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 organism’s 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 enzyme’s 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 protein’s 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 inspector’s 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 couldn’t 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 enzyme’s activity.
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 http://www. uni-leipzig. de/uspdu/docs/Protein%20guide_Storage_Working. pdf 6