BIOTINYLATED HYALURONIC-ACID AS A PROBE FOR IDENTIFYING HYALURONIC ACID-BINDING PROTEINS

The glycosaminoglycans hyaluronan (HA), heparin, and chondroitin sulfa te were biotinylated using biotin-x-hhydrazide (biotin-epsilon-aminoca proyl hydrozyde) in conjunction with N-ethyl-N'-(3-dimethylaminopropyl )carbodiimide hydrochloride, an activating agent for carboxyl groups. The biotin-x-hydrazide was shown to be coupled directly to the glycosa minoglycans in enzymatic digestions and competition experiments. The b iotinylated HA was shown to bind to link protein and receptor for hyal uronic acid-mediated motility, two proteins known to bind HA. The labe led HA was used as a probe to detect known HA-binding proteins in chic ken cartilage extract and to identify new HA-binding motifs in the G3 domain of the proteoglycan aggrecan. The significance of the biotinyla tion of HA, heparin, and chondroitin sulfate A are discussed. (C) 1995 Academic Press, Inc.