MDH Assays - Substrates and Enzyme Dilution

OAA - Diluted in water or buffer, OAA degrades even on ice. HOWEVER, Dr. Chris Berndsen (JMU) with his students found that using 0.1 M HCl to dissolve OAA creates a stable solution of OAA for MDH assays. This OAA/0.1 M HCl solution is stable for many hours. Freeze aliquots and use without a problem. Some folks have used several freeze-thaw cycles without obvious degradation. To be safe, freeze in small volumes and thaw once as needed. Using the 20 mM stock OAA for the 1.0 and 1.5 ml assay protocols, the pH will not change if the enzyme assay buffer is 100 mM Tris or Phosphate (pick your favorite pH). If using a plate reader assay, you may want to check the volumes and potential changes in pH after adding OAA in 0.1 M HCl to your cocktail. Lisa Gentile and her students (St Benedict & St John’s Univeristy) is studying OAA stability using NMR.

NADH - Preparing 10 mM NADH for the assays are often prepared in one of two ways. 1) Pre-weigh ~0.04 to 0.08 g and store in falcon tube in freezer after calculating the volume of water/buffer to add to each tube. Prepare NADH and freeze in 0.5 to 1.0 aliquotes and use as needed. Frozen samples are good for many months or even semesters.

Diluting MDH: Diluting MDH for performing enzyme assays is a concern. Diluting most enzymes from a 0.2-1.0 mg/ml at 1:200 in assay buffer will result in a 5-10% loss of activity in three hours and ~20% activity after 4 hours or longer. Higher dilutions (1:1000) of purified MDH can result in loss of even 50% of the initial activity over 2-3 hours. Diluting in 5% glycerol will limit the loss of enzyme activity. However the Provost group found that diluting the enzyme in Enzyme Dilution Buffer (10% glycerol, 0.1 mg/ml BSA Fatty Acid Free dissolved in enzyme assay buffer) will maintain activity with very little loss even over several hours.

Storing MDH: Once purified - do NOT freeze your MDH until you dialyze the imidazole away. Most MDH isoforms are very stable in the Ni-affinity elution buffer containing imidazole. Store at 4oC for 2-3 months with minimal loss of protein. For longer-term storage, dialyze with several changes using 10% Glycerol, 50 mM NaCl, 1 mM beta-ME in 10 mM K or Na Phosphate, pH 8.0. This will freeze and store for months with minimal loss of activity. Some proteins will aggregate at around 1.0 mg/ml protein.

Protocols: MDH 1.0 ml assay MDH 1.5 ml assay MDH Storage Instructions