home / homework help / questions and answers / science / biology / lab 5: enzyme kinetics provided m… Show more home / homework help / questions and answers / science / biology / lab 5: enzyme kinetics provided materials: •1 … Question Lab 5: Enzyme Kinetics Provided materials: •1 tablet of lactase enzyme, 3,000 to 9,000 FCC per group •30 clean 13×100 mm test tubes per group •250 mL of chilled 0.1 M PO4 buffer, pH 7 aliquoted per group ?TA will double check with pH stick, needs to be precise •10 mL for each group of each 0.1 M PO4 buffer at each of the five following pH values, stock solutions located in beaker at front of room: pH 5.0, pH 5.5, pH 6.0, pH 6.5, pH 7.5, and pH 8.0 TA: make two solutions and mix – see guide in references •40 mL per group stock solution of 1.0 mM ONP aliquoted 69.55 mg in 500 mL of PO4 buffer, pH 7 for 12 groups; CAS# 88-75-5 •30 mL per group stock solution of 200 ?M ONPG (sometimes 2-NPG) aliquoted 24.08 mg in 400 mL of PO4 buffer, pH 7 for 12 groups; CAS# 19710-96-4 •30 mL 4% K2CO3 aliquoted per group 4 g in 400 mL for 12 groups •Mortal and pestle, 1 per group •Spectrophotometer, digital or analog, 1 per group •30°C water bath, 1 per table. The water bath can get crowded with pipetting, so they need to be individual water baths. Use a hot plate and 400 mL beaker filled with ~150 mL tap water and a thermometer stick Potential hazards: •None, all materials can go down the drain Objectives: •Prepare a standard curve of a product •Calculate the extinction coefficient for the product using either absorbance or concentration •Determine concentration of product formation in an enzyme assay •Calculate the reaction velocity, Vmax, and KM for an enzyme •Determine the optimal pH of an enzyme BIOL 301, Enzyme Kinetics Lab Last Modified Feb 12, 2013 Page 1 of 9 Introduction: Michaelis-Menten Kinetics Living systems depend on chemical reactions which, on their own, would occur at extremely slow rates. Enzymes are catalysts which reduce the needed activation energy so these reactions proceed at rates that are useful to the cell. Various enzymes have very different rates of catalysis, or activity. To determine the rate of activity for a given enzymatic reaction, one generally monitors either the disappearance of the substrate or the appearance of the product of the reaction, thus allowing the measurement of the rate of catalysis by the enzyme. In this laboratory exercise, you will analyze product formation to determine enzyme activity. As demonstrated in last week’s lab on protein assays, spectrophotometry can be used to determine the concentrations of a compound of interest. This is dependent on two criteria: (1) the substrate and the product do not absorb light at the same wavelength, and (2) the Beer-Lambert Law holds true for the substance or product. Two essential conditions for any enzyme assay are: 1.Controls must be performed to show that the assay’s response (product formation, in this case) is due only to the action of the enzyme. 2.The assay response must be linearly related to the time of the assay and to the quantity of enzyme assayed under the experimental conditions used. Controls are performed along with the experimental assays to compensate for activity (assay response), which may not be due to the action of the enzyme. Two types of controls are generally needed for enzyme assays: 1.One type of control has substrate in the reaction mixture, but no enzyme. This controls for the amount of the reaction, which may proceed without the aid of the enzyme. 2.The second type of control contains the enzyme, but no substrate. This controls for possible background reaction in the enzyme preparation itself. When the proper controls are performed with the enzymatic assay, one can use the data generated to determine the enzyme kinetics. In this laboratory, the substrate concentration will be altered to determine the maximum rate or velocity (Vmax) for the enzymatic reaction. If you measure enzyme velocity at many different concentrations of substrate, the graph generally looks like Figure 1. The initial rate of reaction or V0 is computed by taking the moles of product formed per volume per time of reaction or moles/mL/sec. BIOL 301, Enzyme Kinetics Lab Last Modified Feb 12, 2013 Page 2 of 9 Figure 1. Enzyme saturation in terms of initial reaction rate (or velocity), V0, substrate concentration, [S], maximum velocity, Vmax, and the Michaelis constant, KM. An equation derived in 1913 by Leonor Michaelis and Maud L. Menten describes quantitatively the rate of velocity of an enzyme catalyzed reaction. The equation for the curve is: V = V max?