Effects of Peroxidase Dilutions and Substrate Concentrations on Enzyme Reactions, Lab Reports of Biochemistry

Download Effects of Peroxidase Dilutions and Substrate Concentrations on Enzyme Reactions and more Lab Reports Biochemistry in PDF only on Docsity! 1 BCH3033L ENZYMES, Part I Purpose: This multi-week lab will demonstrate how to extract and assay the enzyme peroxidase from plants. You will use this knowledge to study the effect of enzyme and substrate concentration, pH, inhibitors and temperature on the rate of a peroxidase catalyzed reaction. Key Concepts and Terms: [Enzyme] [Substrate] Active site Catalyst E.C. 1.11.1.7 Inhibition – reversible/non-reversible inhibitors Lysosome Peroxisome Vmax Vo Background: Enzymes are usually proteins that act as catalysts in biochemical reactions. Catalysts cannot initiate reactions that would not happen in their absence, but can, and do, radically affect reaction rates with the result that the cell can carry out rapid and complex chemical activities at relatively low temperatures. Most enzymes are highly specific. They tend to accelerate only one or a group of related reactions. The result is that many different enzymes may be present in a cell and may act simultaneously without mutual interferences. Here we demonstrate the characteristics of enzyme catalyzed reactions by examining peroxidase (E.C. 1.11.1.7) from plants. What does the first “1” in EC 1.11.1.7 mean? Hydrogen peroxide (H2O2) is a common end product of oxidative metabolism and, being a strong oxidizing agent, would be toxic if allowed to accumulate. To prevent this, eukaryotic cells have enclosed the enzymes producing peroxides within a membrane-bound organelle, the peroxisome, which is similar in size and appearance to a lysosome. Peroxisomes also contain high concentrations of peroxidase – the enzyme that functions to reduce the peroxide to water, rendering it harmless. A variety of electron donors can be used, including aromatic amines, phenols, and enediols like ascorbic acid. A dye like o-dianisidine can be used as the electron donor (colorless) to easily detect peroxidase in vitro because its oxidized product is highly colored (Extinction coefficient is 11.3 mM-1cm-1). The rate of appearance of this colored pigment can be measured colorimetrically and is equivalent to the rate of reaction. H2O2+ Colorless Dye (reduced) peroxidase > H2O + Colored Dye (oxidized) When first measuring enzyme activity from a tissue (in this case turnip or horseradish root) one must first “range find” to determine the amount (extent) of enzyme activity in the tissue. So, right after grinding up the root to make a crude enzyme extract, we will dilute the extract (enzyme) and measure the rate of reaction. This will tell us what amount of enzyme to use. In these assays, the tubes will be made up with buffer and substrates (completely mixed). This tube is used to blank the spectrophotometer BEFORE adding enzyme dilution. Then the tube is removed, the enzyme dilution is added and immediately returned to the cuvette chamber of the spectrophotometer. OD readings are made immediately and then at 30 second intervals to get a rate 2 of reaction from a plot of the OD (ordinate or y-axis) against time (abscissa or x-axis). At low dilutions (highest amount of enzyme, the rate may go to fast to accurately measure, at high dilutions (lowest amount of enzyme) the rate may be too slow. Thus, the first range finding will allow each group to find a reasonable rate for future experiments: the amount of enzyme that will produce a change in OD of 0.4 to 0.7 in 5 minutes. Then using this amount each time, we can examine the effect of substrate concentration on this rate; that is do the classical Michaelis-Menten experiment. Materials and Reagents: Equipment Needed: • Spectronic 20 spectrophotometer set at 460 nm. • One box of spec 20 tubes per group. • P200 and P1000 pipets and tips. • Vortex mixers – one per group. • Blender. • Small Buchner funnel, Whatman #1 filter paper, single hole stopper. • 1 500 ml side arm flask. • Timers. • 13x100 glass test tubes & racks. • 1 500 ml Beaker for o-dianisidine waste Reagents Needed: • Fresh turnip or horseradish root. 40 grams needed per group. • 1 liter 0.10M phosphate buffer, pH 7.0 • 200 ml each of 0.10M phosphate buffer, pH 2.0, 4.0, 5.0, 6.0, 8.0 and 10.0. • 200 ml of 8.8mM H2O2 substrate one. • 50 ml 0.5% w/v o-Dianisidine dye (Sigma #D-3127) in methanol, substrate two. WARNING: O-Dianisidine is a proven carcinogen and toxic. Avoid contact with skin. Dispose of material as directed by TA. MW o-dianisidine (=3,3-dimethoxybenzidine) is 244.3 • Acid-alcohol bath. Procedures: I. Extraction of Horseradish Peroxidase 1. Peel, wash and cut ~40g fresh turnip or horseradish root (best to use) into ~1 inch cubes. 2. Homogenize with 100 ml water in a blender. 3. Filter with buchner funnel and Whatman filter paper by vacuum into a sidearm. 4. Keep filtrate cool, on ice during the experiment. II. Reaction Time Course for Enzyme Dilutions