ACTIVITY AND STABILITY OF NATIVE AND MODIFIED ALANINE AMINOTRANSFERASE IN COSOLVENT SYSTEMS AND DENATURANTS

Alanine aminotransferase (ALT) is used in clinical diagnostics, amino acid synthesis and in biosensors. Here we describe the stabilization o f soluble porcine ALT by chemical modification with mono- and bis-imid ates. The apparent transition temperatures ('T-m', the temperature whe re 50% of initial activity was lost in 10 min) for native and DMS-modi fied ALT were 46 and 56 degrees C respectively. The effects of water-m iscible organic solvents (methanol, dimethylformamide, dimethylsulphox ide and 1,4-dioxane) on the activity/stability of native and modified forms were determined. In all systems studied, an abrupt decrease in A LT catalytic activity was observed on reaching a certain threshold con centration of the organic solvent. The modified derivatives were more organotolerant than native enzyme. Comparison of the apparent V-max an d K-m for 2-oxoglutarate as substrate, determined in 10% (v/v) organic solvent, with the results of thermal inactivation studies showed that the solvents have different effects on ALT's catalytic parameters and on its conformational stability. At 35 degrees C with no organic solv ent the dimethylsuberimidate (DMS)-modified derivative's half-life was 16 times greater than that for native enzyme; in 30% (v/v) solvent at 35 degrees C, the DMS-modified ALT's half-life was up to 4.6 times gr eater than native enzyme's. DMS-modified ALT was also more stable in u rea and guanidine HCl, and its refolding was more noticeable, than tha t of native enzyme.