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The combination of several drugs is necessary, especially during long-term therapy. A competitive binding of the drugs can cause a decrease in the amount of drugs actually bound to the protein and increase the biologically active fraction of the drug. Here, the interaction between 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) and 2,4-Dinitrophenol (DNP) with Hemoglobin (Hb) was investigated by different spectroscopic and molecular modeling techniques. Fluorescence analysis was used to estimate the effect of the DIDS and DNP on Hb as well as to define the binding properties of binary and ternary complexes. The distance r between donor and acceptor was obtained by the FRET and found to be 2.25 and 2.13 nm for DIDS and DNP in binary and 2.08 and 2.07 nm for (Hb-DNP) DIDS and (Hb-DIDS) DNP complexes in ternary systems, respectively. Time-resolved fluorescence spectroscopy confirmed static quenching for Hb in the presence of DIDS and DNP in both systems. Furthermore, an increase in ellipticity values of Hb upon interaction with DIDS and DNP showed secondary structural changes of protein that determine to disrupt of hydrogen bonds and electrostatic interactions. Our results showed that the Hb destabilize in the presence of DIDS and DNP. Molecular modeling of the possible binding sites of DIDS and DNP in binary and ternary systems in Hb confirmed the experimental results.

The effect of postirradiation conditions on the haemolysis of y-irradiated (2.1 kGy) human erythrocytes was studied. Haemolysis was inhibited by incubation in mannitol and sucrose instead of saline, by hypertonicity of the medium and by calcium chelators. Dithiothreitol, butylated hydroxytoluene, deferoxamine, DIDS (in inhibitor of anion exchange) and furosemide (an inhibitor of K/Cl and K/Na/Cl cotransport) did not slow down the haemolysis. Apparently, the radiation-induced haemolysis is due to the formation of membrane pores leaky for electrolytes. From the inhibition of haemolysis by mannitol, apparent pore radius was estimated to be about 0.7 nm. The pores appear to be transient, the average pore number per cell being much less than unity.