EQUILIBRIUM SEC MASS SPECTROMETRY FOR STUDY OF CARBOHYDRATE-PROTEIN BINDING

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Carbohydrates play major roles in mediating biological processes through their interactions with different extracellular matrix proteins. To understand the physiological significance of these interactions, it is necessary that relationships between carbohydrate structure and binding be analyzed. Mass spectrometry has been demonstrated to be a powerful method to study the structures of carbohydrates. The combination of equilibrium size exclusion chromatography (SEC) with MS for measuring carbohydrate-protein binding affinities overcomes traditional limitations, i.e. the need to purify oligosaccharides. Binding interactions between heparin, the most highly sulfated member of the heparan sulfate family of glycosaminoglycans (GAGs), and biologically relevant peptides has been studied in this report. A Superdex peptide (3.2mm x 30cm) SEC column (from Amersham Pharmacia) was equilibrated with buffer containing 0.1 M ammonium acetate (pH 7) and a known concentration of heparin using a flow rate of 0.1 mL/min. Peptides were dissolved in mobile phase buffer and 10 ¿L of this solution was then injected onto the SEC column. The effluent solution was continuously monitored at 214nm using a Hewlett Packard 1090 Series HPLC. Five peptides of interests were analyzed using mobile phases containing buffer only and those containing heparin. In a second series of experiments a smaller diameter SEC column (1mm x 30cm) and a physiological buffer (0.1 M Tris, 0.15 M NaCl, pH 7) were tested. Using equilibrium SEC, the carbohydrate-protein binding affinity can be calculated by the following equation: A=(E0-E)/E0, A=A?/(1+Kd/[heparin]), where E = the elution volume and A is the factor whereby the elution volume is observed to decrease in the presence of heparin in the mobile phase. A? is taken to represent the maximum value of A (when the heparin concentration is saturating). Acquiring mass spectra on the eluting peptides allows analysis of complex mixtures with identification of components that bind heparin in the mobile phase. The five peptides used in these experiments are: fibronectin peptides FN-C/H II, (KNNQKSEPLIGRKKT) and FN-C/H V (WQPPRARI), substance P, gastrin and hirudin. The fibronectin peptides are known to bind heparin and are positive controls. Substance P peptide is a basic neuropeptide, and it was of interest to determine its binding to heparin to test the hypothesis that such binding is an important mechanism in its biological activity. The last two peptides are acidic and serve as negative controls. A series of peptide injections were made using the SEC column equilibrated with heparin concentrations of 8pmol/¿L and 80pmol/¿L, respectively. The results demonstrate that FN-C/H II eluted with a significantly lower volume with increasing the concentration of heparin. The binding of this peptide was distinct from FN-C/H V in that it required a lower concentration of heparin to shift the elution volume. Using this system, substance P is clearly shown to bind heparin, a result that is of considerable biological interest. As expected, the elution volumes of gastrin and hirudin remained unchanged as a function of heparin concentration in the mobile phase. These results demonstrate that equilibrium SEC chromatography is a useful means for identifying biologically relevant binding interactions between peptides and hepar