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. Introduction: The study of Chondroitin Sulfate (CS), a repeating polymer consisting of GlcA(1,3)-GlcNAc(1,4) disaccharide units with various sulfation patterns at the 4- and 6-O-positions of GalNAc, and 2-O- position of GlcA (CSA, CSC, and CSB), is hindered by the large size and heterogeneity of these glycans. Enzymatic depolymerization of the CS chains has been a useful technique to obtain structural information but has proven difficult to control. The lyase cleaves smaller CS fragments faster than large fragments, producing populations containing more disaccharide than information rich oligosaccharides. In addition, each substrate has different susceptibility to the lyase, requiring different reaction conditions. In this work, controlled enzymatic depolymerization is achieved using porous chromatography beads to sequester oligosaccharides from the further degradation. Methods: Chondroitinase ABC (of various amounts) was added to solutions containing 100 ?g CS and monitored on a Beckman DU640 UV-Vis detector at 37 ?C until the reaction was completed. Identical reactions were exposed to varying amounts and types of hydrated size-exclusion resin to determine the optimal pore size for the desired cleavage profile. Reactions were filtered and washed to remove the resin and elute the digestion products. After lyophilization the reactions were separated by SEC (Beckman Gold 125 solvent module). The eluant was monitored at 232 nm using a Beckman Gold 166 UV detector. The fractions were analyzed on a Bruker Esquire 3000 Ion Trap mass spectrometer. Results CS type-C was digested in the presence of various amounts of size-exclusion resin (SEC). The use of a 10,000 molecular weight cut-off dialysis membrane was also tested. The removal or sequestering of digestion fragments allows for the analysis of larger subunits than those typically observed after complete digestion. Resins were chosen by their pore sizes, with fractionation ranges from 700 to 30,000 Da, to determine the optimal pore size for an informative distribution of polysaccharide fragments, while excluding substrate (50 kDa) and enzyme (120 kDa). Results indicate that the distribution of digested CSC is significantly be altered in the presence of resins designed to fractionate 700-7000 Da. Interestingly, the resin utilized for the separation of larger biomolecules (30,000 Da) did not have a significant effect on the digestion. In experiments where the complete digestion of CSC was observed in the control reaction, digestions performed in the presence of SEC resin possessed one-third the quantity of both disaccharide and tetrasaccharides and significant increases in the presence of hexamer, octamer and decamers species, while leaving little to no undigested CSC. The presence of these saccharides was confirmed by mass spectrometry. These results indicate that this may be useful as a general method for enzymatic depolymerization of GAG substrates. A preliminary digestion performed using dialysis membranes (mwt c.o. 10,000 Da) did not significantly alter the distribution of the resulting fragments. However, no digestion products were observed in the buffer chamber indicating that there may not have been free exchange between chambers. This system is currently under further investigation.