihilation factor analysis (RAFA) method was used for estimation of the equilibrium constants by annihiling contribution of equilibrium concentrations of one or more species (protonated and deprotonated forms of neutral red), simultaneously. Instead of a minimum point for the residual standard deviation (R.S.D.), a minimum line was acquired, because the system was suffered from rank deficiency in the concentration mode. To circumvent the ambiguity, which is resulted from rank deficiency, experiments were performed in two different conditions (phosphate buffers at pH 7.20 and 7.40). The parameters were determined from the cross point of two minimum lines of R.S.D. This is the first report of the simultaneous determination of the complex formation constants for both protonated and deprotonated forms of NR with DNA. The merit of this procedure is using the information contents of two sets of data, without augmentation of them.