Enzyme Kinetics Practical Report: BBSPS Proforma Analysis 2017-18

This practical assignment, based on the BBSPS proforma, focuses on determining the enzyme kinetic parameters Vmax and Km using a continuous enzyme assay involving alkaline phosphatase. The experiment involves analyzing the relationship between initial rates and substrate concentrations, resulting in a rectangular hyperbola. The assignment includes data analysis, conversion of absorbance measurements to concentrations, and understanding the role of enzymes and pH in product formation. The report presents data tables, calculations, and graphs, including a calibration curve and Lineweaver-Burk plot, to analyze enzyme activity and kinetics. Students are expected to understand the concepts of enzyme kinetics, including Michaelis constant, and apply them to interpret experimental results.

BBSPS Proforma for the practical in Enzyme Kinetics 2017-18
Student Number (SRN) __________________ Date ____/______/2019
1.
This practical aims to determine the enzyme kinetic parameters V max and
Km. In order to do this, a continuous enzyme assay will be performed and a
graph of initial rates and substrate concentration will be drawn. The result is a
rectangular hyperbola. The rate of reaction and the effect of substrate
concentration on the initial rate will be analyzed. In addition, Michael's
constant will also be analyzed.
This practical also aims to convert the absorbance measurements into
concentrations, absorbance per unit time measurements into mole product
produced per unit time or molarity change per unit time. Through this
practical, we will be able to understand the role of enzymes and pH in product
formation.
Number of words=115
2.
Table
Tube Volume of
PNP Stock
(mL)
Conc. of
PNP
(mmol.dm-
3)
Abs. 1 at
410 nm
Abs. 2 at
410 nm
Mean
Absorbance
at nm
1 0 (blank) 0 0 0 0
2 0.2 0.1 0.065 0.07 0.0675
3 0.4 0.1 0.14 0.13 0.135
4 0.6 0.1 0.2 0.21 0.205
5 0.8 0.1 0.285 0.27 0.2775
6 1.0 0.1 0.34 0.35 0.345
a)
1

3) a)
Table 2:
Cell Volume of PNPP used
(mL)
Final Concentration of
PNPP in cell
(M)
1 1.3 0.65
2 1.1 0.55
3 0.9 0.45
4 0.7 0.35
5 0.5 0.25
6 0.3 0.15
7 0.1 0.05
Replicate 1 0.9 0.45
Replicate 2 0.9 0.45
b)
Final Concentration of the PNPP = Initial concentration x Initial Volume
Final Volume
=0.1x0.9
0.45
=0.2 mol/dm3
=0.1x1.1
0.55
=0.2 mol/dm3
c) M or mol/L or mol/dm3
2

4.
Time
(sec) volume of PNPP used (mL)
0.05 0.1 0.3 0.5 0.7 0.9 1
absorbance at 410
nmetres
5 0.142 0.393
10 0.005 0.027 0.067 0.088 0.123 0.154 0.394
15 0.121 0.162 0.397
20 0.007 0.021 0.079 0.112 0.132 0.178 0.417
25 0.144 0.179 0.421
30 0.038 0.102 0.128 0.160 0.183 0.441
35 0.186 0.202 0.457
40 0.009 0.031 0.119 0.142 0.195 0.233 0.473
45 0.191 0.233 0.482
50 0.011 0.046 0.119 0.163 0.210 0.245 0.496
55 0.234 0.257 0.479
60 0.018 0.044 0.151 0.194 0.247 0.281 0.489
65 0.249 0.304 0.508
70 0.018 0.058 0.125 0.205 0.260 0.308 0.519
75 0.281 0.315 0.529
80 0.018 0.055 0.129 0.221 0.289 0.336 0.528
85 0.300 0.348 0.546
90 0.023 0.055 0.165 0.215 0.280 0.366 0.559
95 0.308 0.381 0.578
100 0.021 0.059 0.192 0.238 0.327 0.574
105 0.322 0.597
110 0.018 0.069 0.218 0.260 0.344 0.630
115 0.355
120 0.026 0.077 0.253 0.290 0.378
125
130 0.027 0.079 0.274 0.314
135
140 0.030 0.080 0.301 0.323
145
150 0.032 0.093 0.330 0.354
CALIBRATION CURVE DATA
PNP
(100uMOLARstock
)VOL mL
ABS
410
REP1
ABS
410
REP
2
0.2 0.065 0.07
0.4 0.14 0.13
0.6 0.2 0.21
0.8 0.285 0.27
1 0.34 0.35
5.
3

6)
Y-axis: nm
X-axis: min
7) a)
b)
4

1/velocity ws 1/substrate concentration
c)
Hyperbolic Plot Lineweaver-Burk plot
Km 0.35 2.857142857142857
Vmax 0.2 5
e)
The hyperbolic plot:
Strengths- easy to read presented data
Weakness- time consuming to input data
The Lineweaver-Burk plot:
Strength- presented data is easy to analyze
Weakness-time consuming
8. a)
= 0.2*3.25
1.36+3.25
=0.65
4.61
=0.14099783080260
= 0.14%
b)
Role –to catalyse hydrolysis of mono phosphate esters
Function – to catalyse reactions
Location- in the cells of living tissue
5

c)
Six buffer compounds will be required
6