[S ] Equation 1 V = [S ]+ KM Equation 1 where V is the current reaction rate, Vmax is the maximum velocity of the reaction when the enzyme is saturated (horizontal dotted line in Figure 1), [S] is the substrate concentration, and KM is the Michaelis’ constant. The Michaelis’ constant reflects the affinity of an enzyme for its substrate (that is, the strength by which the enzyme binds to its substrate). Generally, the lower the value of the KM the greater the affinity of the enzyme for its substrate. If the substrate concentration [S] is adjusted so that it equals KM, KM can be substituted for [S] in Equation 1. Vmax?K M 1 V = = ?V max Equation 2 V = K M + K M = ?V max Equation 2 K M + K M 2 Since this algebraic manipulation shows that V = ½ Vmax when KM = [S], the reverse statement is also true: KM = [S] when V = ½ Vmax. Therefore, KM is equal to the substrate concentration when an enzyme-catalyzed reaction is running at half of its maximum velocity. Figure 1 graphically depicts the relationship between V, Vmax, [S], and KM. However, in practice, it is difficult to use experimental results to generate a saturation curve of the type shown in Figure 1 because many points must be plotted to draw the curve of this type accurately. It is also difficult to determine the saturation level, or Vmax, directly from the experiment. To solve these problems, a conversion called the Lineweaver-Burke plot is often used. For this plot, reciprocals are taken of both sides of Equation 1: 1 = [S ]+ K M = ( K M K M )? 1 + 1 Equation 3 V = V ?[S ] = ( V max )? [S ] + V max Equation 3 V V ?[S ] V max [S ] V max max max max Equation 3 has the form of a linear equation (y = mx + b) with a slope equivalent to KM/Vmax and the intercept of the y-axis equivalent to 1/Vmax. This mathematical technique converts the hyperbola of Figure 1 into a straight line that can be accurately drawn from relatively few experimental points (Figure 2). Since y- intercept is equivalent to 1/Vmax, and the x-intercept – 1/KM, the values for Vmax and KM can be determined quickly and accurately. BIOL 301, Enzyme Kinetics Lab Last Modified Feb 12, 2013 Page 3 of 9 Figure 2. Lineweaver-Burke plot. A plot of the reciprocal of the reaction rate (or velocity, 1/V) and the reciprocal of the substrate concentration (1/[S]). This plot, the Lineweaver-Burke plot, produces a straight line with the y-intercept equivalent to 1/Vmax and the x-intercept equal to -1/KM . The slope of the line is equivalent to KM/Vmax. In this lab experiment, you will be able to calculate the Vmax using the above equations 1 and 2. Lactase Assay Lactose, (milk sugar) is a disaccharide formed from galactose and glucose. Its ?-glycosidic linkage must be hydrolyzed to yield its component monosaccharides before they can be absorbed by the body. In humans, lactase — a member of the ?-galactosidase family of enzymes — performs this task (Figure 3). Figure 3. Natural ?-galactosidase reaction of lactose to galactose and glucose. Many individuals lose the ability produce lactase, and therefore cannot digest lactose as they enter their teens. As a result, they suffer GI distress (e.g., nausea, diarrhea) when they consume milk products. This is clinically known as lactose intolerance. Lactase tablets may help reduce this problem. In part one of the lab, we will determine the enzyme kinetics of over-the-counter lactase tablets to catalyze the breaking of the ?-glycosidic linkage. We will not be using lactose as a substrate because we want to obtain a product that can be quantitated using a spectrophotometer. Instead, the substrate used in the assay of the lactase tablets is ortho-nitrophenyl-?-D galactoside (ONPG; M.W. 301.249), which, upon hydrolysis of the ?-glycosidic linkage, yields galactose and ortho-nitrophenol or 2- nitrophenol (ONP; M.W. 139.11), a yellow compound with a maximum absorbance at 420 nm (Figure 4). Enzyme activity is therefore proportional to the increase in absorbance at 420 nm during incubation. Figure 4. Artificial ?-galactosidase reaction of ONPG to galactose and ONP producing a yellow color. In part two of the lab, we will determine the pH optimum of the enzyme lactase. Enzymatic activity is strongly dependent on protein conformation. Since pH determines whether an amino acid’s side chain is charged or not, and ionic interactions affect tertiary protein structure, pH has a pronounced effect on a protein’s conformation and therefore on its enzymatic rate. Typically, the maximum rate of action of an enzyme is found only when it is folded in a precise fashion. The pH which produces this precise folding is termed its pH optimum. An enzyme’s pH optimum may be determined by performing multiple assays, each identical except for the pH at which it is run. Graphic display of the resulting data (reaction rate versus pH) demonstrates the enzyme’s pH optimum. As in many enzyme assays, adjustments in concentrations and volumes may be needed for optimum results. Keep careful track of how you set up your experiment. BIOL 301, Enzyme Kinetics Lab Last Modified Feb 12, 2013 Page 5 of 9 Experimental Procedure Part 1: Standard curve for ONP In order to convert absorbance readings into product, you must first construct a standard curve for the product ONP. You will be provided with a stock solution of 200 ?M ONP. 1)Prepare 7 tubes containing 0.0, 0.5, 1.0, 2.0, 3.0, 4.0, and 5.0 mL of an ONP solution, and bring each tube to a total volume of 6.0 mL by adding 0.1 M PO4 buffer, pH 7.0. 