Report On Purifying LDH Laboratory

The LDH enzyme is used by the body to catalyze the change of lactate into pyruvate as it changes NAD+ to NADH. LDH is a very significant enzyme for energy production in the cells. In this experiment the LDH from a deer heart was purified and the concentration was found using a Bradford assay. The techniques used for the first part of the experiment included homogenization, centrifugation, and ammonium sulfate precipitation. The homogenization process began by taking 128. 178 g of deer heart and blending it with 380 ml of 0. 05 M phosphate buffer. 0. 5 ml of each fraction isolated was assayed for enzyme activity using the Genesys 10S uv-vis spectrophotometer. The activity per minute consistently increased as the concentration of the protein increased with each additional step of purification. The absorbances of each purified fraction was plotted on a standard curve graph to determine the concentration of LDH in the original fractions of deer heart. The 65% pellet contained the highest concentration of proteins of all the fractions at 9. 9 mg/ml.

Introduction

Lactate dehydrogenase is a common enzyme found in many bodily cells. The LDH enzyme is used by the body to catalyze the change of lactate into pyruvate as it changes NAD+ to NADH. LDH also converts pyruvate to lactate when oxygen levels are depleted. It is a very significant enzyme for energy production in cells. The enzyme is found in many diverse bodily cells, with higher concentrations found in the heart, liver, kidneys, and muscle cells. When cells with a high concentration of LDH die or become ruptured from damage, the enzyme enters the blood stream. Many diseases of the body may cause the levels of LDH to increase in the blood stream. LDH is composed of four key subunits that form a tetramer. The two subunits combine to form a total of five different forms. The five different isoenzymes are found in different tissues of the body, which aides in diagnoses of diseases. LDH-1 consist of four subunits of the heart isoform. In this experiment the LDH from a deer heart was purified and the concentration was found using a Bradford assay.

Methods

The first part of the experiment consisted of collecting and partially purifying the LDH fragments from a deer heart. The techniques used for this part of the experiment included homogenization, centrifugation, and ammonium sulfate precipitation. The homogenization process began by taking 128. 178 g of deer heart and blending it with 380 ml of 0. 05 M phosphate buffer with a pH of 7. After the homogenization was completed, 45 ml of the crude homogenate was placed in a centrifuge tube. The crude homogenate was spun at 20000 x g for 15 minutes at 4 degrees Celsius. 25 ml of supernatant was collected from the 20000 x g sample, and the pellet was discarded.

Next, ammonium sulfate was added to complete a 40% cut of the sample. The powdered ammonium sulfate was added at a ratio of 0. 230 g/ml, for a total of 5. 753 g added slowly to the sample. This 40% sample was centrifuged at 15000 x g for 15 minutes at 4 degrees Celsius. 20 ml of the 40% supernatant was recovered after centrifugation was complete, and the pellet was discarded.

Next, a 65% cut was preformed, using ammonium sulfate added at a ratio of 0. 166 g/ml. A total of 3. 32 grams of ammonium sulfate was added for the 65% cut. The 65% sample was centrifuged at 15000 x g for 15 minutes at 4 degrees Celsius. 20 ml of the 65% supernatant was recovered, and the pellet was resuspended in 5 ml of TRIS buffer. 0. 5 ml of each fraction isolated was assayed for enzyme activity. The reaction cocktail consisted of 1. 9 ml of 0. 14 M caps buffer, 0. 5 ml of NAD+ and 0. 15 M lactate, 90 ul of water, and 10 ul of fraction to be tested. The Genesys 10S uv-vis spectrophotometer was set a 340 nm for the assay of enzyme activity. For the second part of the experiment the concentrations of the LDH fractions was determined using a Bradford assay. The standard curve for the Bradford assay was completed by adding 1 ml of Bradford protein reagent to each cuvette, along with an increasing amount of LDH standard. The LDH standard stock was 1 mg/ml, it was added in increments of 0, 2, 4, 6, 8, 10, 15, and 20 ul into each cuvette. The spectrophotometer was set at 540 nm for the Bradford reagent. Each cuvette was run in the spectrophotometer and the absorbances were plotted to produce a standard curve for the LDH concentration.

Next, each of the fractions collected previously were run in the spectrophotometer. 2 ul of each fraction was added to 1 ml of Bradford reagent. The absorbances collected were plotted on the standard curve to determine the concentration of LDH in each fraction.

Results

The activity per minute consistently increased as the concentration of the protein increased with each additional step of purification. The 65% supernatant had the lowest concentration of protein, with an activity of only 0. 016.

Conclusion

The process of purifying LDH was completed by the processes of homogenization, centrifugation, and salting out by ammonium sulfate precipitation. The processes of centrifugation and salting out efficiently removed the LDH from all the other cell debris. After the purification process was completed, a standard curve of LDH stock was made using a Bradford assay. Two microliters of each fraction were assayed in the spectrophotometer to find the absorbance. The absorbances of each purified fraction was plotted on graph 1 to determine the concentration of LDH in the original fractions of deer heart. The 65% pellet contained the highest concentration of proteins of all the fractions at 9. 9 mg/ml. To further purify the fractions of LDH a combination of chromatography techniques could be used. These techniques could include ion exchange, affinity chromatography, and gel chromatography. One place of possible error in the experiment could come from adding the ammonium sulfate to fast, this would cause error in the purification process by causing the proteins to clump up unevenly.