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Name: ________________________________________ Date: __________________________ Worksheet for Enzyme Kinetics Lab (Part I)
- Complete the table (1 point) Note: All volumes are in mL Buffer Substrate 0.5 0. 25 0. 1 0. 05 Enzyme Total Volume 1.5 1.5 1.5 1.
- Complete the following table using the data obtained from the experiment described in Section II. (4 points) Time (sec) Abs (^) 475 nm Substrate 5 mM Abs (^) 475 nm Substrate 2.5mM Abs (^) 475 nm Substrate 1 mM Abs (^) 475 nm Substrate 0.5 mM 30 60 90 120 150 180
- Complete the following table using the data obtained previously. (4 points) Time (sec) Abs (^) 475 nm Substrate 5 mM Abs (^) 475 nm Substrate 2 .5mM Abs (^) 475 nm Substrate 1 mM Abs (^) 475 nm Substrate 0.5 mM 90 - 30 120 - 60 150 - 90 Add buffer then substrate and mix Put it in spectrophotometer Blank then add the enzyme and add timer
Sophia Spalding
O 0. 3 0. 6 0. 9
1 >. 2 mL · ~^3 mL
- 8mL 0. 8m> 0.^ 8mL^0.^ 8 mc 2 L 2MCe^ - 2Mc-
30mM
(30mM) (^
18
B.
G
~
~ 5 (2ML)(v)
- (^0). 01 S (^0). (^008 ).
0190.^022
017 0.^026 0. 0380. 044
0.^009 0. 0690. 080 0. 096 O.^027 0.^091
- 102 0. (^124)
- 0.^019 O^.^111. 126-0. 147
210-150 210 0.^069
- 132 0. 146 0. 179
I 0. 201
S 3
. 5
9 4.^5
L-0.^ 006- (-^0.^ 016)^0.^012 0.^038 0.^040 . 048
.^0 - 0. 027 0 . 043 0. 042 0. 052
033 0.^0430.^043 0.^034 (-0.^ 006)
- 072-0. 027
0.^ 091-
I I^ I
0. 049 00 a^0.^054
A= ExCX(
Average Δ Abs (^) ( nm)/min
- Determine the Δ Abs (475 nm)/min for the 0.25 mM, 0.5 mM, 1 mM and 0.2 mM substrate reactions by using the data recorded in number 3 above. ( 4 points) a. Δ Abs (^) (475 nm)/min (5 mM Substrate) = ____________________________________ b Δ Abs (475 nm)/min (2.5 mM Substrate) = _____________________________________ c. Δ Abs (^) (475 nm)/min (1.0 mM Substrate) = _____________________________________ d. Δ Abs (^) (475 nm)/min (0.5 mM Substrate) = _____________________________________
- Using the Lambert-Beer equation, calculate the molar concentration of dopachrome represented by each Δ Abs (^) (475 nm)/min for each of the 4 concentration of substrate used. The molar extinction coefficient of dopachrome is 3,600 M-^1 cm-^1. Show your calculations. ( 4 points) a. [dopachrome] for 5 mM substrate rxn = ___________________________________ b. [dopachrome] for 2. 5 mM substrate rxn = ___________________________________ c. [dopachrome] for 1.0 mM substrate rxn = ___________________________________ d. [dopachrome] for 0. 5 mM substrate rxn = ___________________________________ 18 mM 13.5 mM 9mM 4.5 mM -
- 04 0.^04 0.^05 0. 051 0.^033 0.^0430 . 0430 . 052 S 0.^033 S/min B .^5
AbS/min
- (^0).^043 /Min 4.^5 ~ 0.^043 /Min (^1).^2
- 167x M/min
0.^9
- 6
- 194 105M/min 0.^3 -^1.^44 10 M/min A (^). 0.^033 T = 9.
- 167x10 (^) /min (3600 (m/m) (^) (1) B. 0.^043 =
- (^) 194x10M/min (3600cm/m) (1) 0.^043 C.. ~men)(1)
9410M/min
- Draw the graph using the data from the above table. Title and label the graph properly and write proper units. (8points) TITLE: N Lineweaver (^) Bork Plot to (^) Find (^) Umax and Kim
For
Tyrosinase 30 I & 25 20 15
·^20 . (^2) ,^8. 45)
- (^) (0.^1 , 7. (^) 89) e -interO
-o S ·
I (^) I
- 3 &
- 2 I
- (^1) -inter O I^2
[S]MM =^
- Determine the Km and Vmax values of Tyrosinase from the graph. (4 points) X-intercept: ____________ Y-intercept: ____________ Km = __________________ Vmax = ________________
ImM 0. 167 mol/min
Xint
= /m (^) Jint
= /Umax
- 1 mM = 1/ km = (^) ImM (UMO/min) = max Vmax=C (^) (amol/min) O
ImM 0. 167 mol/mid
