Investigation on Effects of DOPA pH on Enzyme Activity Essays

Investigation on Effects of DOPA pH on Enzyme Activity Essays Investigation on Effects of DOPA pH on Enzyme Activity Paper Investigation on Effects of DOPA pH on Enzyme Activity Paper How do the different pH buffers affect activity of potato enzyme/extract? Introduction: Proteins are polymers that are made up of smaller units/monomers called amino acids. There are 20 different types of amino acids, thus make up many different combinations in types, numbers of amino acids as well as their orders an explanantion for why there are so many proteins. Every protein, due to various reactions of amino acids to each other, have its own three dimensional structures and therefore, function (Reece JB and others 2011). Proteins are fundamental substance that perform various range of metabolism in the organisms body such as fghting pathogens, sending signals, catalyzes reactions, storing substance, or acting as building blocks of body parts (Reece JB and other 2011). Proteins are grouped into four structures includin primary, secondary, tertiary, and quarternary. Primary structure of protein is composed of a single chain of amino acid without any foldings (The Medical Biochemistry Page, 2013). However, most proteins are active under secondary, tertiary, or quarternary structures. These strucures involve foldings due to he attachment between amino acids including hydrogen bonds, disulfide bridges, and Van de Waals force (The Medical Biochemistry Page, 2013). Proteins could be denatured due to the effects of temperature or PH. A decrease in pH level causes the environment surrounding to have a more positive charge while an increase in pH level causes the surroundings to have a more negative charge. (Aune, Salahuddin, Zalengo and Tanford 1967). Enzyme is a type of proteins that, by decreasing the activation energy needed for a chemical reaction, can therefore gives a start to that chemical reaction. Either atabolic or anabolic reactions need an enzyme to occur (Reece JB and others 2011). Every enzyme has an active site (Reece JB and others 2011). Enzymes metabolism occurs as enzyme binds to a substrate, which is specific to its enzymes structure and function, to form an enzyme2 subtrate complex (The Medical Biochemistry Page, 2013). As an enzyme binds to its substrate, the enzyme slight changes shape. (Reece JB and others 2011) Enzymes are denatured due to changes in temperature and pH as other types of proteins are. A slight change in pH away from the proteins optimal pH level can cause huge loss to he activitiy of an enzyme. In a strong basic or acidic pH solution, enzymes are denatured quickly and lose their functions. For most proteins, the optimal pH is around 7. 2 and 7. 4 (University Leipzeig, 2013). In this experiment, the enzyme activity (which is included in the potato extract) is measured in DOPA of pH 6. 8 and of pH 5. 0. If enzyme activity is measured in environment of DOPA pH 6. 8 and DOPA pH 5. 0, enzyme activity will be higher in DOPA environment that the enzymes are introduced in are shifted away from its optimal pH, causing denaturation and lowering enzyme activity, resulting in lower enzyme ctivity comaring to DOPA pH 6. . Materials and Methods: Gloves and goggles were always on over the course of this lab. Potato extract and four 15mL conical tubes were obtained. One was labeled phosphate buffer and 5mL of 0. 1M phosphate buffer was added in. Another one was labeled dH20 and add 5mL of distilled water. Another conical tube was labeled DOPA 5. 0 and add 3mL of DOPA pH 5. 0. The last one was labeled DOPA 6. 8 and add 3mL of pH 6. 8. Everything was kept on ice until needed. Spectrometer was calibrated followed inspectors instruction. Then 4 cuvettes were obtained. For the first cuvette, 2mL of DOPA 6. and 100pL of enzyme were added (enzymes should always be put last into the cuvettes). This cuvette was then placed in the spectrometer and spectrometer was used to measure enzyme activity. When absorbance data reached 200s in spectrometer, this cuvette was removed. The second cuvette had 2mL of 3 distilled water and 100pL of enzyme added in it and was placed into the spectrometer to measure enzyme activity until it reaches 200s. 2mL of distilled water and 100pL of DOPA 6. 8 was added into the third cuvette. It was then placed in the pectrometer to get measures for enzyme activity. th cuvette had 2mL of DOPA 5. 0 and 100pL of enzyme added in it and was placed into spectrometer to get measurements as for the first three cuvettes. When the experiment is finished, the waste was disposed properly. Results: Through the course of this experiment, the effects of different pH levels on enzyme activity. was examined. It was found that enzyme activity under DOPA of pH 6. 8 was higher than enzyme activity in DOPA of pH 5. 0. The percent change of Dopachrome formed for 50s of the experiment was calculated along with the overall percentage hange of Dopachrome formed (Figure 1). These calculated results were greater for enzyme activity in pH 6. 8 than Percent change of Dopachrome formed (%) that in pH 5. 