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Carbohydrate recognition by lectins often involves the side chains of tyrosine, tryptophan, and histidine residues. These moieties are able to produce chemically induced dynamic nuclear polarization (CIDNP) signals after laser irradiation in the presence of a suitable radical pair-generating dye. Elicitation of such a response in proteins implies accessibility of the respective groups to the light-absorbing dye. In principle, this technique is suitable to monitor surface properties of a receptor and the effect of ligand binding if CIDNP-reactive amino acids are affected. The application of this method in glycosciences can provide insights into the protein-carbohydrate interaction process, as illustrated in this initial study. It focuses on a series of N-acetylglucosamine-binding plant lectins of increasing structural complexity (hevein, pseudohevein, Urtica dioica agglutinin and wheat germ agglutinin and its domain B), for which structural NMR- or X-ray crystallographic data permit a decision of the validity of the CIDNP method-derived conclusions. On the other hand, the CIDNP data presented in this study can be used for a rating of our molecular models of hevein, pseudohevein, and domain B obtained by various modeling techniques. Experimentally, the shape and intensity of CIDNP signals are determined in the absence and in the presence of specific glycoligands. When the carbohydrate ligand is bound, CIDNP signals of side chain protons of tyrosine, tryptophan, or histidine residues are altered, for example, they are broadened and of reduced intensity or disappear completely. In the case of UDA, the appearance of a new tryptophan signal upon ligand binding was interpreted as an indication for a conformational change of the corresponding indole ring. Therefore, CIDNP represents a suitable tool to study protein-carbohydrate interactions in solution, complementing methods such as X-ray crystallography, high-resolution multidimensional nuclear magnetic resonance, transferred nuclear Overhauser effect experiments, and molecular modeling.

The 20 kDa precursor of hevein and its C-terminal 14 kDa domain have been isolated. Sequence analysis of the Cterminal tryptic peptides of these proteins and comparison with the cDNA sequence indicate that they represent mature forms from which a C-terminal propeptide, possibly involved in vacuolar targeting, has been removed. The molar ratio of hevein to the C-terminal domain in the lutoid-body fraction of rubber latex is about 30:1. This indicates that not only the pre-and propeptides but also the 14 kDa domain are removed by proteolysis or other processes in the latex vessel after the processing of hevein has taken place.the hevein domain in this protein is not cleaved off in the mature form.Here we report the isolation of the hevein precursor and its C-terminal domain and present evidence that they are the mature proteins from which a C-terminal propeptide has already been removed. The large molar excess of hevein in the lutoidbody fraction of rubber latex indicates that much proitein turnover occurs in the latex vessels of the rubber tree. Materials and methods