Part C: Change the amount of the substrate
First, the blank was prepared according to table 2 without the enzyme addition. The enzyme was added later after the blank was measured by the spectrophotometer.
Table 2: The amount of Sodium Phosphate Buffer pH 7.0, L-Dopa, and enzyme needed in each cuvette.
Cuvette 1 Cuvette 2 Cuvette 3 Cuvette 4 Cuvette 5
Sodium Phosphate Buffer PH 7.0 (mL) 2.40 2.20 1.80 1.60 1.10
L- Dopa (mL) 0.20 0.40 0.80 1.00 1.50
Enzyme (mL) 0.40 0.40 0.40 0.40 0.40
For example, to prepare the cuvette 1, 2.40 mL of buffer pH 7.0 was measured by the micropipette P-1000, and was added into cuvette labeled #1 for the second set of cuvette. Next, 0.20 mL of L-dopa was measured by the micropipette P-1000 and was added into
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Then, the cuvette that labeled #1 was wiped off with the KimWipe and placed in the single cuvette holder in position 1 in the sample compartment. The position 1 was making sure aligned with the light source. The sample compartment door was closed, and was pressed “auto zero” button on the keypad. Then, after 30 seconds, the absorbance that displayed on the screen was read again and it was 0. The absorbance was read at 0 seconds, at 30 seconds, at 60 seconds, at 90 seconds and at 120 seconds. All the absorbances were remained 0 for the blank. After 120 seconds, the blank was then removed, and the appropriate amount of enzyme Tyrosinase (0.40 mL) was measured and added into the blank (cuvette #1) using the micropipette P-1000 according to the table 2. The final volume in the cuvette was 3mL. The cuvette contained the enzyme sample was wiped off with a KimWipe and was placed into the sample compartment of the machine. The sample compartment door was closed. The absorbance that displayed on the screen was read and documented in table 5. Then, after 30 seconds, the absorbance was read again and documented in table 5. The absorbances were read at 0s, at 30s, at 60s, at 90s, and at 120 seconds. Cuvette 2, 3, 4, 5 were followed the same method as cuvette 1 but different amount of buffer, L-dopa according to table 2. All the absorbances were read and documented in table