0. 73. 7 80. 0 70. 0 60. 0 50. 0 40. 0 19. 7 20. 9 20. 0 5. 9 10. 0 0. 0 PH 6. 8 PH 5. 0 PH of DOPA 50S -100S 100S 1 50S -200S 50s-200s (Total change) The Effects of Different pH on Enzyme Activity Figure 1: The Effects of Different pH on Enzyme Activity 4 Discussion: The purpose of this experiment was to examine the effect of different pH of DOPA on activity of enzymes. Percentage change of Dopachrome formed was calculated. The hypothesis for this experiment was stated that the enzyme activity would be higher in DOPA pH 6. 8 than that in DOPA pH 5. 0. This hypothesis was supported. Percent enzyme in DOPA pH 5. 0, meaning that the enzymes in pH 6. 8 work better than those in pH 5. 0. This likely happens due to the effect of enzyme denaturation. Since most enzymes have optimal pH of 7. 2 to 7. 4 (University Leipzeig, 2013), enzymes are denatured in pH 5. 0 because the environment pH was away from its optimal pH for activity. A decrease in pH level causes a more postive charge to the environment Aune, Salahuddin, Zalengo and Tanford 1967), causing the solution to be too acidic for this enzyme that causes the enzyme to lose its shape, leading to inability to function (Reece JB and others 2011) Several random sources of errors and inconsistences could have involved throughout the experiment including inappropriate storage conditions for enzymes and DOPA, errors in conditioning pH of DOPA, uncertainty of solution conditions such as temperature or light, or unreactive enzyme. Systematic error that could have existed was bits of solution remains in transfer pipet and couldnt be fully tested. Future experiments could be conducted to investigate closely on changing the type of enzyme using or the amount of enzyme added or DOPA added could also be changed. Changing pH level to pH 8, pH 10 or changing temperature could also be extensions for future experiment. However, for any experiment that would be conducted, there is a simple rule which follows: moving away from optimal condition for an enzyme decreases that enzymes activity. Literary Cited Aune KI, Salahuddin AH, Zalengo MA, Tanford CH. 1967. Evidence for Residual Structure in Acid- and Heat- denatured Proteins. J. Biol. Chem. 242 (10): 4486 4489. Reece JB, Urry LA, Cain ML, Wasserman SA, Minorsky PV, Jackson RB. 2011. Campbell Biology, 9th edition. San Francisco: Pearson Education, 1263p. The Medical Biochemistry Page [Internet]. Updated July 21, 2013. Michael W King. [Updated July 21, 2013; cited October 28, 2013]. http://themedicalbiochemistrypage. org/protein-structure. php Universitat Leipzeig [Internet]. Updated 2013. Dr. Thole Zuchner. [Updated 2013; Cited October 22, 2013]. Avaiable from uni-leipzig. de/uspdu/docs/Protein%20guide_Storage_Working. pdf 6

Battle of Valmy in the French Revolutionary Wars

Battle of Valmy in the French Revolutionary Wars The Battle of Valmy was fought September 20, 1792, during the War of the First Coalition (1792-1797). Armies and Commanders French General Charles Franà §ois DumouriezGeneral Franà §ois Christophe Kellermann47,000 men Allies Karl Wilhelm Ferdinand, Duke of Brunswick35,000 men Background As revolutionary fervor wracked Paris in 1792, the Assembly moved towards conflict with Austria. Declaring war on April 20, French revolutionary forces advanced into the Austrian Netherlands (Belgium). Through May and June these efforts were easily repulsed by the Austrians, with the French troops panicking and fleeing in the face of even minor opposition. While the French floundered, an anti-revolutionary alliance came together consisting of forces from Prussia and Austria, as well as French à ©migrà ©s. Gathering at Coblenz, this force was led by Karl Wilhelm Ferdinand, Duke of Brunswick. Considered one of the best generals of the day, Brunswick was accompanied by the King of Prussia, Frederick William II. Advancing slowly, Brunswick was supported to the north by an Austrian force led by the Count von Clerfayt and to the south by Prussian troops under Fà ¼rst zu Hohenlohe-Kirchberg. Crossing the frontier, he captured Longwy on August 23 before advancing to take Verdun on September 2. With these victories, the road to Paris was effectively open. Due to revolutionary upheaval, the organization and command of the French forces in the area were in flux for most of the month. This period of transition finally ended with the appointment of General Charles Dumouriez to lead the Armà ©e du Nord on August 18 and the selection of General Franà §ois Kellermann to command the Armà ©e du Centre on August 27. With the high command settled, Paris directed Dumouriez to halt Brunswicks advance. Though Brunswick had broken through the fortifications of the French frontier, he was still faced with passing through the broken hills and forests of the Argonne. Assessing the situation, Dumouriez elected to use this favorable terrain to block the enemy. Defending the Argonne Understanding that the enemy was moving slowly, Dumouriez raced south to block the five passes through the Argonne. General Arthur Dillon was ordered to secure the two southern passes at Lachalade and les