The environmental factors that effect turnip peroxidase

Four environmental factors of enzymes were tested in lab. The changing of pH, substrate concentrations, temperature, and an inhibitor (NaCl) and the effects it hade on the enzyme turnip peroxidase. Enzymes are biological catalysts which increase reaction rates by lowering the activation energies of substrates. A substrate is a reactant that interacts with the enzyme. The enzyme and substrate can be viewed as the recently discovered “induced fit model”, which suggests enzymes are flexible and dynamic things that change their shape so all these substrates (reactants) can become catalyzed when the activation energy is lowered and the reactions happen a lot faster. Sometimes in cells though it may not need a particular substrate (reactant) so an inhibitor comes into play. Inhibitors are basically regulators that inhibit (disallow) the process of catalysis to take place within a particular substrate.

There are two types of inhibitors that occur in such reactions. Competitive inhibiters are remarkably similar to the substrate, so much that it can match interchangeably with the substrate, thus leading to a halt in the production of the intended product. Another variety of inhibitor is the allosteric inhibitors. Allosteric inhibitors change the shape of the enzyme by binding to a different site other the active site, which is the usual site for catalysis. Usually the allosteric inhibitors make contact with the side of the enzyme opposite the active site. In some occasions however an allosteric activator is introduced and functions by connecting to the enzyme in a way that it allows for easy access for a substrate to the active site. Allosteric activators are the opposite of allosteric inhibitors.

These regulators (both inhibitive and active in function) help keep the cell in homeostasis by not allowing too much or too little of a needed or not needed product to be produced.. Enzyme activity is also regulated by cofactors which are either metal ions (e.g. Zn2+, Mg2+) or small organic molecules (e.g. coenzyme A, NAD, NADP, and many vitamins in our diet like iron). Cofactors main is to work together with enzymes to enhance the catalytic activity and by stabilizing the transition state. The transition state is the second of three steps of catylic action of an enzyme. The transition state and termination state are important in regards to cofactors because the cofactors allow interactions between the enzyme and substrate lowering the activation energy required.

The goal of the following paper is to inform you the reader on how environmental conditions such as temperature, pH, salt (an electrolyte), and how substrate concentration itself effects the rate of reaction and properties of turnip peroxidase. If optimal rates of reaction can be determined for each parameter, perhaps a farmer can grow his turnips more effective and efficiently rather than if he didn’t know that surrounding temperature, pH, cofactors, and inhibitors can effect the outcome of how much crop yielded. Turnip peroxidase may be insignificant to us because it’s just a “part of a turnip” but we must realize that the following experiments can and do affect us just as much if not more than these lifeless turnips. Inside our bodies right now things are being catalyzed and ph, temperature, the amount of a particular substrate, and the presence of electrolytes (or absence of) can effect how we function from day to day.

For instance if you over eat if you over eat, your body can only release so many digestive enzymes to break down this food (which can be seen as the substrate) so rate of reaction doesn’t increase because their isn’t enough digestive enzymes to digest this ridiculous amount of food. Thus an increase a digestive enzymes can catalyze more food leading to higher rate of reaction and less of a tummy ache. So the wise choice would be to eat small portions of food (substrate) so the digestive enzymes can breakdown evenly the food and produce energy for the body rather than wasting energy to breakdown the large amount of food. Thus the common hypothesis is that an increase in substrate concentration will effect peroxidase activity. The null hypothesis is that the concentration of substrate will no effect on activity.

PH is derived from the French puissance “d’ hydrogene, or power of hydrogen”.(biological science). The ph scale is a scale that indicates whether a substance is acidic or basic. The scale ranges from pH zero (acidic) to pH 14(basic). An example of an acidic product is lemon juice and milk of magnesia would be a base. Products are considered acidic because they give up protons during chemical reactions while basic receive protons. (Biological science). Neutral 7 is the point of reference. An example of a neutral solution would be rain water. Knowing the natural pH of turnip peroxidase would be interesting to see whether it’s acidic or basic and to see what affect the two factors (acidic or basic) have on rate of reaction. The following information led the group to believe that the effect of ph activity will increase or decrease the natural pH of turnip peroxidase and effect activity. The null hypothesis is that pH will have no effect on peroxidase activity.

The effect of temperature on an enzyme depends on the plant. Some plants can exist and grow hot climate like the desert and others can grow in cold climates respectively. Plants are said to be “incapable of adjusting to internal temperature so it must be flexible to allow growth to occur when the temperature outside is optimal”. To figure out optimal temperature for turnip peroxidase could lead to better methods of choosing climates for the harvest of these turnips. An important term to know when it comes to temperature and the protein property of the peroxidase is the term denatured. Denaturation occurs when an enzyme (with protein properties) is misfolded and rendered inactive. High temperatures usually lead to denaturations because the increase in temperature essentially “cooks” the protein in the peroxidase. Thus the increase in temperature will either increase peroxidase activity of hinder it. The null hypothesis would that temperature will not effect activity.

Salt is an electrolyte. Meaning the salt molecules (NaCl) will dissolve completely in water and individual and opposite charges will be a byproduct. Thus the amino acid property of peroxidase can be affected by the Na+ and Cl- ions. Denaturation can occur is exposed to a high concentration of salt. Knowing the natural salt content of the turnip peroxidase and adding more or less quantities can tell us when the peroxidase becomes highly active and eventually denatured. The concentration of salt effects peroxidase activity. The null hypothesis is that it doesn’t.

