Tyrosine lab.edited

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Tyrosine lab Tyrosinase is a compound that contains Oxidoreductase in a copper mixture. Tyrosinase catalyzes catechols aerobic oxidation and monophenols ortho hydroxylation. To convert L-dopa to the Dopaquinone, a catalyze in the form of an enzyme must be present. In this week one lab, enzyme activity will be assayed. It will be done through 3,4-dihydroxyphenylalanine oxidation and observing the color change to the dopachrome, which is red-colored. The lab will be performed in three parts, namely part one, which will entail determining the concentration of Tyrosinase, part two will be on the optimization of Tyrosinase, and L and D-dopa kinetic constant will be determined in part three. Hypothesis The hypothesis for this experiment is that L or D dopa is naturally occurring due to that one of the substrates, L- dopa, a naturally occurring substrate, will have kinetic properties superior to the D-dopa, which is the unnatural substrate. Materials The following materials will be needed for the success of the experiment, 0.05 M of sodium phosphate buffer at pH 7.0. Mushroom Tyrosinase, both L-dopa and D -dopa in 1.5mg/ml in the buffer of sodium phosphate, Cuvettes, UV-Vis spectrometer that contains a recorder, glass, and quartz. Part one: determination of the concentration of Tyrosinase Problem In this lab, the concentration of Tyrosinase was to be determined via running an A 280 assay, and the E 1% obtained was used to calculate the concentration of Tyrosinase. Variables In this experiment, the independent variable was the 50 mm of potassium phosphate buffer at the pH of 7.0, while the dependent variable was the [Tyrosinase], and the control was the blank employed in zeroing the spectrophotometer Procedure:  The spectrophotometer was set to 280nm.  50 mm of Potassium phosphate of pH 7.0 buffer was employed to blank spec.  The spectrophotometer was run after the insertion of the sample into the cuvette. Qualitative result No observable color change since this was an A280 assay which is mainly concentrated in determining or offering Absorbance and E 1% Calculation

E 1% ACl, where c= concentration, while l = pathlength C =? L = 1cm E 1% = 24.9 m-1cm-1 A = 0.2 C= E 1% /(A*l) C= 24.9m-1cm-1/ (0.2*1cm) C = 124.5 M C = 1.245*10^8 uM Part two: determination of the optimal concentration of Tyrosinase to be employedAssay Problem In this lab, the optimal concentration was determined to offer the right concentration of enzyme to be employed Assay. Understanding the amount of enzyme to be employed is crucial since when the amount is high, a fast reaction is noticed, and it isn't easy to measure it with more ease. Furthermore, when the enzyme is low or negligible, a significantly slower reaction is observed. Variables In this lab, the reaction rate is the dependent variable which depends on the independent variable, which is the tyrosinase amount used Assayssay. Before the reaction rate was measured and before the Tyrosinase was added, the rate of hydrolysis represented the control rate. Throughout the experiment, the value of L- Dopa, and D- Dopa was held constant. Procedure:  A Cuvette was prepared according to the table below, and the Tyrosinase was left out. ( it is not added as it will commerce the reaction, hence assed last)  The spectrophotometer was set to 475 nm.  Kinetics programming was prepared, and the reaction time was set to two minutes, and absorbance measured after every 0.1 seconds.  Hydrolysis rate was then measured ( it is crucial to perform this as it informs one that he/she does not measure buffer substrate interaction).  Tyrosinase was added to the cuvette left in the spectrophotometer. Reagent Assay 1 Assay 2 Assay 3 Assay 3 Assay 4 Phosphate buffer 0.48mL 0.47mL 0.43mL 0.4mL 0.37mL L or D-Dopa 0.5mL 0.5mL 0.5mL 0.5mL 0.5mL Tyrosinase 0.02mL 0.03mL 0.07mL 0.1mL 0.13 mL

Qualitative result Calculate Measure Assay 1 Assay 2 Assay 3 Assay 4 Assay 5 Rate ΔA/min 0.05 0.05 0.1 0.125 0.2 Assay 3 gave 0.1 ΔA/min Volume = chosen is 0.7 mL as assay 3 gave a great rate. Part three; L and D – dopa kinetic constant determination Statement of the problem In this lab, the kinetic constants (Km and Vmax) were to be determined by holding the tyrosine constant while varying the volume of both L and D- dopa and using the value obtained to determine a superior substrate that is more compatible with Tyrosinase. Variables In this experiment, both L and D – dopa volume were the independent variables as they changed. At the same time, the kinetic constants (Km and Vmax) were the dependent variables, while the control was the volume of Tyrosinase which was held constant. Procedure  A cuvette was prepared according to the table below, and the Tyrosinase was left out. Since its addition at this point may lead to the initiation of the reaction.  At 475 nm, the spectrophotometer was set.  The programming kinetics was then prepared, and the time for reaction set to two minutes, and at every 0.1 seconds, the absorbance was measured.  The hydrolysis rate was then measured. Its prominent role in helping in ensuring that one does not measure the substrate interaction buffer.  Tyrosinase was then added to the cuvette left in the spectrophotometer. Reagent Assay 1 Assay 2 Assay 3 Assay 4 Assay 5 Phosphate buffer (mL) L or D dopa (mL) 0.1 0.2 0.4 0.8 1 Tyrosinase (mL) 0.20 mL 0.20 mL 0.20 mL 0.20 mL 0.20 mL

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Substrate used Assay 1 (ΔA/min) Assay 2 (ΔA/min) Assay 3 (ΔA/min) Assay 4 (ΔA/min) Assay 5 (ΔA/min) L – Dopa 0.2 0.4 0.5 0.9 1.2 D – Dopa 0.0752194 0.1067616 0.1337494 0.1535836 0.1581861

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