A Guaiacol Dye-Coupled Reaction Reports That Catalytic Activity of Peroxidase Isolated from Fresh Turnip (Brassica Rapa) Increases as Temperature Rises.

Enzymes are proteins which serve to reduce the activation energy required for biological reactions (Russell and others 2010). This allows biologically important chemical reactions to occur rapidly enough to allow cells to carry out their life processes (Russell and others 2010). Enzymes are made of one or more polypeptide strands, which individually or as an associated complex take on a three-dimensional shape. When properly associated, these shapes form the active site and other supporting structures that allow enzymes to be effective catalysts (Nelson and Cox 2005).
Temperature represents the average kinetic energy in an object or solution (Russell and others 2010). This energy causes rapid movement of dissolved particles, such as enzymes and substrate molecules, increasing the chances that they’ll contact each other in such a way as to allow a chemical reaction to occur (Nelson and Cox 2005). The kinetic energy may also influence the folding of the enzyme. If the weak and strong bonds involved in stabilizing the protein structure are disrupted, denaturation of the protein can occur, eliminating the enzyme’s effectiveness (Nelson and Cox 2005; Russell and others 2010).
This experiment will investigate the effects of temperature on the enzyme kinetics – that is, the rate of an enzyme’s catalysis – of peroxidase isolated from turnip. Plant peroxidases are involved in lignin formation, which is part of the cell wall (Cosio and Dunand 1985). Turnip roots contain peroxidases which are enzymes that can be easily extracted, and because peroxidases can liberate oxygen from hydrogen peroxide, their activity can easily be measured in the laboratory (Pitkin 1992). The rate of oxygen release is followed by measuring the rate of oxidation of guaiacol, which turns brown in the presence of oxygen and thus can be quantified in a spectrophotometer (Nickle 2009). We hypothesize that as we increase the temperature of reaction, kinetic energy will increase the frequency with which peroxidase engages hydrogen and the rate of guaiacol oxidation will increase. Because turnips grow in cool climates, often below 24oC (Pollock 2009), we expect that the optimal temperature for enzyme activity will be around room temperature or cooler, and temperatures in excess of this will cause denaturation of the enzyme and a concurrent loss of enzyme activity.
METHODS AND MATERIALS A store-purchased turnip was scrubbed and rinsed with tap water. A razor blade was used to cut a 0.5 g piece of tissue from the cortex. This was placed in a mortar along with 50 ml phosphate extraction buffer (0.1 M, pH 7) and a pinch of sand. The tissue was ground to a slurry and then filtered through cheesecloth to form the extract used for all experiments after standardization.
To ensure peroxidase was extracted from the turnip and that the reagents were suitable for the experiment, a positive control was performed. 2 ml of enzyme was added to a test tube containing 3 ml buffer, 2 ml H2O2, and 1 ml guaiacol dye. After quickly inverting twice to mix the fluids, the contents darkened.
Standardization was performed to correct for differences in extraction techniques and tissue enzyme content. Three volumes of enzyme (0.5, 1.0, and 2.0 ml) were tested. To ensure reactions did not begin prematurely, reaction components were placed into two separate test tubes. These were labelled “a” and “b” for each volume of extract, where “i” contained 0.5 ml (dilute), “ii” held 1.0 ml (medium), and “iii” had 2.0 ml (concentrated) extract each (Table 1). The contents of paired tubes were combined in the tube containing the enzyme at “time zero”. This tube was mixed by inverting twice before 1 ml was transferred to a cuvette which was placed into a Genova spectrophotometer so the rate of absorbance change at 500 nm could be calculated. The concentration which gave the largest constant absorbance change (as shown by plotting absorbance over time) was used for subsequent experiments. The slope of each line in the plot was measured to determine the rate of guaiacol oxidation. The sample containing 0.5 ml fulfilled this criterion (data not shown). For all trials, the “a” tubes contained 2.0 ml H2O2 and 1.0 ml guaiacol, and “b” tubes contained 4.5 ml buffer and 0.5 ml enzyme extract. These were placed into the appropriate equilibrated water bath (see below) for 5 minutes prior to mixing and measuring their absorbance changes.
For the temperature experiment, water baths were equilibrated at the desired temperatures of 4.5oC, 10oC, 22.5oC, 50oC and 80oC. To create the 4oC temperature, a beaker of water was placed in the refrigerator. Both “a” and “b” tubes were placed in racks in the appropriate water bath for 5 minutes prior to the time for them to be mixed together. Mixing was performed as described above, and the spectrophotometer was again used at 500 nm light. To determine if high temperature will alter results by degrading reagents (such as causing H2O2 to spontaneously release oxygen or make guaiacol oxidize independently of enzyme activity), we created a duplicate control tube (Table 1) and heated it to 80oC for 15 minutes. This negative control did not show an increase in absorption compared with the unheated control tube, so we concluded that the temperatures only affect molecule movements in the experiment. A similar test was done with the 4oC temperature and again no difference was measured. Three replicates were for each temperature. Rate of absorption change was established for each, and standard deviations between trials at each temperature were determined using Excel 2000 software.
RESULTS The positive control turned brown / beige constantly and continuously over about 1.5 minutes. This was quite apparent to the naked eye. Controls exposed to high or low temperature without enzymes present did not show a different absorption than the control that remained at room temperature (data not shown).
Reaction rate at lower temperatures was lowest at 4.5oC at 0.25 A500/min. This increased as temperature rose until a peak rate of 0.52 A500/min at room temperature (22.5oC) was noticed. At 50oC, the rate of oxidation declined to0.39 A500/min and a reaction rate of 0.05 A500/min was measured at 80oC (Figure 1).
DISCUSSION The results indicate that enzyme activity does indeed increase as the temperature of the reaction is raised. The optimal temperature must lie between 10 and 50oC, but most likely is near temperature, possibly slightly cooler as turnips naturally grow in temperate climates (Pollock 2009). Enzymes are typically structured to function in a particular environment; usually one in which it normally functions (Russell and others 2010).
The large standard deviation noticed for values collected at 10oC suggests that the true optimum may lie below room temperature. More measurements at this temperature could refine these values, giving a more precise average at this temperature. To find the optimal reaction temperature more accurately, a series of temperature intervals, perhaps 2oC apart and spanning 10oC to 50oC could be measured. It would be interesting to compare the precise optimal temperature for turnip peroxidase activity to the average temperature at which turnips naturally grow. A study that compares this to a peroxidase extracted from a tropical plant might also prove to be interesting. Investigating the reversibility of a weak thermal denaturation might also prove interesting.
Thermal energy probably affects weak bonds, such as the hydrophobic, hydrophilic, and ionic associations, to the largest extent (Russell and others 2010). Denaturation might be prevented by stabilization with covalent linkages within and between polypeptide strands (Anfinsen and Haber 1961). Enzymes that are particularly susceptible to thermal damage are often supported by chaperonins or other proteins which can repair the denatured enzymes (Morimoto and others 2009). We plan to next identify the effects of denaturation, and whether it can be reversed by subsequent cooling.
SUMMARY Plant peroxidases cause peroxides to break down and release oxygen. The rate of oxygen release can be calculated by observing the amount of oxidation that occurs with guaiacol insolution with peroxidase and its substrate. Enzyme activity was determined at 4oC, 10oC, 22oC, 50oC, and 80oC by measuring darkening of guaiacol. The highest amount of oxidation was recorded at 22oC. Enzyme activity was absent at 80oC, suggesting the enzyme denatured at this temperature. Enzyme activity correlates with the cool temperate conditions natural for this plant.