N-HYDROXYSUCCINIMIDE-ACTIVATED GLYCINE-SEPHAROSE - HYDROLYSIS OF ACTIVATED GROUPS AND COUPLING OF AMINO-COMPOUNDS

Glycine-Sepharose CL 6B, activated with 1-ethyl-3-(3-dimethyl-aminopro pyl)-carbodiimide (EDC) and N-hydroxysuccinimide (NHS), was used as a model compound to study the hydrolysis and aminolysis of immobilized N HS-activated groups. For comparison, the soluble analog N-t-BOC-glycin e-NHS has been used. Coupling of amino compounds, such as aminoethanol , aminohexane, amino acids, and esters of amino acids, is fast and eff icient in both organic medium and buffered aqueous medium, e.g., coupl ing of aminoethanol is complete within 1 min. Hydrolysis of the activa ted groups in aqueous medium is general base catalyzed and is, particu larly in berate buffer and at higher pH (> 9.0), accelerated by additi on of salt. The NHS-activated glycine-CL 6B is less sensitive toward h ydrolysis as compared to N-t-BOC-glycine-NHS. When amino compounds are coupled to NHS-activated Sepharose in aqueous medium, the use of a lo w buffer concentration and a pH of 8.5-9.0, without salt, is recommend ed. In combination with salt, phosphate buffer is preferred.