Experiment 2 EFFECT OF EXTRACTION CONDITIONS AND TITRIMETRIC ASSAY OF ENZYME ACTIVITY

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1 Experiment 2 EFFECT F EXTRACTIN CNDITINS AND TITRIMETRIC ASSAY F ENZYME ACTIVITY Some enzymes are present in extracellular fluids, secretions and excretions; assay and purification of the enzyme may be done directly on those fluids. ther enzymes are contained in cells either in the cytoplasm or in subcellular organelles. The cellular and organelle membranes must be ruptured by grinding, blending, pressure or ultrasonic oscillation in order to release the enzyme. The ph and ionic strength, as well as dielectric constant, of the extracting fluid is often quite important in releasing and stabilizing the enzymes. We will use isobutanol in Experiment 11 to solubilize alkaline phosphatase from its adsorption on the fat globules of milk. Plant cells concentrate acids in the vacuoles. When the cells and vacuoles are ruptured the acids are released and will denature enzymes not stable at lower phs. Denaturation is prevented during extraction by use of a buffer to maintain the ph in a range where the enzyme is stable. Enzymes often are attached to membranes and cell walls and are released only at higher ionic strengths. In this experiment we shall look at the effect of three extraction conditions on the amount of pectinesterase activity recovered from the albedo (white) of an orange. During many enzyme reactions a proton is either a substrate or product of the reaction. In the presence of a buffer with adequate capacity the ph is maintained constant and one does not observe a change in the [H + ]. If the buffer is eliminated, or is in low amount, the ph will change in such reactions. If NaH or HCl is added to the reaction at a rate that keeps the ph constant, then the rate of addition of titrant measures the rate of the enzyme reaction. All one needs to obtain the rate of reaction is a ph meter, a burette filled with titrant of known concentration, a timer and lots of patience. A ph stat, designed to automatically add and record the amount of titrant needed to keep the ph constant, greatly simplifies the experimental work. Pectinesterase is found in most higher plants and is in particularly large amounts in the albedo of the orange. Pectinesterase catalyzes the hydrolysis of the methyl ester bond of pectin giving pectic acid and methanol as products (Eqn. 1). Assuming a pk of 4 for the carboxyl group, above ph 6 an equivalent amount of H + will be liberated for each ester bond hydrolyzed.

2 CCH 3 H CCH 3 H 2 H 2 C - H C - H H Pectin H n H Pectic acid H n REAGENTS: + 2 CH 3 H + 2 H + Extraction fluids: The solutions have been prepared for you. Chill them in an ice bucket. They are 0.5% NaCl 0.1 M sodium phosphate, ph M sodium phosphate, 0.5% NaCl, ph 7.5 ranges (1 case for two laboratory sections) Crude pectinesterase: Peel two oranges so as to just remove the flavedo (orange-colored skin). Then strip the albedo from the pulp of the orange. The albedo will be used as described below. Pectin: 1% solution in 0.1 M NaCl. Pectin (high methoxy) or any good grade purified pectin may be used. The pectin must be added slowly to the NaCl solution, with stirring, to dissolve the pectin. NaH or 0.1 N, standardized. The more dilute NaH is used with low enzyme activities. PRCEDURE: Extraction of enzyme. Weigh out 10.0 grams of albedo and add to it 50 ml of cold 5% NaCl. Blend at top speed in a Waring blender for only 10 sec. Blending too long will give time for partial enzymatic hydrolysis of the orange pectin to pectic acid and the pectin will gel. Filter on a small Buchner funnel under low vacuum. You will not need to filter all the material to obtain the amount of enzyme needed for your experiments.

3 Repeat this extraction procedure with 0.1 M phosphate, ph 7.5, and 0.1 M phosphate, ph 7.5, containing 5% NaCl as extracting fluids. Activity determinations. I. Place 20 ml of 1% pectin solution containing 0.1 M NaCl into a 50 ml beaker. Place a magnetic stirring bar in the beaker, and with the beaker on a magnetic stirring motor lower the ph electrodes so that they are in the liquid but do not touch the stirring bar. Turn on the stirrer. Adjust the ph to about ph 6 by adding 0.1 M NaH dropwise. Add 0.1 to 2.0 ml of one of the enzyme solutions and immediately readjust the ph to At this time start the timer. Add NaH from the 50 ml burette to maintain the ph essentially constant. Take readings of the amount of NaH added at approximately one min intervals for 10 min. Be sure the ph reads exactly 7.50 when the reading is taken and record the exact time of the reading. You can overshoot the titration slightly, then record the time when the ph reaches 7.5. The amount of enzyme used should be sufficient to cause a reasonable rate of ester hydrolysis but not so much as to cause the ph to change faster than can be compensated for by NaH addition. Repeat the procedure for each of the enzyme preparations. Record the temperature of the reaction as determined in the reaction vessel. Definition of unit of activity and avtivity yield. ne unit of activity is the amount of enzyme that will hydrolyze one millimole of ester per min under the conditions used. Activity yield is the number of units extracted per gram of albedo. Activity determinations. II. A more convenient procedure to determine [H + ] produced by hydrolysis of the methyl ester is to use a ph indicator dye in an unbuffered solution. We will use bromthymol blue (also called bromothymol blue), which has a pk a of It is blue above ph 7.6, and yellow below ph 6. We will measure the decrease in the blue color by measuring the absorbance at 620 nm in the spectrophotometer, which means that we will have a continuous assay, unlike the titration described above. The system is calibrated by using a standard curve obtained from samples containing bromthymol blue and several concentrations of galacturonic acid. The method is summarized in the following table:

4 A: Enzymatic Analysis Kinetic Assays Tube A B C Compound All volumes are mls Water % Pectin ph 7.5/0.1M NACL % Bromthymol Blue ph A: enzyme prep: 1M NACL 0.10 NA NA B: enzyme prep: 100 mm phosphate ph 7.5/1M NACL NA 0.10 NA C: enzyme prep: 100 mm phosphate ph 7.5 NA NA 0.10 Note: Do not add enzyme prep until at spectrophotometer! Kinetic Mode: Program: Pectinesterase Wavelengh: 620 nm Cycle time=1sec Total time=100 sec Scale= Absorbance Referance Blank is Type I H 2 B. Standard Curve Photometric Assay TUBE Compound All volumes are mls Type I Water % Pectin ph 7.5/0.1 M NACL % Bromthymol Blue ph mm galacturonic acid Type I Water Photometric Mode: Program: Galacturonic wavelengh: 620 nm Referance Blank is Type I H 2 QUESTINS: 1. Describe some other variables that might be investigated in a more complete experiment on factors affecting efficiency of extraction of an enzyme. 2. Suppose the titration version of the experiment had been performed at ph 5.0. Show how the measured milliequivalents (millimoles) of NaH uptake relate to the milliequivalents of ester hydrolyzed.

5 3. Describe at least two other experimental procedures that might be used to follow pectinesterase activity. 4. Calculate the ionic strength of each of the extracting fluids.

6 EXPERIMENT 2 Effect of Extraction Conditions and Titrimetric Assay of Enzyme Activity Laboratory Report Name: Date Performed: A. Extraction 1. Record data on the exact amount of albedo, extracting fluid and blending conditions used. B. Assay of enzyme activity 1. Submit a graph in which millimoles of ester hydrolyzed (y axis) is plotted versus time for each of the reactions. Indicate the temperature of the reaction and the amount of extract used. 2. Determine initial velocities, units of activity and activity yield for each preparation and report the values in a table. Discuss the reasons for the different results obtained with the different extracting fluids. 3. Submit answers to the questions (previous page).

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