Enzyme Used | Lysozyme | |
Thermal Denaturation | ||
Temp (Celsius) | Abs (325 nm) | |
40 | 0.106 | |
50 | 0.086 | |
60 | 0.109 | |
65 | 0.161 | |
70 | 0.409 | |
75 | 2.06 | |
80 | 2.173 | |
90 | 0.821 | |
Urea Denaturation | ||
M of urea | Abs (325 nm) | |
0 | 0.464 | |
1 | 0.07 | |
2 | 0.056 | |
4 | 0.05 | |
6 | 0.035 | |
8 | 0.028 |
Please help me calculate the fraction denatured at each urea concentration and plot ΔG vs [urea] graph and Calculate the ΔG of denaturation in water and What is the concentration of urea where half of the protein is denatured?
Temperature | Absorbance | |
40 | 0.106 | |
50 | 0.086 | |
60 | 0.109 | |
65 | 0.161 | |
70 | 0.409 | |
75 | 2.06 | |
80 | 2.173 | |
90 | 0.821 | |
Urea denaturation | ||
M | ||
0 | 0.464 | |
1 | 0.07 | |
2 | 0.056 | |
4 | 0.05 | |
6 | 0.035 | |
8 | 0.028 | |
The temperature T of denaturation is determined using the following graph as 70 C. and is 343.15 K
The concentration of Urea when half denature is about 0.5M
Denature fraction, fD =
Y is the observe absorbance, Yn is in native state, Yd is in denatured state
Yn = 0.464, Yd =0.03 = .434
Example. fD =
KD = fD/(1-fD)
G
= -RTlnKD
R = 8.314, T =343K,
fD | KD | |
0.464 | 0 | 0 |
0.07 | 0.908 | 9.87 |
0.056 | 0.94 | 9.79 |
0.05 | 0.954 | 10.15 |
0.035 | 0.988 | 82.33 |
0.028 | 1.005 | 201 |
KD | lnKD |
0 | 0 |
9.87 | 2.289 |
9.79 | 2.281 |
10.15 | 2.317 |
82.33 | 4.410 |
201 | 5.303 |
lnKD | GD |
0 | 0 |
2.289 | -6530 |
2.281 | -6610 |
2.317 | -6610.28 |
4.410 | -12581.5 |
5.303 | -15129.19 |
Accordingly,
G(H2O) = -2400
Enzyme Used Lysozyme Thermal Denaturation Temp (Celsius) Abs (325 nm) 40 0.106 50 0.086 60 0.109...