Thermodynamic and kinetic analysis of porphyrin binding to Trichosanthes cucumerina seed lectin

The interaction of several metallo-porphyrins with the galactose-specific l ectin from Trichosanthes cucumeirna (TCSL) has been investigated. Differenc e absorption spectroscopy revealed that significant changes occur in the So ret band region of the porphyrins upon binding to TCSL and these changes ha ve been monitored to obtain association constants (K-a) and stoichiometry o f binding (n). The dimeric lectin binds two porphyrin molecules and the pre sence of the specific saccharide lactose did not affect porphyrin binding s ignificantly, indicating that the sugar and the porphyrin bind at different sites. The K-a values obtained for the binding of different porphyrins wit h TCSL at 25 degreesC were in the range of 2 X 10(3)-5 X 10(5) M-1. Associa tion constants for meso-tetra(4-sulphonatophenyl)porphyrinato copper(II) (C uTPPS), a porphyrin bearing four negative charges and meso-tetra(4-methylpy ridinium)porphyrinato copper(II) (CuTMPyP), a porphyrin with four positive charges, were determined at several temperatures; from the temperature depe ndence of the association constants, the thermodynamic parameters change in enthalpy and change in entropy (DeltaH degrees) and change in entropy (Del taS degrees) associated with the binding process were estimated. The thermo dynamic data indicate that porphyrin binding to TCSL is driven largely by a favourable entropic contribution; the enthalpic contribution is very small , suggesting that the binding process is governed primarily by hydrophobic forces. Stopped-flow spectroscopic measurements show that binding of CuTMPy P to TCSL takes place by a single-step process and at 20 degreesC, the asso ciation and dissociation rate constants were 1.89 X 10(4) M-1.s(-1) and 0.2 9 s(-1), respectively.