The equipment needed to test the parameters of the enzyme activity include a spectrophotometer set at 500nm, cuvettes, pipettes with pipettes , pipette tips, parafilm squares, blender, Kim wipes. To get the spectrophotometer ready to read our reaction, we need to first set the wavelength to 500nm.then set front left knob to 0%(no light).insert the reference blank (with water) then set to 100%(no light block).then finally you can insert the sample tube into the chamber and red absorbance from the lower scale and the front of the spectrophotometer. (Note: be sure to use Kim wipes on cuvettes to prevent fingerprints that could throw off data).

Regents or chemicals needed include hydrogen peroxide (1 percent H202 from 3% stock solution), turnip peroxidase, guiaicol, ph buffers, and NaCl. To obtain turnip peroxidase the lab instructor blended 5g of turnip into 500ml of water then filtered through a p2 filter. To obtain the correct amount of substrate (h202) and Nacl, the c1v1=c2v2 equation was necessary. C equals concentration and v equals volume in the equation.

To test the effect the effect of ph on reaction rate we prepared 4 sets of reaction mixtures that contained guiaicol, h2o2, peroxidase. And 4 cuvettes containing PHS 2, 5, 7, and 10. (Note: mix peroxidase last because the reaction happens immediately). The cuvette with h2o2 and guiaicol are mixed and added to a blank. The ph 2 is then poured in the blank as well. then finally the peroxidase is added. With parafilm covered over the cuvette the mix is inverted a couple of time to mix. The solution will turn brown due to the loss of hydrogens of guiaicol. Immediately after mixture insert the cuveete into already set spectrophometer.once set begin recording absorbances (start at zero) every 15 seconds until three minutes have elapsed.after reading values for three minutes discard the mixture appropriately in the waste beaker and clean cuvettes.now you can test PHS 5,7,10.

To test the effect of substrate concentration on peroxidase activity different concentrations of substrate, peroxidase guiaicol and h2o2 (the substrate) are needed. The procedures of adding chemicals (different amounts for h2o2) and peroxidase apply. Begin recording absorbances (start at zero) every 15 seconds until three minutes have elapsed. After reading values for three minutes discard the mixture appropriately in the waste beaker and clean cuvettes.now you can test PHS 5,7,10.

To test the effect of temperature the same amounts of peroxidase, guiaicol, h2o2, are used instead of using different pHs we used just ph 7.to regulate temperatures we used water/ice baths a 0,23,47,and 71 degrees Celsius. All solutions but the indicator (guiaicol) need to be at the temperatures. When ready the tubes sitting in the baths can be mixed (1 cuvette with h2o2 and ph 7 buffer, guiaicol, and finally peroxidase) and set in the spectrophotometer. Begin recording absorbances (start at zero) every 15 seconds until three minutes have elapsed. After reading values for three minutes discard the mixture appropriately in the waste beaker and clean cuvettes

To test the effects of Nacl activity simply get 1ml solutions of 5%,4%,%3 and 2 percent concentrations of 5% stock solution using c1v1=c2v2. Follow same instructions as done for the temperature experiment and remember to mix the enzyme last so you get accurate results. Begin recording absorbances (start at zero) every 15 seconds until three minutes have elapsed. After reading values for three minutes discard the mixture appropriately in the waste beaker and clean cuvettes.

Figure 1. Graph of absorbances of tubes in which salt was added to the assays that already contained a pH buffer (2.1 ml), h2o2 (.2 ml), turnip peroxidase(1 ml) and guaiacol(.2ml).

Discussion

Ph2 had the lowest absorbance and had ph 10 had the highest. Thus peroxidase is more prone to being a base. In the varying concentration levels of hydrogen peroxide on peroxidase activity showed 2% to be the least active while 1% had the highest rate of reaction.This shows that an increase of the toxic h2on results in a decrease of inactivity due to denaturation. In the affect of temperature on enzyme activity as temperatue went up so did the reaction but at the expence of denaturing of the peroxidase.For the nacl effect on peroxidase activity showed increase for absorbance for all percentages and at 3% of .1ml of salt had the greatest absorbance.this shows that an increase or decrease of 3 hundreths of a ml of nacl solution is going to denature the turnip peroxidase. The hypothesis of each parameter was approved and the null was nulled. Nacl concentration indeed have an effect on the peroxidase activity based on the grapghs.

The temperature did effect whether protein became denatured or not. The higher the ph the more activity occurred in peroxidase and showed ph7 to be optimal for turnip peroxidase. The increase of substrate effected the peroxidase activity by denaturing the toxic h2o2. some problems were encountered in all the perameters of experimentation. The time between the the tranfsfer of the assays to the spectrophotometeraffects the absorbencies. This could be easily solved by having the assay right by the spectrophotometer to make sure the sample gets in before the enzyme reacts. For each perameter there are future questions that arise from the results of the experiments.for instance what if the wave lengths were changed?or what would have happended if we used a peroxidase from a different plant? Does gravity have an effect on enzyme activity.

References

1.Weinheimer, T. White,D. 2003. Using peroxidase to demonstrate enzyme kinetics.The American Biology Teacher:pp116-121.

2.Helser,T 2006. Enzyme catalyzed reactions.

Employee.oneonta.edu/helsert/enzyme.html

3.Pitkin,R.B. 1990. Introductory biology manual.Shippensburg university,Shippenburg. PA.