Nural Absar

Ultraviolet difference spectroscopic analysis of the saccharide-binding properties of Ricinus communis agglutinin.

The nature of the binding of saccharides to Ricinus communis agglutinin was studied by ultraviolet difference spectroscopy. Upon binding of galactose and galactose-containing saccharides, R. communis agglutinin displayed difference spectra with an extreme maximum at 291-293 nm and a smaller maximum at 284-285 nm. Such difference spectra suggest that the environment of a tryptophan residue located at or near the saccharide-binding site of R. communis agglutinin is being changed by an interaction between a tryptophan residue and the bound saccharides. The value of the difference spectra (delta epsilon) increased upon progressive addition of saccharide until the saccharide binding site was saturated with ligand. From the increase in delta epsilon at 291-293 nm, the association constants were obtained for the R. communis agglutinin-saccharide interaction over the temperature range 5-35 degrees C and various pH values. The results clearly demonstrate that the association constants are nearly equal in the range of pH 5-8, but decrease beyond the above pH range and with elevation of temperature. From the thermodynamic parameters for the binding of various saccharides to R. communis agglutinin, we suggest that there exists a subsite structure in the saccharide-binding site of the R. communis agglutinin molecule.

Chemical Modification of Castor Bean Hemagglutinin with Diethylpyrocarbonate

Chemical modification of histidine residues in castor bean hemagglutinin (CBH) with diethylpyrocarbonate (DEP) was studied with regard to saccharide binding. The analytical data clearly indicate that 6 out of 14 histidine residues in CBH are eventually modified with DEP at pH 6.1 in the absence of specific saccharides. Modification of histidine residues greatly decreased the cytoagglutination by CBH, and only 10 % of the residual activity was found in the derivative of CBH in which 2 histidine residues/mol were ethoxyformylated. Upon binding with galactose, the modified CBH containing 2 ethoxyformyl histidine/mol displayed an UV-difference spectrum with a maximum at 291 nm owing to the red shift of tryptophan, similar to native CBH, but its ability to bind galactose was one-fourth that of native CBH. On binding with galactopyranosides, the number of histidine residues eventually modified with DEP decreased by 2 per molecule, and the cytoagglutinating activity was retained. Hydroxylamine removed the ethoxyformyl groups from the inactive derivative of CBH which contained 6 ethoxyformyl histidine/mol, restoring the saccharide-binding ability. The results suggest that in the saccharide-binding site on each B-chain of CBH, there exists a histidine residue essential for interaction with a galactopyranosyl residue at the non-reducing end of saccharides.

Chemical Modification of Tryptophan Residues in Castor Bean Hemagglutinin

Modification of tryptophan residues in castor bean hemagglutinin (CBH) with N-bromosuccinimide (NBS) was investigated in detail. Tryptophan residues accessible to NBS increased with lowering pH and six tryptophan residues/mol were oxidized at pH 3.0, while two tryptophan residues/mol were oxidized at pH 5.0. From the pH-dependence curve for tryptophan oxidation, we suggest that the extent of modification of tryptophan in CBH is influenced by an ionizable group with pKa = 3.6. The saccharide-binding activity was decreased greatly by modification of tryptophan concomitantly with a loss of fluorescence. A loss of the saccharide-binding activity was found to be principally due to the modification of two tryptophan residues/mol located on the surface of the protein molecule. In the presence of raffinose, two tryptophan residues/mol remained unmodified with retention of fairly high saccharide-binding activity. The results suggest that one tryptophan residue is involved in each saccharide-binding site on each B-...