The lactate dehydrogenase is an important enzyme involved in fermentation process. The objective this experiment is to extract, purify and characterize the kinetic behavior of lactate dehydrogenase from rabbit muscle. Ammonium sulfate suspension, Affi-Gel Blue chromatography and dialysis techniques are used to purify the enzyme. The enzyme assay is used study about the activity while Lowry assay is used to determine the protein concentration and purification efficiency. The LDH purification factor is 14 fold while the recovery of the protein is 59% based on Lowry standard curve. An unknown inhibitor behavior also studied and determined to competitive. The Vmax, Km and KI values of pyruvate is successfully quantified as such 0.36A340/min,0.14 …show more content…
mM and 0.01M. Lactate dehydrogenase (LDH) is an isoenzyme which converts the glycolysis products pyruvate into lactate and NADH into NAD+ via fermentation.
When animal tissues are facing an anaerobic condition, pyruvate enters fermentation and reduced to lactate. This provides the NAD+ which needed for oxidation of Glyceraldehyde -3-phospahte to run the glycolysis in anaerobic conditions. The above reaction strongly favor lactate formation because standard –free energy is large and negative (ΔG’0= -25.1 kJ/mol) ( Lehniger ).Although LDH is found everywhere and perform the same function in all living system, its polypeptide subunits are different in each part of the body. LDH is first perfect example of an isoenzyme found in history ( ).In this lab, we will be purifying M4 type pyruvate which has four identical subunits. This type of LDH is predominately found in muscle. The LDH is also involved in Cori Cycle (reverse reaction) and pyruvate to lactate conversion in aerobic condition such as erythrocytes which cannot do oxidative respiration because of lack of …show more content…
mitochondria. In this experiment LDH is isolated and purified using three methods such as ammonium sulfate suspension, Affi-Gel Blue method and dialysis. The ammonium sulfate is known as salting out where LDH is partially purified from the crude LDH .The salting out technique exploits the solubility property. The ammonium sulfate suspension concentration varies upon each protein’s distribution of surface charges. The salt ions neutralize the surface charges so interaction between the aqueous environments was reduced. The decreased solubility force the proteins to self-associate and aggregate (Asenjo). The purpose of method this methods to decrease volume and increase purity of the protein before chromatographic purification in the next step. Affi-Gel Blue is an affinity based chromatography .The beads in the column has covalently attached chemical group called ligand which binds to protein of interest. The Affi-Gel Blue has agarose gel linked to cibarcon blue FG3A dye that mimics NPA nucleotide. The NADH binding site of LDH will be bind to cibarcon F3GA dye. As the LDH bind to the affinity column, the will eluted. This can be done with repeated centrifugation. The specific elution buffer will remove LDH from the Affi-Gel Blue using the NADH which has more affinity to LDH than the cibarcon F3GA.This is how Affi-Gel Blue purifies LDH specifically out of LDH suspension. After the Affi-Gel Blue purification enzyme activity will be measured using enzyme assays. There two terms are convenient to quantify and characterize the enzyme activity .The first term is total activity which refers to the total units of enzyme in a solution. This could be determined from LDH assay where reaction dilute solutions purified and unpurified LDH .This is performed at 340nm where reaction velocity is determined by decreased absorbance resulting from oxidation of NADH .The total activity of the enzyme is low for purified form because enzyme loss by inactivation and non-ideal interaction with chromatographic column during purification. The second term is specific activity which indicates the enzyme’s purity. The specific activity is calculated from enzyme assay and protein concentration determined by Lowry’s assay. The Lowry method is based protein quantifying techniques introduced by chemist Oliver Howe Lowry in 1940’s ( ).Lowry discovered the Folin phenol reagent will bind ready to copper treated proteins and will change color from yellow to blue. This color change can be quantifying using spectrophotometry at 660nm where disappearance of yellow can be correlated with protein concentration. The steady state kinetic behavior of LDH can be studied using its substrate pyruvate and unknown inhibitor using Michaelis –Menten plot. The pyruvate concentration is plotted against initial concentration, where there is a point where substrate saturation does not increase the initial velocity of substrate binding to active site of enzyme. This value is called Vmax which explains the limitation of binding sites in the enzyme. Michaelis Menten constant Km is a significant kinetic term describes the concentration of substrate which permits the enzyme to achieve half of the Vmax. The lower the value of km, the greater the affinity to substrate to its binding site. The LDH isoenzyme analyzed is in this experiment has lowest Km among the LDH. The accurate Vmax, km and inhibition model will be determined from Lineweaver-Burk plot which the double reciprocal of Michaelis –Menten plot. This is a graphical method developed to calculate the above kinetic terms conveniently and accurately The mechanical extraction of LDH from the rabbit muscle is done with a food processor and filtration.
