Spectroscopic studies of AFP, Anti AFP antibody and AFP/125I- anti AFP antibody complex.

The aim of this work is to characterize spectrophotometrically the isolated Alpha fetoprotein from human colorectal tumor homogenates and the molecules of both AFP antibody and the complex of AFP/anti AFP antibody.Gel filtration technique was used to separate 125I-anti AFP antibody bound to human AFP from unbound (free) 125I-anti AFP antibody. The characterization of human-AFP, anti-AFP, and (AFP/Anti-AFP) complex were carried out through the ultraviolet (U.V) spectroscopic studies.Factors affecting the light absorption properties of the molecules under investigation in this work such as pH, solvent polarity (solvent perturbation technique), spectrophotometric pH titration and thermal stability have been studied.The spectrophotometric pH titration for h-AFP, anti AFP, and (AFP/anti-AFP) complex showed that pKa for tyrosine was 9.5, 10.2, and 9.9, while for histidine was 5.7, 6.0, and 5.9 respectively. Spectrophotometric pH titration and several spectral changes were obtained in the presence of different polar and non-polar solvents, like the alteration of max position and intensities of protein spectrum, and the appearance of new chromophores on the surface of protein molecule. These chromophores where embedded in an interior region of the protein in the absence of the solvent.The difference in pH and polarity of the solvents is very important thing to characterize the protein molecules spectrophotometrially because they change the positions and values of molecules λmax in the UV region

Kinetic and Thermodynamic Studies on Afp Binding to Afp Antibody in Colorectal Tumor Homogenates

Kinetic and thermodynamic parameters associated with the binding of 125I- anti AFP Antibody to AFP in both crude colorectal homogenate and partially- purified fractions were investigated. It was shown that the reaction in all studied groups follow pseudo- first order reaction kinetics.The value of kinetic parameters Ka, Kd, Kobs, K+1, K-1, (t1/2) ass., (t1/2)diss. and maximal binding capacity (Bmax) at 25oC for the binding of anti 125I-AFP antibody with its cytosolic antigen in benign colorectal tumor were found to be : 0.0543 x 1010 M-1, 15.064 x 10-10M, 0.0158 min-1, 4.086 x 106 M-1.min-1, 64.76x10-4 min-1, 43 min, 104.28 min and 29.14 Pmol/mg protein respectively, while 0.0538 x 1010 M-1, 15.142 x 10-10 M, 0.0163 min-1, 4.301 x 106 M-1.min-1, 67.63 x 10-4 min-1, 40 min, 79.66 min and 46.25 Pmol/mg respectively for the binding of anti 125I-AFP antibody with its cytosolic antigen in tissues of malignant colorectal tumor and at 4oC.  The maximum binding of partially purified AFP occurred at 25oC. The values ofKa and K+1 increased with increasing temperature. The Van’t Hoff plot demonstrated linear relationship between lnKa and 1/T, using crude colorectal homogenate partially purified AFP as AFP source. Plotting between ln K+1 and 1/T gave linear relationship called Arrheniuns relationship. The thermodynamic parameters ΔΗ˚, ΔG˚ and ΔЅ˚ for the formation of (125I- anti AFP Antibody / AFP) complex at the standard state had been determinedas well asEa, ΔΗ*, ΔG* and ΔЅ* which representing the transition state