Question

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?

Answer 1

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, f_D =

Y is the observe absorbance, Yn is in native state, Yd is in denatured state

Yn = 0.464, Yd =0.03 = .434

Example. fD =

K_D = f_D/(1-f_D)

G = -RTlnK_D

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