1. Oxidation of Alkaline Phosphatase from TCEP Denaturation
By Joaquin Amaro
Feel Gu Kang

2. Introduction/Background
Alkaline Phosphatase loses activity with TCEP denaturation as previous work in lab demonstrated
At least 0.7mM concentration of TCEP is needed at 45oC
Copper sulfate has reduction potential of +0.34
Potassium ferricyanide has reduction potential of +0.36

3. Objective
Regain enzyme activity by oxidation of Alkaline Phosphatase after TCEP denaturation
Oxidation with Copper Sulfate (CuSO4) and Potassium Ferricyanide (K3[Fe(CN)6])

4. Materials/Methods 0.25mM of AP in Equilibration Buffer
Denaturation with 0.7mM of TCEP in Equilibration Buffer at 45oC
Equilibration Buffer (5mM Tris-HCl, 5mM MgCl2, 5mM ZnCl2 in pH7.4)
Centrifugation in Centricon tubes at 8K rev/min for 20 minutes (3x)
Elution from Centricon tubes at 3K rev/min for 5 minutes
Enzyme Kinetics
Read at wavelength of 410nm with extinction coefficient of 0.02μM-1cm-1.

5. Enzyme Activity [V0 (μM/min)]
Results
Copper Sulfate Oxidation
Enzyme Activity [V0 (μM/min)]
1st
2nd
Without TCEP
7.223
7.7692
n/a
TCEP
10 Min in 45oC
0.453
0.324
6.167
5.546
Addition of Copper Sulfate
No Addition
3x Centricon
0.456
0.468
6.234
5.437
Overnight
0.344
0.211
6.37

6. Enzyme Activity [V0 (μM/min)] Enzyme Activity [V0 (μM/min)]
Results cont.
Potassium Ferricyanide Oxidation
Enzyme Activity [V0 (μM/min)]
1st
2nd
With TCEP
0.034
n/a
Addition of Ferricyanide
3x Centricon
0.454
0.65
Overnight
0.456
0.3
Enzyme Activity [V0 (μM/min)]
1st
2nd
Without TCEP
7.967
n/a
TCEP
10 Min in 45oC
0.53
0.324
Addition of Ferricyanide
3x Centricon
2.11
0.468
Overnight
1.972
1.678

7. Discussion Regained about 1/3 of activity
Results show Potassium Ferricyanide is better oxidizing agent compared to Copper Sulfate

8. References
Ruegg, U.T and Rudinger, J. "Reductive cleavage of cystine disulfides with tributylphosphine". Methods Enzymol.. Methods in Enzymology 47: 111–126 (1977). Print.
Nelson, David L., Michael M. Cox, and Albert L. Lehninger. Principles of Biochemistry. New York, NY: Freeman, Print.