Islettes. Meanwhile, Dumouriez and his main force marched to occupy Grandprà © and Croix-aux-Bois. A smaller French force moved in from the west to hold the northern pass at le Chesne. Pushing west from Verdun, Brunswick was surprised to find fortified French troops at les Islettes on September 5. Unwilling to conduct a frontal assault, he directed Hohenlohe to pressure the pass while he took the army to Grandprà ©. Meanwhile, Clerfayt, who had advanced from Stenay, found only light French resistance at Croix-aux Bois. Driving off the enemy, the Austrians secured the area and defeated a French counterattack on September 14. The loss of the pass forced Dumouriez to abandon Grandprà ©. Rather than retreat west, he elected to hold the southern two passes and assumed a new position to the south. By doing so, he kept the enemys forces divided and remained a threat should Brunswick attempt a dash on Paris. As Brunswick was forced to pause for supplies, Dumouriez had time to establish a new position near Sainte-Menehould. The Battle of Valmy With Brunswick advancing through Grandprà © and descending on this new position from the north and west, Dumouriez rallied all of his available forces to Sainte-Menehould. On September 19, he was reinforced by additional troops from his army as well as by the arrival of Kellermann with men from the Army du Centre. That night, Kellermann decided to shift his position east the next morning. The terrain in the area was open and possessed three areas of raised ground. The first was located near the road intersection at la Lune while the next was to the northwest. Topped by a windmill, this ridge was situated near the village of Valmy and flanked by another set of heights to the north known as Mont Yvron. As Kellermanns men began their movement early on September 20, Prussian columns were sighted to the west. Quickly setting up a battery at la Lune, French troops attempted to hold the heights but were driven back. This action did buy Kellermann sufficient time to deploy his main body on the ridge near the windmill. Here they were aided by Brigadier General Henri Stengels men from Dumouriezs army who shifted north to hold Mont Yvron. Despite the presence of his army, Dumouriez could offer little direct support to Kellermann as his compatriot had deployed across his front rather than on his flank. The situation was further complicated by the presence of a marsh between the two forces. Unable to play a direct role in the fighting, Dumouriez detached units to support Kellermanns flanks as well as to raid into the Allied rear. The morning fog plagued operations but, by midday, it had cleared allowing the two sides to see the opposing lines with the Prussians on the la Lune ridge and the French around the windmill and Mont Yvron. Believing that the French would flee as they had in other recent actions, the Allies began an artillery bombardment in preparation for an assault. This was met by return fire from the French guns. The elite arm of the French army, the artillery, had retained a higher percentage of its pre-Revolution officer corps. Peaking around 1 PM, the artillery duel inflicted little damage due to the long distance (approx. 2,600 yards) between the lines. Despite this, it had a strong impact on Brunswick who saw that the French were not going to break easily and that any advance across the open field between the ridges would suffer heavy losses. Though not in a position to absorb heavy losses, Brunswick still ordered three assault columns formed to test the French resolve. Directing his men forward, he halted the assault when it had moved around 200 paces after seeing that the French were not going to retreat. Rallied by Kellermann they were chanting Vive la nation! Around 2 PM, another effort was made after artillery fire detonated three caissons in the French lines. As before, this advance was halted before it reached Kellermanns men. The battle remained a stalemate until around 4 PM when Brunswick called a council of war and declared, We do not fight here. Aftermath of Valmy Due to the nature of the fighting at Valmy, the casualties were relatively light with the Allied suffering 164 killed and wounded and the French around 300. Though criticized for not pressing the attack, Brunswick was not in a position to win a bloody victory and still be able to continue the campaign. Following the battle, Kellermann fell back to a more favorable position and the two sides began negotiations regarding political issues. These proved fruitless and the French forces began extending their lines around the Allies. Finally, on September 30, Brunswick had little choice but to begin retreating towards the border. Though the casualties were light, Valmy rates as one of the most important battles in history due to the context in which it was fought. The French victory effectively preserved the Revolution and prevented outside powers from either crushing it or forcing it to even greater extremes. The next day, the French monarchy was abolished and on September 22 the First French Republic declared. Sources: History of War: Battle of ValmyBattle of Valmy