Part 6a

1. Values are already converted into absorbance values

2. Sample calculation using tube 2:

A=exlxc

0.382 = 6220 M-1cm-1 x 1 cm x c

c = 61.4 uM

Corrected Concentration = concentration x (Final volume / volume of reaction)

 0.05

Optimal pH

0.0375
Enzyme Velocity (umol/min/mL)

0.025

0.0125

0
0 3 7 10 13 16
pH

Figure 1: pH-rate profile of LDH solutions used to determine the optimum pH of the enzyme.
Enzyme velocity was calculated for each of the eight solutions which contained different pH buffers.
Absorbance values obtained for each tube were converted to concentration (uM) and subsequent
velocity values using Beers law. Optimal pH was determined to be 10.5.

= 61.4 uM x (3.40 / 2.40)

= 87.0 uM

3. (87.0 umol / L) x (1.00L / 1000 mL)

= 0.087 umol/ mL

v = 0.087 umol/mL / 4 min

= 0.0218 umol/min/mL

Table 1: The velocity of Sigma (L-1254) LDH solutions recorded when various pH buffers
are added.

Each of the eight tubes contained 1.92 mL of pH buffer, 0.200 mL of 0.300 M lactate, and
0.200 mL NAD+ with 0.08 mL of LDH added to start the reaction. After 4 minutes, the
reaction was stopped with the addition of 1.00 mL of 9.0 M urea and transmittance values were
taken against the blank from a spectrometer set at 340 nm.

pH buffer Absorbance Concentration (uM) Enzyme Velocity

(mol/min/mL)

5.5 0.036 5.79 0.0021

6.5 0.382 61.4 0.0218

7.5 0.398 64.0 0.0227

8.5 0.552 88.7 0.0315

9.5 0.712 115 0.0405

10.5 0.750 121 0.0428

 11.5 0.710 114 0.0405

12.5 0.690 111 0.0393

Part 6b

1. Values are already converted into absorbance values

2. Sample calculation of tube 2:

0.658 = 6220 M-1cm-1 x 1 cm x c

c = 106 uM

Corrected Concentration = concentration x (Final volume / volume of reaction)

= 106 uM x (3.40 / 2.40)

= 150 uM

3. (150 umol / L) x (1.00L / 1000mL)

= 0.150 umol / mL

v = 0.150 umol/mL / 4 min

= 0.0375 umol/min/mL
 Optimal
0.05375
temperature
= 60 C

0.043
Enzyme Velocity (umol/min/mL)

0.03225

0.0215

0.01075

0
0 25 50 75 100 125
Temperature (C)

Figure 2: Temperature-rate profile of LDH solutions used to determine the optimum

temperature of the enzyme. Enzyme velocity was calculated for each of the eight solutions which
Table 2: The velocity of Sigma (L-1254) LDH solutions recorded when the temperature of
were reacted under different environmental temperatures. Absorbance values obtained for each tube
were converted to concentration (uM) and subsequent velocity values using Beers law. Optimal
temperature was determined to be 60 .
the solutions are altered.
Actual Temperature Absorbance Concentration (uM) Enzyme Velocity
(C) (mol/min/mL)
4 0.598 96.1 0.0341
23 0.658 106 0.0375
37 0.698 112 0.0397
50 0.734 118 0.0418
60 0.750 121 0.0427
70 0.730 117 0.0416
100 0.613 98.6 0.0349
Each of the seven tubes contained 1.92 mL of GE buffer, 0.200 mL of 0.300 M lactate, and 0.200
mL NAD+. After 5 minutes of equilibration in the correct temperature, the reaction started with
the addition of 0.08 mL of LDH and finished after 4 minutes with the addition of 1.00 mL of 9.0
M urea. Transmittance values were taken against the blank from a spectrometer set at 340 nm.

Appendix

LDH Activity Assay pH

pH Abs

5.5 0.036

6.5 0.382

7.5 0.398

8.5 0.552

9.5 0.712

10.5 0.750

11.5 0.710

12.5 0.690

LDH Activity Assay Temperature

Temperature Abs

4 0.598
Temperature Abs

23 0.658

37 0.698

50 0.734

60 0.750

70 0.730

100 0.613