2)Calculate the total amount of mole present in the solution. 3)Using the sample with only phosphate buffer as the blank to calibrate the spectrophotometer. Save this blank for the other experiments. 4)Read the absorbance of these standard tubes at 420 nm. It is best to blank the spectrophotometer after each reading. 5)Plot absorbance against moles of ONP in the standard samples in your lab report Complete the following table (with the exception of the absorbance at 420 nm) BEFORE lab to help the experiment proceed more efficiently. Some blank have been filled in for you. If you think you need the molecular weight, you are doing it wrong. Tube # ONP(mL) PO4 Total Volume (mL) ONP (umol) Absorbance (420 nm) 1 0.0 6 6 0 0 2 0.5 5.5 6 0.1 .01 3 1.0 5.0 6.0 0.2 .02 4 2.0 4 6 0.4 .03 5 3.0 3 6 0.6 .05 6 4.0 2.0 6 0.8 .05 Use transmittance and convert to absorbance on the analog spectrophotometer. Part 2: Enzyme Assay In this experiment, the initial rate of product formation (V0) will be measured under various substrate concentrations [S]. The substrate concentrations used will vary from those that are rate-limiting to those that are excess. Subsection 2A: Enzyme Preparation 1)Get a lactase tablet 2)Using the box, record the brand of lactase, number of units of lactase per tablet and the expiration date. 3)Note whether you have 9,000 FCC [Food Chemical Codex] or 3,000 FCC units per tablet. Ask the TA to see the Lactase box for the number of units. 4)Grind in a mortar and pestle until finely ground. 5)GOAL: We want to get a final solution of exactly 1 unit/mL, but we have to do it in two steps: 6) Dissolve the powdered lactase pellet in chilled 0.01 M PO4 buffer so that the final concentration is 100 units/mL. If you have 9,000 unit pellet dissolve in 90 mL of buffer. If you have 3,000 unit pellet dissolve in 30 mL of buffer. (Solution will be cloudy because of undissolved powder.) 7) Now, dilute the 100 units/mL solution by a factor of 100 with 0.01 M PO4, pH 7. Final volume of diluted enzyme should be 30 mL with a concentration of 1 unit/mL. Use the DF = V1 / V2 equation; solve for V1; the calculate V(dilutent). see Lab 1 for review. 8) You will use this diluted enzyme in both parts 2 and 3. Keep on ice until ready to use. Subsection 2B: Enzyme Assay 1)Gather and label tubes 1–10: 2)Set up the following reaction tubes, omitting the lactase. Tube # PO4 (mL) ONPG (mL) Lactase(mL) Total Vol (mL) Transmittance/Absorbance (420 nm) 1 5.5 0.0 0.50 6.0 0 2 5.25 0.25 0.50 6.0 0.01 3 5.00 0.50 0.50 6.0 0.01 4 4.50 1.00 0.50 6.0 0.07 5 3.50 2.00 0.50 6.0 0.16 6 1.50 4.00 0.50 6.0 0.53 7 0 5.50 0.50 6.0 0.60 Table 2: Amount of sample to add to each tube. * denotes control samples, # do NOT add lactase until instructed. 3)Place these tubes in a 30°C water bath for about 3 minutes to warm up. (Be sure to check the temperature of the water bath before putting in your tubes). 4)Because you cannot add enzyme to each tube at the same time, we will do it in a set time interval. 5)At 15 second intervals, add 0.5 mL (500 ?L) of diluted lactase suspension (enzyme) to tubes 2-10 and mix. DO NOT ADD ENZYME TO TUBE 1. Start a stopwatch when the 1st tube is returned to the 30°C water bath. Note: you should add enzyme to each tube ONLY ONCE. BIOL 301, Enzyme Kinetics Lab Last Modified Feb 12, 2013 Page 7 of 9 6)After exactly 10 minutes, add 1.0 mL 4% K2CO3 to the first tube to stop the reaction, mix and remove from water bath. At 15 second intervals, repeat 4% K2CO3 addition for each of the successive tubes, mix and set aside. Making sure that tube 2-10, each had exactly 10 minutes of active enzyme. 7)Using a tube with only phosphate buffer as the blank to calibrate the spectrophotometer. Read the absorbance at 420 nm, record in your notebook table. 8)Use your standard curve relating absorbance to moles of ONP from Part 1, determine the amount of product (ONP) formed in each reaction tube. Analysis of Results Include the following in your lab report: 1. Plot the standard curve obtained in Part 1. Does the absorbance of light by ONP obey the Beer- Lambert Law? 2. Data tables of absorbance, moles of product formed, and initial reaction velocities for all samples. The initial rate of reaction, V0 is the moles of product formed per milliliters of total reaction per minute (V0 is in units of mol/mL/min). 3. Plot the initial reaction rate, V0, against the substrate concentration [S]. 4. On a separate graph, plot your data as a Lineweaver-Burke plot. 5. Determine KM and Vmax for your data from the slope and intercept of this graph. Can anyone please do these last 5 problems of this lab. It is due today and I am just so lost. I need this data to do my lab report. I am so lost and its more math and graphing then I expected. I will reward with a lot of points. • Show less