Laboratory 10 Enzyme chemistry - reaction kinetics Introduction The effect of substrate concentration on the rate of an enzyme catalyzed reaction is described by the Michaelis-Menten equation for most enzymes. The Michaelis-Menten equation relates substrate concentration [S] to reaction rate (v) and the kinetic constants V max and Km. Once these kinetic constants have been determined for a given set of conditions (pH, buffer, temperature etc.), the Michaelis-Menten equation can be used to predict the reaction rate given knowledge of the substrate concentration. In this manner, enzyme reactions can be used and rates predicted for both food processing applications and for analytical purposes. The objectives of this exercise are to observe the enzymatic hydrolysis of sucrose and to obtain data that permit the calculation of Km and Vmax for invertase from bakers yeast. Materials - 0.1 M sodium citrate buffer pH 4.65 with 0.01 M sodium azide - 2.0 N sodium hydroxide - 0.5% (w/v) crude invertase (ß-fructosidase prepared from bakers yeast) in citrate buffer (pH 4.65) - sucrose solutions in pH 4.65 citrate buffer (5,10,20,40,80,100 mM sucrose) - 1% (w/v) 3,5-dinitro salicylic acid in 1.43 M sodium potassium tartrate - spectrophotometer at 546 nm Procedures 1. Pipet 0.5 mL of each sucrose solution into duplicate 20 mL screw cap test tubes. Also pipet 1.0 mL citrate buffer into a screw cap tube to serve as a blank. 2. To each sucrose tube, add exactly 0.5 mL of enzyme suspension and mix well. Note: Do not add enzyme to the blank. 3. After 15 minutes at room temperature, add 1.0 mL of the 3,5-dinitro salicylic acid color reagent to all tubes (reaction tubes and blank). Add 1 mL of 2 N NaOH, mix well, and place in a 90C water bath for 10 minutes. This stops the reaction. Cool with tap water to room temperature. 4. Add 9 mL distilled water to each tube and mix well. Read the absorbance at 546 nm after adjusting the zero with the blank. Data for the standard curve will be provided. Calculations and discussion 1] Calculate the concentration of total reducing sugar in each tube from the standard curve (or from the regression analysis provided). Plot the rate of reducing sugar formation (v, micromoles per 15 minutes) versus substrate concentration (sucrose concentration in the 1.0 mL reaction mixture). Remember to decrease the sucrose concentration appropriate to the dilution in the reaction mixture. 2] Plot 1/v vs. 1/s and determine the Km and Vmax from each plot. Enzyme chemistry - activity and inhibition of enzymes in cabbage Introduction Peroxidase and phenoloxidase are two enzymes involved in the deterioration of plant products. Reactions catalyzed by these enzymes and treatments to inhibit the reactions will be examined. Peroxidases catalyze the following reaction: H2O2 + AH2 2 H2O + A (or polymeric A) where AH2 is a hydride donor. In plant products, peroxidase activity may cause the development of off-flavors during frozen storage. The primary way of deactivating enzymes prior to freezing or dehydration is blanching. Because of the thermal stability of peroxidase compared to other enzymes, peroxidase activity is used as an indicator of the adequacy of blanching. Phenol oxidases are enzymes in fruits and vegetables that catalyze enzymatic browning. The reactions are complex since, depending on the plant material, the enzyme may catalyze the reactions of monophenols, polyphenols or both. The reactions are shown in the following figure: 1. monophenol + O2 + AH2 o-diphenol + A + H2O 2. 2 (o-diphenol) + O2 2(o-quinone) + H2O Many phenolic compounds act as substrates for phenol oxidases and thus a wide variety of products are formed. The brown color characteristic of enzymatic browning is due to the polymerization of the o- quinones and the reaction of o-quinones with amines and phenolic compounds. Procedures A. Enzymatic browning observation 1. Slice an apple into 6 pieces. Immediately place the slices into one of the following: a. water (control) b. 0.2% ascorbic acid c. 0.2% catechol d. 0.1% EDTA e. 0.2% sodium metabisulfite 2. Observe the amount of browning after 30 minutes after dipping. Rate the brown color on a scale of 0- 5 (0=none). Explain the effect of each treatment. B. Effect of blanching on enzyme activity Materials - cabbage (finely chopped) - cheese cloth - 0.1 M potassium phosphate buffer (pH 7) - Waring Blender - 3.6 mM guaiacol (peroxidase substrate) - 8 mM hydrogen peroxide (peroxidase substrate) - spectrophotometer 1. You will blanch samples ( 60 g) and also have a control (0 time). Place the samples in a boiling water bath for various heating times (0, 10, 20, and 40 sec). At the end of the time-interval, dump the cooked sample into a colander in the sink, and immediately place into an ice bath to stop the reaction. Drain the sample and then assay for enzyme activity as in “step 2.” 2. Weigh 25 g cabbage sample, place in Blender and blend with 200 mL distilled water for 1 minute. 3. Filter a portion using Whatman No. 1 paper in a funnel collecting 5-10 mL on ice. These filtrates are used to assay enzyme activity. 4. For each filtrate, prepare the following mixture in a test tube precisely: 5.0 mL phosphate buffer pH 7.0, 0.5 mL guaiacol solution, and 0.5 mL cabbage filtrate 5. Transfer an aliquot of one filtrate into a cuvette and zero spec at 436 nm then pour it back into its test tube. 6. To start the enzyme reaction, add 0.1 mL of the hydrogen peroxide solution, mix thoroughly, pour the mixture into the cuvette, and record the absorbance at 436 nm each 30 seconds for 5 minutes. Repeat this for all of your samples. Calculations and discussion 1] Plot the absorbance vs time. 2] From the plots in 1], determine the initial rate (absorbance change per minute) for each sample. 3] Make a plot of blanching time vs enzyme activity. What blanching time is required to completely inactivate the cabbage peroxidase? Questions for discussion and conclusion 1. What do the Km and Vmax tell you about the invertase reaction under the conditions examined ? 2. How could pH effect a chemical reaction? 3. What factors are important to consider when calculating kinetic parameters (Vmax, Km) from the plots as above? 5. Where on the velocity substrate concentration curve is it best to measure enzyme rates and why? 6. What are four reasons why a measured enzyme activity could be zero? 7. In your report, discuss the importance of measuring initial rate when determining enzyme activity.