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1 Skinner

Purification of Lactate Dehydrogenase (LDH) from Chicken Breast Muscle: Enzyme Kinetics

Author: Jaise Skinner Lab Partner: Alyssa Klancher November 23, 2020

2 Skinner Introduction Lactate dehydrogenase (LDH, E.C. 1.1.1.27) is an enzyme that catalyzes the anaerobic production of energy from glucose.1 It is present in many of the body’s cells and most prominently in the heart, muscles, kidneys, lungs, and liver.2 When tissue damage occurs, the body releases LDH into the bloodstream – making it an identifiable marker for injuries. Multimeric crude LDH can be extracted from tissues like chicken breast muscle. Dehydrogenase enzymes like LDH transfer hydrides between different molecules. LDH usually exists in two different forms of isozymes due to its composition of protein subunits from two separate genes. However, there are five different forms of LDH isozymes existing in specific concentrations throughout the body. In addition, LDH is essential for cellular respiration and is involved in the reciprocating reaction of lactate and pyruvate. LDH allows for nicotinamide adenine dinucleotide (NAD+) -- a non-protein co-catalyst -- to assist in the redox portion of the interconversion reaction.3 Subsequently, LDH is essential in allowing NAD+ to reform when NADH (reduced form of NAD+) is unable to progress along the electron transport chain. This allows for the mitochondria to continue the production of ATP. Essentially, LDH is the driving force behind an athlete’s ability to continue ATP production despite the presence of oxygen. The rate of the interconversion reaction of pyruvate to lactate is associated with the formation of NADH and is proportional to the amount of LDH present. Thus, pure LDH is required in order to study the kinetics and effectiveness of the conversion of lactate to pyruvate. 1 Berg, J.M., Tymoczko, J.L., Stryer, L. (2002). The Purification of Proteins Is an Essential First Step in Understanding Their Function. Biochemistry 5th Edition. 4.1. Retrieved from: https://www.ncbi.nlm.nih.gov/books/NBK22410/ 2 Wolf, E. (1988). The Partial Purification and Characterization of Lactate Dehydrogenase. Biochemical Education 16(4), 231-234. Retrieved from: https://iubmb.onlinelibrary.wiley.com/doi/pdf/10.1016/03074412%2888%2990136-7 3 Department of Chemistry and Biochemistry. (2020). Weeks 2&3: Purification of Lactate Dehydrogenase (LDH) from chicken breast muscle. University of Minnesota- Duluth.

3 Skinner Protein purification is essential to determine the specific properties and structures of different enzymes. In addition, protein purification is what allows biochemists to study how different enzymes and catalysts contribute to biological processes. Affinity chromatography is an effective method to purify proteins. It purifies proteins based off of an individual’s affinities for specific moieties.4 An elution column is packed with a resin that contains resin beads that specific proteins have high affinities for. The beads will bind to specific protein molecules that one wishes to extract from a mixture. The desired protein, LDH has a histidine rich region (histag) that creates a high affinity for metals like nickel and cobalt that make up the beads of the affinity resin.5 LDH will bind to these immobilized metals due to the his-tag region and all other proteins within the homogenous mixture will pass through the column. The desired proteins bound to the column require an elution buffer to be extracted from the column. The elution buffer usually contains a molecule that will have a higher affinity for the resin than the desired protein; therefore, they will replace the protein on the resin. Another purification method that is effective at separating proteins of different shapes and sizes is size exclusion chromatography. Controlling the pore sizes of resins is the basis of size exclusion chromatography. Size exclusion chromatography resins have a molecular weight (MW) cut off that is the approximate MW of a spherical protein that is too large to penetrate the pores.6 Mixtures containing proteins that are added to size exclusion resins usually separate in three different ways. Large molecules flow around the beads of the resin while smaller molecules 4 Department of Chemistry and Biochemistry. (2020). Week 2 & 3 Part II: Purification of chicken LDH by affinity chromatography. University of Minnesota- Duluth. 5 Chaga, G., Hopp, J., Nelson, P. (1999) Immobilized metal ion affinity chromatography on Co2+carboxymethylaspartate–agarose Superflow, as demonstratedby one-step purification of lactate dehydrogenase from chickenbreast muscle. Biotechnol. Appl. Biochem. (29), 19-24. Retrieved from: https://pubmed.ncbi.nlm.nih.gov/9889081/

6 Department of Chemistry and Biochemistry. (2020). Weeks 4&5: Size Exclusion Chromatography of Chicken LDH. University of Minnesota-Duluth.

4 Skinner enter the pores with ease. Thus, the largest molecules will elute first because they travel the shortest path through the beads in the resin.7 Conversely, the smallest molecules will elute last due to their longer travel path through the beads. Furthermore, the shape of a protein will also be affected by the pores of the beads which allows size exclusion chromatography to be used to separate proteins of different shapes. To understand enzymes like LDH, it is important to understand how it participates in different reactions. Kinetic assays allows one to study how an enzyme will interact with other substrates, products, and other molecules that effect activity. The use of a purified enzyme allow for more accuracy in kinetics experiments as more variables can be controlled for. This allows for information and conclusions to be drawn with ease and simpler evaluation of kinetic parameters.8 Enzymes catalyze reactions and can exist in different intermediate states. For this reason, it is most common to observe the kinetics of a catalyzed reaction. The data obtained through kinetic experiments are essential for Michaelis-Menten kinetic analysis and LineweaverBurk plots. Using these schemes allows for the Michaelis constant (Km) and the maximum reaction rate (Vmax) of the enzyme to be found. Enzyme kinetics is being performed on a previously purified sample of LDH. The kinetics of the enzyme is being evaluated by changing the concentration of NAD+ used. It is expected that the steady-state reaction will produce linear data that will allow for the Km of the enzyme to be determined. In addition, the inhibitor used is expected to display competitive inhibitor characteristics. 7 Loa, C.C., Lin, T.L., Wu, C.C., Bryan, T.A., Thacker, H.L., Hooper, T., Schrader, D. (2002). Purification of turkey coronavirus by Sephacryl size-exclusion chromatography. Journal of Virological Methods 104(2), 187-194. Retrieved from: https://www.sciencedirect.com/science/article/abs/pii/S0166093402000691 8 Department of Chemistry and Biochemistry. (2020). Weeks 8&9: Introduction to LDH Michaelis-Menten Enzyme Kinetcs. University of Minnesota-Duluth.

