Studies on conformation of soluble and immobilized enzymes using differential scanning calorimetry. 1. Thermal stability of nicotinamide adenine dinucleotide dependent dehydrogenases.
The technique of differential scanning calorimetry (DSC) has been applied to the study of temperature-induced irreversible denturation and thus to the heat stability of soluble and Sepharose-bound liver alcohol dehydrogenase (LADH, EC 1.1.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) in the presence of various coenzymes or coenzyme fragments. The transition temperature (Ttr) of 82.5 degrees C obtained for soluble LADH was increased by 12.5 degrees C in the presence of a saturating concentration of NACH. In the presence of NAD+, Ttr increased by 8.5 degrees C, whereas ADP-ribose and AMP caused an increase in Ttr of only 2 and 1 degree C, respectively. The Ttr of 85.5 degrees C obtained for Sepharose-bound LADH was increased by about 12 degrees C after the addition of free NADH. However, when the enzyme was immobilized simultaneously with a NADH analogue (which also binds to the matrix), a broad endotherm with a Ttr of 91.5 degrees C was obtained, indicating the presence of immobilized enzyme molecules both with, and without, associated NADH. Corresponding increases in heat stability were observed for LDH in solution in the presence of NADH, NAD+, and AMP, leading to increases in Ttr from 72 to 79.5 and 74 and 73 degrees C, respectively. The addition of pyruvate and NAD+ to the enzyme to form an abortive ternary complex led to the same stabilization as that observed with NADH, attendant with a large increase in the enthalpy of transition, deltaHtr. In these studies the technique of DSC was utilized because it is applicable both to soluble and immobilized enzymes and (1) provides rapid information about Ttr and thus thermal stability of enzymes, (2) different energetic states of an enzyme molecule can be identified, and (3) an overall picture of the thermal process is rapidly obtained.
D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D002151:Calorimetry; D004355:Drug Stability; D004800:Enzymes, Immobilized / Q000378:metabolism; D006736:Horses; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008099:Liver / Q000201:enzymology; D009206:Myocardium / Q000201:enzymology; D009243:NAD; D011487:Protein Conformation; D011489:Protein Denaturation; D012685:Sepharose; D013552:Swine; D013696:Temperature
D004800:Enzymes, Immobilized; D009243:NAD; D012685:Sepharose; D000429:Alcohol Oxidoreductases; D007770:L-Lactate Dehydrogenase