The purification of LDH accomplished with ammonium sulfate suspension and affi-gel blue suspension. The protein is dialyzed to remove the ammonium salt ions and the kinetic behavior of LDH was studied using an unknown inhibitor type. The pyruvate’s Vmax and km for LDH are reported in the result section of this report. The Vmax and km obtained from the Michaelias Menten plot and lineweaver plots are different as expected. The estimation of Vmax and Km from Michaelias Menten plot is not accurate as double reciprocal plot because the Vmax is an asymptote in Michaelias Menten plot. However, the Vmax found from double reciprocal plot based of y intercept while Km is calculated based of the respective
slopes. According to the results, double reciprocal plot reveals the unknown inhibitor is a competitive inhibition. The y-intercepts are almost the same while the slope of the inhibitor trend line is greater for the “with inhibitor”. This is typical of a competitive inhibition. In competitive inhibition, the intercept on the y-axis of the plot of Lineweaver-Burk plot is the same in the presence and in the absence of inhibitor, although the slope is increased. The y-intercept is unchanged is because a competitive inhibitor does not alter Vmax. The inhibited enzyme will have same Vmax although it approaches it slowly than the uninhibited. This happens because pyruvate molecules are interrupted by inhibitor because competition for the same binding site. However as the pyruvate concentration getting saturated, the effect of inhibitor interruption vanishes. Since Vmax is unaffected by competitive inhibitors the inverse of Vmax also doesn't change. The increase in the slope of the double reciprocal plot indicates the strength of binding of competitive inhibitor. The constant α which is the factor the slope of the double reciprocal graph increase which is one for competitive inhibition. The caluclated α’ is 1.008 which very close to one. This confirms the inhibition type is competitive. The B11 solution which is the protein purified by affi-blue gel suspension. The protein is usually dialyzed to purify the protein from small solutes such as ions. In this experiment the initial purification was done by salting out the protein which contains ammonium sulfate ions. In this experiment, we calculated the purification efficiency of the LDH from it stock solution by calculating purification factor which is 15. This value is obtained from specific activity of B11 after dialysis. Since the dialysis is a purification method which exploits the size of protein and equilibrium of small solutes, it is not possible to remove all the small solutes. However dialyzing specific elution and subtracting its absorbance from the B11 absorbance will give corrected absorbance for dialysis purified protein. Purification factor after dialysis is satisfactory because between ranges of normal purification fold (10-25).The recovery of the protein from the dialysis is 59% which is also in the acceptable range (40% to 80%). However above calculation are not completely reliable because protein concentration and activity are caluclated based on standard curve obtained based of Lowry assay. Lowry assay has limitation because the protein concentration determined from absorbance quality exhibited by aromatic amino acids such Tryptophan and tyrosine. Each protein has different compositions of aromatic amino acids, so that the absorbance is not proportional to actual protein concentration. In addition, the color development in Lowry assay is interrupted by ions such as ammonium sulfate (Folin.O).
