Comparing the Interaction of Cyclophosphamide Monohydrate to Human Serum Albumin as Opposed to Holo-Transferrin by Spectroscopic and Molecular Modeling Methods: Evidence for Allocating the Binding Site

The interaction between cyclophosphamide monohydrate with human serum albumin (HSA) and human serum transferrin (hTf) was studied with UV absorption, fluorescence and circular dichroism (CD) spectroscopies as well as molecular modeling. Based on the fluorescence quenching results, it was determined that HSA and hTf had two classes of apparent binding constants and binding sites at physiological conditions. The KSV1, KSV2, n1 and n2 values for HSA were found to be 8.6×108 Lmol-1, 6.34×108 Lmol-1, 0.7 and 0.8, respectively, and the corresponding results for hTf were 6.08×107 Lmol-1, 4.65×107 Lmol-1, 1.3 and 2.6, respectively. However, the binding affinity of cyclophosphamide monohydrate to HSA was more significant than to hTf. Circular dichroism results demonstrated that the binding of cyclophosphamide to HSA and hTf induced secondary changes in the structure and that the α-helix content became altered into β- sheet, turn and random coil forms. The participation of tyrosyl and tryptophan residues of proteins was also estimated in the drug-HSA and hTf complexes by synchronous fluorescence. The micro-environment of the HSA and hTf fluorophores was transferred to hydrophobic and hydrophilic conditions, respectively. The distance r between donor and acceptor was obtained by the Forster energy according to fluorescence resonance energy transfer (FRET) and found to be 1.84 nm and 1.73 nm for HSA and hTf, respectively. This confirmed the existence of static quenching for both proteins in the presence of cyclophosphamide monohydrate. Site marker competitive displacement experiments demonstrated that cyclophosphamide bound with high affinity to Site II, sub-domain IIIA of HSA, and for hTf, the C-lobe constituted the binding site. Furthermore, a study of molecular modeling showed that cyclophosphamide situated in domain II in HSA was bound through hydrogen bonding with Arg 257 and Ser 287, and that cyclophosphamide was situated in the C-lobe in hTf, presenting hydrogen bonding with Asp 625 and Arg 453. The modeling data thus confirmed the experimental results.