The_Effects_of_Salt_on_Turnip_Peroxidase_Activity

Abstract The Effects of Salt on Turnip Peroxidase activity. Functional Biology, Lab section 1262, Texas State University, San Marcos, Texas 78666 The Purpose of the experiment was to find the optimal salt concentration in a solution before it begins to denature the protein. The effects of salt on peroxidase activity were studied in our experiment. Ten cuvettes were prepared with varying concentrations of salt (0%, 4%, 7%, 13%, 15%), we mixed 5 blank solutions, then five experimental cuvettes were mixed after the spectrophotometer had been cleared with the blank of that particular concentration. As salt concentration increased we noted that the reaction rate also increased up to 7%. After reacting the optimal level of 7% the solutions began to denature and the reaction rate decreased. We concluded that salt has a significant effect on enzyme activity. Introduction The purpose of this experiment was to find the optimal salt concentration in a solution before it begins to denature the protein. Enzymes are biological catalysts, Many of the reactions in catabolism are favorable (they have a negative DG). What this means is that these reactions will occur spontaneously even outside of a living organism. The problem is, they are way too slow to be of any use in a biological system. If cells did not have ways of speeding up catabolism, life would be nearly impossible. Enzymes accelerate almost all biological reactions. (Paustian, 2000) They are also highly specific in that they only bind to substrates to aid in reactions. Lower activation energy of reactions, so they go faster, also they act as a reactant that gets turned into a product. After a certain point (in our case after 7%) the enzymes will begin to break down, or denature, after being exposed to much salt for a period of time. Guaiacol was reduced, and peroxide was oxidized in our experiment. The benefits of this study were to learn the tolerance range for enzymes with salt present and salt intake before it hinders the level of functioning. As we increase the amount of salt in the solution the reaction rate will peak and then steadily decrease. Materials and Methods Dilutions of 15% salt solution, 0% (no salt), 4%, 7%, 13%, and 15% (stock solution), were mixed to compare the effects of salt on turnip peroxidase. The turnip peroxidase was created by taking a teaspoon of inner turnip root then mixing it with a buffer until there were no large pieces of turnip visible. The 0 % salt concentrations was the control because it showed the natural rate of reaction with no slat added. We mixed the solutions the same for every blank and experimental cuvette, each containing 0.3 ml of pH 7 buffer (tap water), 2.0 ml of peroxidase, a 1 ml dilution of 15% salt concentration, and 0.2 ml of 1% peroxide which was added last because it started the reaction and would break down if exposed to light. The difference between the blanks and the experimental cuvettes was the addition of 0.02ml of guiaiacol into the experimental tubes; the guiaiacol was added next to last. The spectrophotometer had to be blanked for each of the different solutions, so that we could view the different reaction rates. Once the spectrophotometer had been blanked, the experimental cuvettes were mixed, whipped clean, and immediately put into the spectrophotometer. Absorbance measurements were recorded as soon as the lid was shut, and, then every 15 seconds for two minutes. This process was repeated for all of the salt concentrations. Time allowed for only one trail of each solution. Results As the salt concentrations were increased the absorbance rates increased until the solution reached its highest absorbance level at 7%. The lowest absorbance level was recorded at 0%, because no salt was in that solution. Absorbance of Different Salt Solutions Over Time The reaction rate was determined from the slopes in the 1st graph. Once again 7% was the highest reaction rate, and 0% had the lowest reaction rate. The Reaction Rate of Varying Salt Solutions Discussion We found that as time passed and the concentrations changed that the absorbance rates increased, until it reached the peak of 7% concentration. The top tolerance range we recorded was at 7%. At that concentration the solution had reached the optimal salt concentration, however it might be somewhat higher since in our experiment we jumped from 7% to 13%. So it is possible that there might have been a higher absorption rate just not in our experiment. The amount of salt was the reason for the solution to denature. Yes the data supported our hypothesis, in that as we added salt we found that the solution peaked at 7% and then decreased at 13% then increased again at 15%. For future experiments one could test what really happened between 7% and 13%, test to if see if there was a higher reduction rate. A few things that my lab partners and I could have done, was to do further test or to do two trails on each experimental tube, due to time restraints we only went through each concentration once. Another thing we could have done would have been to be more efficient, it took us a while to understand what we needed to do for our experiment, and to pick the concentrations of salt that we wanted to use. References Abed Shalata and Moshe Tal. (17 Jan 2002). The effect of salt stress on lipid peroxidation and antioxidants in the leaf of the cultivated tomato and its wild salt‐retrieved tolerant relative Lycopersicon pennellii. Physiologia Plantarum. 104. 169 – 174. Retrieved: March 22, 2009 From: http://www3.interscience.wiley.com/journal/119112276/abstract'CRETRY=1&SRETRY=0 Frederick Albert and Anne J. Anderson. (Oct., 1987). American Society of Plant Biologists, 537-541. Available: http://www.jstor.org.libproxy.txstate.edu/stable/4270945'&Search=yes&term=effects&term=peroxidase&term=salt&list=hide&searchUri=%2Faction%2FdoBasicSearch%3FQuery%3Deffects%2Bof%2Bsalt%2Bon%2Bperoxidase%26wc%3Don%26dc%3DAll%2BDisciplines&item=4&ttl=2995&returnArticleService=showArticle Melina A. Talano, Elizabeth Agostini, María I. Medina , Silvia Milrad De Forchetti and Horacio A. Tigier. (May - Jun., 2003). Tomato (Lycopersicon esculentum cv. Pera) Hairy Root Cultures: Characterization and Changes in Peroxidase Activity under NaCl Treatment. Society for In Vitro Biology. 354-359. Available : http://www.jstor.org.libproxy.txstate.edu/stable/info/4293623'seq=1&Search=yes&term=effects&term=peroxidase&term=salt&list=hide&searchUri=%2Faction%2FdoAdvancedSearch%3Fq0%3Dthe%2Beffects%2Bof%2Bsalt%2Bon%2Bperoxidase%26f0%3Dall%26c0%3DAND%26q1%3D%26f1%3Dall%26c1%3DAND%26q2%3D%26f2%3Dall%26c2%3DAND%26q3%3D%26f3%3Dall%26wc%3Don%26Search%3DSearch%26sd%3D%26ed%3D%26la%3D%26jo%3D&item=1&ttl=2995&returnArticleService=showArticle&resultsServiceName=doAdvancedResultsFromArticle M. Muthukumarasamy, S. Dutta Gupta and R. Panneerselvam. (October 28, 2004). Enhancement of Peroxidase, Polyphenol Oxidase and Superoxide Dismutase Activities by Triadimefon in NaCl Stressed Raphanus Sativus L. Springer Netherlands. 43. 317-320. Retrieved: March 25, 2009 From: http://www.springerlink.com/content/u514lp8741247488/ Paustian, Timothy. Sep 21, 2000. Enzymes- The Biological catalysts. Available: http://lecturer.ukdw.ac.id/dhira/Metabolism/Enzymes.html