In the lab, Inhibiting the Action of Catechol Oxidase we had to investigate what type of enzyme inhibition occurs when an inhibitor is added. Catechol oxidase is an enzyme in plants that creates benzoquinone.Benzoquinone is a substance that is toxic to bacteria. It is brown and is the reason fruit turns brown. Now, there are two types of inhibitors, the competitive inhibitor and non-competitive inhibitor. For an enzyme reaction to occur a substrate has to bind or fit into the active site of the enzyme. In competitive inhibition there is a substrate and an inhibitor present, both compete to bind to the active site. If the competitive inhibitor binds to the active site it stops the reaction. A noncompetitive inhibitor binds to another region
Data table 1 Well plate Contents Glucose concentration A 3 drops 5% sucrose + 3 drops distilled water Negative B 3 drops milk+3 drops distilled water Negative C 3 drops 5% sucrose +3 drops lactase Negative D 3 drops milk +3 drops lactase 15+ E 3 drops 20% glucose +3 drops distilled water 110 ++ Questions B. In this exercise, five reactions were performed. Of those reactions, two were negative controls and one was a positive control.
In this experiment the enzyme peroxidase and the substrate hydrogen peroxide were not mixed initially, instead they were both placed in separate tubes and were incubated at a specific temperature, to prevent hydrogen peroxide from undergoing any reaction with peroxidase until they both acquire the required temperature.
Living organisms undergo chemical reactions with the help of unique proteins known as enzymes. Enzymes significantly assist in these processes by accelerating the rate of reaction in order to maintain life in the organism. Without enzymes, an organism would not be able to survive as long, because its chemical reactions would be too slow to prolong life. The properties and functions of enzymes during chemical reactions can help analyze the activity of the specific enzyme catalase, which can be found in bovine liver and yeast. Our hypothesis regarding enzyme activity is that the aspects of biology and environmental factors contribute to the different enzyme activities between bovine liver and yeast.
One of the most primitive actions known is the consumption of lactose, (milk), from the mother after birth. Mammals have an innate predisposition towards this consumption, as it is their main source of energy. Most mammals lose the ability to digest lactose shortly after their birth. The ability to digest lactose is determined by the presence of an enzyme called lactase, which is found in the lining of the small intestine. An enzyme is a small molecule or group of molecules that act as a catalyst (catalyst being defined as a molecule that binds to the original reactant and lowers the amount of energy needed to break apart the original molecule to obtain energy) in breaking apart the lactose molecule. In mammals, the lactase enzyme is present
The affects of pH, temperature, and salt concentration on the enzyme lactase were all expected to have an effect on enzymatic activity, compared to an untreated 25oC control. The reactions incubated at 37oC were hypothesized to increase the enzymatic activity, because it is normal human body temperature. This hypothesis was supported by the results. The reaction incubated to 60oC was expected to decrease the enzymatic activity, because it is much higher than normal body temperature, however this hypothesis was not supported. When incubated to 0oC, the reaction rate was hypothesized to decrease, and according to the results the hypothesis was supported. Both in low and high pH, the reaction rate was hypothesized to decrease, which was also supported by the results. Lastly, the reaction rate was hypothesized to decrease in a higher salt concentration, which was also supported by the results.
The purpose of this experiment was to see if phenylthiourea (PTU) is a non-competitive or competitive inhibitor. Catechol, a phenolic compound found in the potato extract used will play the part of the substrate. Competitive inhibitors are known to bind to the active site of an enzyme and mimic the job of a substrate. This in turn causes the substrate to compete for a position at the active site and increase the concentration of substrate but the inhibitor is still at a constant level. If PTU were a competitive inhibitor the test tube carrying the extract would turn dark brown. Non- competitive inhibitors are known to bind on the enzyme and prevent the substrate from attaching
The purpose of this experiment was to discover the specificity of the enzyme lactase to a spec...
The Effect of pH on the Activity of Catalase Planning Experimental Work Secondary Resources Catalase is a type of enzyme found in different types of foods such as potatoes, apples and livers. It speeds up the disintegration of hydrogen peroxide into water because of the molecule of hydrogen peroxide (H2O2) but it remains unchanged at the end of the reaction.
