Volume 35, number 2 FEBS LETTERS September 1973 SEPARATION OF THE ISOENZYMES OF LACTATE DEHYDROGENASE BY AFFINITY CHROMATOGRAPHY USING AN IMMOBILIZED AMP-ANALOGUE Peter BRODELIUS AND Klaus MOSBACH l~'ochemical Division, Chemical Centre, University of Lund, P.O. Box 740, S-220 07 Lund 7, Sweden Received 17 July 1973 1. Introduction The use of affinity chromatography [ 1] or biospe- cific aff'mity chromatography [2] for the separation and purification of enzymes is by now a widely ac- cepted technique. In order to test whether this tech- nique will permit separation of the multiple molecular forms of an enzyme as lactate dehydrogenase, the present investigation was initiated with the object of separating this enzyme into its five different isozymes. In previous studies the advantages involved, using a general ligand for affinity chromatography, have been demonstrated. Thus Sepharose-bound N6-(6-ami - nohexyl)-AMP, was used for separation and purifica- tion of a number of NAD÷-dependent enzymes [3-5]. The general underlying principle is that adenosine-5'- monophosphate (AMP) as inhibitor for a great number of enzymes will eliminate the need of binding a differ- ent inhibitor ligand for each enzyme to be separated. Subsequent application of specific eluents will then permit elution of one enzyme at a time [5]. The tech- nique used in the present study involves application of the above Sepharose-bound AMP-analogue, elution subsequently being carried out with a weak gradient of NADH. 2. Experimental Crystalline beef heart L(+) -lactate dehydrogenase (LDH) (type III), crystalline beef muscle L(+)-LDH (type X), pyruvate (type II, sodium salt), DL-lactic acid (grade DL-V, sodium salt),/3-NADH (grade Ill), /~-NAD ÷ (grade III), nitro-blue tetrazolium (grade III) and phenazine methosulfate were purchased from Sigma Chemical Co., St. Louis, Mo., USA. 'AMP- Sepharose' was prepared as described elsewhere and contained 200 ~umoles of nucleotide per g of dry poly- mer [6]. All other chemicals were of analytical grade and were used without further purification. LDH activity was measured by the Warburg's assay [7], modified as follows: In the assay were included sodium phosphate (300/a moles); pyruvate (5/a moles); NADH (0.5/~ moles) and test solution (50/al) in a to- tal volume of 3.0 ml, pH 7.5. Preparation of the isoenzyme mixture from beef heart LDH (containing mainly H 4 and H3M) and beef muscle LDH (containing mainly M4) was based on the 'quick-freeze' and 'slow-thaw' method [8]. Heart LDH (0.45 mg) and muscle LDH (0.55 mg) were dis- solved together in 0.1 M sodium phosphate buffer pH 7.0, 1 mM/3-mercaptoethanol (0.4 ml) and dialysed against the same buffer (500 ml) for 5 hr. The dia- lysed solution was diluted with the same buffer con- taining 2 M NaC1 to give a protein concentration of 1mg/ml. This solution was rapidly frozen over a mix- ture of acetone and dry ice. It was then allowed to thaw slowly at room temperature. About 90% of the enzyme activity remained after hybridisation. The sample applied to the affinity column was pre- pared by mixing the above hybridised LDH (0.3 mg) with heart LDH (0.035 mg) and muscle LDH (0.040 mg). This was done in order to obtain a solution con- taining roughly equal amounts of each of the five iso- enzymes. A concave gradient of NADH was obtained using two parallel-sided containers of a cross-sectional area North-Holland Publishing Company - Amsterdam 223