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  • Investigation on the interaction between cyclophosphamide and lysozyme in the presence of three different kind of cyclodextrins: determination of the binding mechanism by spectroscopic and molecular modeling techniques.

Investigation on the interaction between cyclophosphamide and lysozyme in the presence of three different kind of cyclodextrins: determination of the binding mechanism by spectroscopic and molecular modeling techniques.

Molecules (Basel, Switzerland) (2013-01-25)
Mahdieh Mansouri, Malihe Pirouzi, Mohammad Reza Saberi, Maryam Ghaderabad, Jamshidkhan Chamani
ABSTRACT

The interactions between cyclophosphamide (CYC) and lysozyme (LYZ) in the presence of different cyclodextrins (CDs) were investigated by UV absorption, fluorescence spectroscopy, circular dichroism (CD), and molecular modeling techniques under imitated physiological conditions. The UV absorption results showed the formation of complexes between CYC and LYZ in the presence of different CDs. Fluorescence data show that CYC has a stronger quenching effect on LYZ, and the red shifts suggested that the microenvironment of Trp residues was changed and became more hydrophilic. The interaction of CYC with LYZ and quenching properties of the complexes caused strong static fluorescence quenching in binary and ternary systems. The binding affinities as well as the number of binding sites were obtained from interaction between CYC and LYZ in the presence of different CDs as binary and ternary systems by modified Stern-Volmer plots. The Resonance Light Scattering (RLS) technique was utilized to investigate the effect of drug and CDs on conformational changes of LYZ as separate and simultaneous. The results suggested that the enhancement of RLS intensity was attributed to the formation of a complex between drug and protein in absence and presence of CDs. The effect of CYC and cyclodextrins on the conformation of LYZ was analyzed using synchronous fluorescence spectroscopy. Our results revealed that the fluorescence quenching of LYZ originated from the Trp and Tyr residues, and demonstrated conformational changes of LYZ with the addition of CYC and CDs. The molecular distances between the donor (LYZ) and acceptor (CYC and CDs) in binary and ternary systems were estimated according to Forster's theory and showed static quenching for protein with CYC in the presence of CDs. The CD spectra indicated that the binding of the CYC induced secondary structural changes in LYZ in binary and ternary systems. Molecular modeling suggested the binding sites of CYC in the ternary systems differ from those in the binary systems. estimated the distance between CYC and Trp residues in binary and ternary systems in the presence of CDs and confirmed the experimental results.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Lysozyme human, Lysobac, recombinant, expressed in rice, ≥100,000 units/mg protein, lyophilized powder
Sigma-Aldrich
Lysozyme from human neutrophils, ≥95% (SDS-PAGE), lyophilized powder, ≥100,000 units/mg protein (E1%/280)
Sigma-Aldrich
Lysozyme from chicken egg white, BioUltra, lyophilized powder, ≥98% (SDS-PAGE), ≥40,000 units/mg protein
Sigma-Aldrich
Lysozyme from chicken egg white, 10 mg/mL
Sigma-Aldrich
Lysozyme from chicken egg white, powder (crystalline), 70000-140000 U/mg
Sigma-Aldrich
Lysozyme from chicken egg white, dialyzed, lyophilized, powder, ~100000 U/mg
Sigma-Aldrich
Lysozyme chloride form from chicken egg white, Grade VI, ≥35,000 units/mg protein (E1%/282)
Sigma-Aldrich
Lysozyme from chicken egg white, aseptically filled
Sigma-Aldrich
Lysozyme from chicken egg white, For use as a marker in SDS-PAGE
Sigma-Aldrich
Lysozyme from chicken egg white, powder or granules, ≥90 %, ≥39,000 units/mg protein
Supelco
Lysozyme from chicken egg white, VETRANAL®, analytical standard