Abstract: Enzymes are catalysts therefore we can state that they work to start a reaction or speed it up. The chemical transformed due to the enzyme (catalase) is known as the substrate. In this lab the chemical used was hydrogen peroxide because it can be broken down by catalase. The substrate in this lab would be hydrogen peroxide and the enzymes used will be catalase which is found in both potatoes and liver. This substrate will fill the active sites on the enzyme and the reaction will vary based on the concentration of both and the different factors in the experiment. Students placed either liver or potatoes in test tubes with the substrate and observed them at different temperatures as well as with different concentrations of the substrate. Upon reviewing observations, it can be concluded that liver contains the greater amount of catalase as its rates of reaction were greater than that of the potato.
According to the graph on amylase activity at various enzyme concentration (graph 1), the increase of enzyme dilution results in a slower decrease of amylose percentage. Looking at the graph, the amylose percentage decreases at a fast rate with the undiluted enzyme. However, the enzyme dilution with a concentration of 1:3 decreased at a slow rate over time. Additionally, the higher the enzyme dilution, the higher the amylose percentage. For example, in the graph it can be seen that the enzyme dilution with a 1:9 concentration increased over time. However, there is a drastic increase after four minutes, but this is most likely a result of the error that was encountered during the experiment. The undiluted enzyme and the enzyme dilution had a low amylose percentage because there was high enzyme activity. Also, there was an increase in amylose percentage with the enzyme dilution with a 1: 9 concentrations because there was low enzyme activity.
Alkaline Phosphatase (APase) is an important enzyme in pre-diagnostic treatments making it an intensely studied enzyme. In order to fully understand the biochemical properties of enzymes, a kinetic explanation is essential. The kinetic assessment allows for a mechanism on how the enzyme functions. The experiment performed outlines the kinetic assessment for the purification of APase, which was purified in latter experiments through the lysis of E.coli’s bacterial cell wall. This kinetic experiment exploits the catalytic process of APase; APase catalyzes a hydrolysis reaction to produce an inorganic phosphate and alcohol via an intermediate complex.1 Using the Michaelis-Menton model for kinetic characteristics, the kinetic values of APase were found by evaluating the enzymatic rate using a paranitrophenyl phosphate (PNPP) substrate. This model uses an equation to describe enzymatic rates, by relating the
Many factors, for example, pH and temperature affects the way enzymes work by either increasing the rate or determining the type of product produced (). The report, therefore, analyses the effects of the enzyme peroxidase in metabolic reactions and determining its optimum temperature in the reactions.
In our Biology Lab we did a laboratory experiment on fermentation, alcohol fermentation to be exact. Alcohol fermentation is a type of fermentation that produces the alcohol ethanol and CO2. In the experiment we estimated the rate of alcohol fermentation by measuring the rate of CO2 production. Both glycolysis and fermentation consist of a series of chemical reactions, each of which is catalyzed by a specific enzyme. Two of the tables substituted some of the solution glucose for two different types of solutions. They are as followed, Table #5 substituted glucose for sucrose and Table #6 substituted the glucose for pH4. The equation for alcohol fermentation consists of 6 Carbons 12 Hydrogens 6 Oxygen to produce 2 pyruvates plus 2 ATP then finally the final reaction will be 2 CO2 plus Ethanol. In the class our controlled numbers were at Table #1; their table had 15 mL Glucose, 10 mL RO water, and 10 mL of yeast which then they placed in an incubator at 37 degrees Celsius. We each then measured our own table’s fermentation flasks every 15 mins for an hour to compare to Table #1’s controlled numbers. At
...t being the only target of these enzymes they also make changes retinol, steroids, and fatty acids. The collection of different kinds of alcohol dehydrogenase guarantees that there will always be one that is just perfect for the each task. This enzyme size is 80,000 g/mol, its charge is pI = 5.4, and it optimal pH I 8.6.