PURIFICATION OF LACTATE DEHYDROGENASE FROM CHICKEN MUSCLE TISSUE

Shivalee Gujarathi Department of Biology, California State University, Fresno October 10, 2013

Abstract The enzyme lactate dehydrogenase (LDH) catalyzes the last step of anaerobic glycolysis that is important for the normal function of the body. Purification of LDH is essential to understand its structure and function. The purpose of this experiment was to extract and purify LDH enzyme from chicken muscle tissue using a variety of various. Analytical methods such as activity and protein assay were employed to determine the presence and purity of LDH. The cells were initially disrupted and proteins were solubilized. LDH was purified from the ammonium sulfate precipitated protein mixture by affinity chromatography and its activity was studied by spectrophotometric determination of NADH at 340 nm. From Pierce BCA assay of crude homogenate, initial protein concentration was shown to be 100 mg/ml. The final protein concentration of the pooled affinity sample was shown to be 0.2 mg/ml. It was found that the total specific activity of LDH was 58.5 mol/min/mg, and yield of 0.6%. Even though we were successful in purifying LDH enzyme, further steps can be taken to increase the yield.

Materials and Methods Cell Lysis and Extraction of LDH: Approximately 40 g of minced chicken breast meat (40.327 g) is blended with 75ml cold extraction buffer in four 30-seconds bursts for homogenation of the muscle tissue. The extraction buffer contained 10mM Tris-HCl (pH-7.4), 1mM 2-Mercaptoethanol, 1mM Phenylmethylsulfonylflouride (PMSF), 1mM Ethylene diamine tetraacetic acid (EDTA). The homogenization procedure was carried out in the cold room to prevent the denaturation of proteins. The homogenate was centrifuged at 15,000 rpm for 20 minutes at 40 C. The supernatant was filtered through two layers of cheesecloth to remove lipids from the supernatant. The total volume was noted and three 0.5 ml aliquots (crude extract) were stored at -200 C. Ammonium sulfate precipitation: 60% ammonium sulfate concentration was used to precipitate proteins. 0.39 g of ammonium sulfate per ml of the supernatant was added gradually to the supernatant for 15-20 min with continuous gentle stirring at 40 C. The mixture was centrifuged for 20 minutes at 15,000 rpm at 40 C. The supernatant was discarded and the pellet was stored at -200 C. Dialysis: Ammonium precipitation leads to high concentration of salts in protein mixture that can interfere with further purification steps. In order to remove excess salts, dialysis was performed. The pellet was suspended in Tris-PMSF buffer (10 mM Tris-HCl, pH 8.6, 0.5 mM 2Mercaptoethanol, and 1mM ratio of EDTA) and mixed very gently until it dissolved at 4 0 C. Volume of 4ml protein mixture was added in the dialysis tubing and incubated twice overnight with two 1L buffer changes (Same buffer as extraction buffer that was used for cell lysis). After two incubations, protein mixture was resuspended gently and centrifuged for 10 minutes at

15,000rpm at 40C. Pellet was discarded, total volume of supernatant was noted and three 0.1 ml aliquots were collected. Affinity Chromatography: Cibarcon Blue column was used to separate LDH from the other proteins. 5ml fractions were collected in thirteen test tubes. Column was first rinsed with Tris-PMSF buffer followed by addition of protein mixture. Then, 10ml NAD Buffer (10mM Tris-HCl pH-8.6, 0.5mM 2-Mercaptoethanol, 1mM Lithium acetate and 1mM NAD+) was added followed by 10ml NADH (10mM Tis-HCL PH 8.6, 1mM NADH and 0.5mM 2Mercaptoethanol). Between each steps, column was washed with 10ml Tris-PMSF Buffer. Each fraction was subjected to absorbance reading of 280nm. For absorbance above 1.5nm, 1:10 dilutions were carried out. Activity Assay: We used LDH Enzyme assay to measure the amount of LDH activity in our protein mixture. LDH catalyzes the conversion of lactate to pyruvate and NAD+ to NADH. The NADH can be determined spectrophotometrically at 340 nm. The LDH assay was performed in the crude homogenate, desalted fraction and six peak fractions from the Cibacron blue column. A cocktail solution was prepared by mixing lactate stock solution (120 mM lithium lactate, 10 mM Tris-HCl; pH 8.6), NAD+ stock solution (12 mM NAD+, 10 mM Tris HCl; pH 8.6) and bicarbonate stock solution (18 mM NaHCO3, 0.5 M NaCl) in the ratio of 6:4:2 in cuvette. 10 microliters of the sample is then added and the assay absorption is measured at 340nm. If absorbance was above 1.5, samples were diluted. Protein Assay: The Pierce BCA Protein Assay (Thermo Scientific) is a detergentcompatible formulation based on bicinchoninic acid (BCA) for the colorimetric detection and quantitation of total protein concentration. A series of standard solution of Bovine Serum Albumin (BSA) ranging from 0-2000 g/ml was prepared from a stock solution of 2 mg/ml

BSA. 25ul of diluted crude (1:500, 1:250), desalted (1:100, 1:50), and 6 peak fractions from cibarcon blue column (1:10, 1:5) were loaded in microplate along with 175ul of BCA working reagent. Microplate was incubated for 30min at 370C and then the absorbance was measured at 562nm.

