Lactate dehydrogenase isoenzymes from human heart: separation by preparative gel electrophoresis; and a crystalline preparation of isoenzyme--1.

A flat-bed apparatus for preparative gel electrophoresis with intermittent elution [l-31 permits the collection simultaneously of anodeand cathode-migrating components of a mixnue. We employed this first for the separation of isoenzymes of lactate dehydrogenase (EC 1.1.1.27) in crude tissue extracts. Enzyme activity was determined by reaction rate observed at 340 nm in the presence of 0.14 mM NADH. 0.66 mM sodium pyruvate and 100 mM uis-acetate, pH 7.4. Units are according to Wroblewski and La Du [4]. Human hearts were obtained from the autopsy mom within 24 h after death. Pieces of heart muscle (2-3 g) were chilled and washed twice in ice cold 0.15 M sodium chloride. The tissue was subsequently minced and homogenized using a Top-Drive homogenizer (M.S.E.) at top speed for 3-4 min. The resultant suspension was filtered through muslin, the filtrate centrifuged at l00,OOO g for 20 min at 4OC and the supernatant was used for separation by preparative electrophoresis with results shown in Figure 1. Heart muscle revealed three peaks of lactate dehydrogenase activity. The major activity was found in isoenzyme 1. Each of the separated zones gave a single band on analytical electrophoresis after staining for enzyme activity. Thus isoenzymes 1, 2, and 3 are easily and completely separated from each other in a single run.