5 Skinner Materials and Methods Enzyme Kinetics LDH Dilution. A series of LDH dilutions were created using HEPES buffer (pH 8.6) in order to find a dilution that yielded an activity of about 0.25 to 0.4 A/min. 0.3 mL of Lactate stock solution (240 mM lithium lactate, 10 mM HEPES, pH 8.6), 0.2 mL of NAD+ stock solution (24 mM NAD+, 20 mM HEPES, pH 8.6), 0.1 mL Bicarbonate stock solution (36 mM NaHCO3, 1.0 M NaCl), and 0.6 mL of water were added to a cuvette. 10 L of an enzyme dilution sample to be tested was added to the cuvette and activity data was collected using the spectrophotometer at 340 nm. Enzyme Kinetics NAD+ Km Determination. A series of lactate dilutions were made after finding an appropriate LDH dilution. These dilutions were used in place of the LDH stock in the activity assay. 6 different NAD+ dilutions were then ran to create a wide range of velocities. NAD+ concentrations were chosen that bracketed the Km about 3-4 substrate concentrations above and below it. The Km and the Vmax of the enzyme were estimated to check the results of the assay. A rectangular hyperbola was created by plotting the data. Inhibitor effects on LDH were examined by performing LDH assays with varying NAD+ concentrations. Data was collected for initial velocity and substrate concentration and was subsequently plotted. Data was collected for at two additional inhibitor concentrations of 50% concentration and full concentration. The concentrations were low enough for activity to be easily detected, but were also high enough for inhibition to be observed. The inhibition type was deduced and the KI for the inhibitor was calculated. Results

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Lineweaver-Burke Inhibition 25 20

1/v0

15 10 5 0

0

Uninhibited 2

4

Linear 8 (Uninhibited) 10

6

12

1/[S] uM

Figure 1. Lineweaver- Burke inhibition plot. The activity (units/minute) of 11 varying NAD+ substrate concentrations (uM) were measured for different dilutions of inhibitors. The reciprocal of both the activity and the substrate concentration were plotted to establish a linear relationship. A linear trendline was produced for each inhibitor dilution and the equation of the line was found in order to solve for Vmax and Km parameters. Table 1. Km (1/uM) and Vmax (units/min) values for different inhibitor concentrations. Values were calculated using linear trendlines from a Lineweaver-Burke plot.

Km (1/uM) Vmax (units/min)

Uninhibited 1.25 mM 2.5mM 0.1335 0.1125 0.1257

0.3362

0.2549

0.08631

The Km of the uninhibited LDH was calculated to be 0.133 1/uM with a Vmax of 0.3362 units/min (Table 1) using the linear trendlines from Figure 1. These values were obtained using the equations y-intercept= 1/Vmax and slope= Km/Vmax. The Km remained relatively the same at either inhibitor concentration while the Vmax decreased (Table 1). Figure 1 displays linear trendlines that have increasing slopes at higher concentrations of inhibitor as well as increasing y-intercepts. Discussion

7 Skinner It was expected that the data in Figure 1 would form linear trendlines that would allow for the Km of the enzyme to be determined. This was supported by the data in Figure 1 and Table 1. The Km was found using the trendline produced from Figure 1. The inhibitor used was expected to have a competitive effect on the enzyme. This initial expectation was not supported by the collected data. The Km remained the same while the Vmax was reduced which represented the characteristics of a non-competitive inhibitor. It was expected that the Km would increase while the Vmax would remain unchanged to mirror the effects of a competitive inhibitor. The discrepancy in the Vmax values is likely a result of improper pipetting of the enzyme. Any slight variation in the amount of enzyme added will directly affect the Vmax of the reaction. In addition, using the reciprocal of either parameter leads to exasperations in the experimental error. Enzyme assays are known to contain considerable experimental error which leads to larger errors when the reciprocal values are plotted. Thus, using linear trendlines to calculate the parameters leads to inaccurate values. It is common to manipulate the errors values using functions in Microsoft Excel in order to account for the error in the enzyme assays. This minimizes the effects of the error and leads to a more accurate determination of Km and Vmax. In the future, it would be beneficial to manipulate the collected data in Microsoft Excel and compare the Km and Vmax to the untransformed data. In addition, a useful follow up experiment would be to observe the effects of changing lactate concentration while keeping the NAD+ concentration constant. Overall, the initial hypothesis that the inhibitor would act competitively was not supported by the data collected in the experiment. Conclusion It was expected that the collected data would create a linear trendline that would allow for Km to be calculated. In addition, the inhibitor used was expected to behave competitively. The

8 Skinner Km for the uninhibited enzyme was calculated to be 0.1335 1/uM while the inhibitor displayed non-competitive affects. For this reason, the collected data did not fully support the initial hypothesis....