Results/Discussion The purpose of this experiment was to extract and purify LDH enzyme from chicken muscle tissue using a variety of techniques including homogenization, ammonium sulfate precipitation, dialysis, and affinity chromatography. Activity and Protein assay were used to track the overall amount of LDH present in the samples. Crude Extraction: Chicken muscle tissue was homogenized in a blender with cold extraction buffer in order to lyse cells, releasing LDH into slurry of tissue components. Centrifugation separated membranes, nuclei, and other large cellular components to a pellet leaving a supernatant of crude product. Controlling temperature was a major consideration after homogenization since not only did this step releases proteins like LDH from the cell, but it also releases proteases that can now interact to degrade the LDH. Keeping samples on ice, precooling the buffer, and avoiding excess kinetic energy through conservative blending were methods used to minimize activity of these proteases. After filtration through cheesecloth, our final volume of crude homogenate sample 74ml, much more volume than expected. Addition of more than 75ml of buffer volume could have increased the volume. Other possible explanation is that more solid components such as fats were present in the sample and hence, more than 20 minutes of centrifugation was required.

Desalted Sample: 60% ammonium sulfate is added to the crude extract that precipitates LDH proteins. The resulting 40% pellet theoretically contains most of the original LDH, which is re-suspended in very less volume (4ml) to create a more concentrated sample. This process leads to high concentration of salts in protein mixture that can interfere with subsequent purification steps. 4ml protein mixture underwent dialysis procedure that removes excess salts and our final volume after dialysis was 6ml. One possible explanation for increase in our volume could be that extraction buffer got mixed with protein mixture either due to tubing leaking or tubing clips not being properly tightened. Affinity Chromatography: Cibacron Blue column is an affinity column, which is specific to dehydrogenase type proteins, due to a compound structurally similar to NADH being attached covalently attached to the column. 13 fractions were collected and absorbance was measured at 280nm to check presence of LDH protein in the fractions. 1:10 dilution was performed if absorbance reading was above 1.5nm since it spectrophoretically indicates saturation and less than 1% light reaching the detector. During the addition of protein mixture (fraction# 4), high absorbance reading of 10nm was obtained (Fig.1). This could be due to lot of non-dehydrogenase-type proteins present in our sample that got eluted first during affinity chromatography. Second peak was seen after NAD+ was added since NAD solution results in the removal of the loosely bound protein. Third peak was seen after NADH was added since NADH solution results in release of maximum LDH proteins (Fig. 1). Enzyme Activity Assay: The LDH activity was measured spectrophotometrically by measuring the absorbance of NADH at 340 nm. Three peak fractions were selected for this assay

based on their absorbance values obtained after adding NAD+ (fraction# 6, 7, 8) and other three after adding NADH in the affinity chromatography step (fraction# 10,11,12). A huge activity of 141umol/min/ml was seen at fraction# 7(PF1) which indicated that we had lot of proteins present in our sample. Second peak activity was seen at fraction #10 indicating that more LDH proteins is present in this fraction than in fraction# 11 (PF2) (fig.1). Based on this information, we selected fraction #10 as for our protein assay. Desalted showed highest activity among all the samples (Table1) possible due to errors occurring during dialysis explained previously.

10

8 6 4 2 0 0 5

Test Tube Number

10

15

Figure 1. Absorbance readings of elutes obtained from affinity chromatography with LDH activity for 6 peak fractions. The desalted fraction was loaded to the Cibarcon blue column and proteins were eluted with Tris-PMSF, NAD+ and NADH wash subsequently. The absorbance at 280 nm of elutes were measured after each collected fractions. The LDH activity was calculated from the absorbance values obtained at 340nm. Protein Assay: We used BCA Pierce Assay to determine protein concentrations in our protein mixture. BSA standard curve was created for series of dilutions ranging from 0-2000 g/ml and linear graph equation was used to calculate protein concentrations for the samples (Table 1). Based on Table 1, with each subsequent purification step, protein concentration decreases as sample become more concentrated with only LDH protein. Specific activity should increase and total activity should decrease with every purification step as samples get less and less diluted. Similar trend was observed in our study as well. However, exception is PF1 that has

Activity (umol/mim/ml)

12

160 140 120 100 80 60 40 20 0

A280 [nm]

higher specific activity due to high activity suggesting more loosely bound proteins were eluted after NAD+ was added.

Step

Activity Protein conc. (mol/ min/ml) (mg/ml)

Total Volume (ml)

Total Total Protein Activity (mg)

Specific Activity

Fold Purific -ation

Yield (%)

(mol/ (mol/min min) /mg) 102 100 74 7400 10200 1.02 1 100 Crude 37 6 222 936 4.2 4 9 Desalted 156 141 1.8 5 9 705 78.5 77 7 PF1 11.7 0.2 5 1 58.5 58.5 57 0.6 PF2 Table 1: Purification Table indicating successful purification of LDH protein. Activity of 10L LDH was determined via time course measurements using the UV-Vis Spectrophotometer, monitoring the NADH production by following the absorbance at 340nm. Protein concentration was determined using the equation of the BSA standard curve determined through the Pierce BCA Assay (y = 0.0012x + 0.1057). Total Activity = Activity x Total volume. Specific Activity = Activity/ Protein Concentration. Fold Purification = Specific LDH Activity at given step/ Crude Homogenate Specific Activity. % Yield = (Total LDH Activity/ Crude Homogenate Total Activity) *100.

Increase in fold purification suggested that we were able to successfully purify LDH with every step (Table 1.) However, steps can be taken to increase the yield by using different cell lysis method such as sonication to reduce surface denaturation of LDH proteins, using desalting column instead of dialysis to avoid leakage, and/or using different affinity column to better separate LDH from other proteins. Further experiments, like SDS PAGE analysis and western blot should also be done to characterize LDH for purity and physical properties. The enzyme kinetic study can also be done to study the functional activity of LDH.