title: Immobilised lipoamide dehydrogenase. 2. Properties of the enzyme immobilised to agarose through spacer molecules of various lengths.
abstract: 1. Pig heart lipoamide dehydrogenase (NADH: lipoamide oxidoreductase, EC 1.6.4.3) has been immobilised to Sepharose by thiol-disulphide interchange via a series of thiolated spacer molecules of increasing length. A number of properties of the immobilised enzyme have been investigated in order to ascertain the effects of proximity to the matrix backbone. 2. Proximity to the matrix backbone reduced the specific activity for lipoamide as substrate but enhanced by 3-8-fold the diaphorase activity with 2,6-dichloroindophenol. These observations are explained in part by an increase in the apparent Km for lipoamide when the enzyme is covalently attached to Sepharose via a short spacer molecule. 3. Both the thermal stability at 90 degrees C and the stability in 30% (v/v) dioxane are enhanced by up to 200% when the enzyme resides close to the matrix but approach those of the native enzyme as the length of the spacer molecule is increased. 4. These data have been correlated with measures of the accessibility of the enzyme as the nominal length of the spacer arm was increased. Thus, as the chain length increased, the rate of cleavage of the disulphide linkage between the enzyme and spacer increased and the enzyme became more susceptible to proteolysis by thermolysin. In contrast, increasing the chain length of the spacer made the enzyme less amenable to inhibition by a specific antibody. 5. These data are discussed in terms of the effect of the matrix on the conformation of the bound enzyme.
mesh_terms: D000818:Animals; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D004146:Dioxanes / Q000494:pharmacology; D004220:Disulfides; D004355:Drug Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D009206:Myocardium / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D011485:Protein Binding; D012685:Sepharose; D013329:Structure-Activity Relationship; D013552:Swine; D013696:Temperature
chemical_list: D004146:Dioxanes; D004220:Disulfides; D004800:Enzymes, Immobilized; D010446:Peptide Fragments; D008623:Mercaptoethanol; D012685:Sepharose; D008058:Dihydrolipoamide Dehydrogenase