Effect of Reduction and Subsequent Oxidation of Critical Disulfide Bonds

in Alkaline PhosphataseAlec Coffman, Susan Olds

Purpose

• Determine if enzyme activity could be regained after disulfide bonds reduced by TCEP

• Further oxidize disulfide bonds using potassium ferricyanide (K3[Fe(CN)6]). – Ellman’s test

Background• Enzyme Activity

– Performed using para-Nitrophenylphosphate (PNPP) as a substrate, at concentrations significantly higher than that of AP (V0=Vmax). At a pH of 8, the product produced by the activity of AP on PNPP will be strongly absorbing at 400 nm, and can be used to quantify the enzyme concentration of a solution(since Vmax = kcat[E]).

• Ellman’s Test– A Test used to quantify thiol concentration using the reagent

5,5’-dithiobis-(2-nitrobenzoic acid) (DTNB). Thiols react with the disulfide of DTNB, and form a dianionic compound, 2-nitro-5-thiobenzoate (NTB), which absorbs light at 412 nm. With the knowledge of the cysteine composition of AP, the fraction of cysteines involved in disulfide bonds can be determined after performing an Ellman’s test.

Background

• Disulfide bond between Cys-286 and Cys-336 has been proven critical to enzymatic activity of AP.

• Disulfide bond between Cys-168 and Cys-178 has no relationship to enzyme activity.

AP Subunit

Figure 1: PyMol representation of AP monomer, with Cys-286/Cys-336 Disulfide bond (cyan) and Cys-168/Cys-178 disulfide bonds depicted (blue). Zn ions are also shown (grey), and the magnesium ion is noted (magenta)

Methods• Eight concentrations of TCEP/AP were prepared in 1 mL of DI water.• Before addition of TCEP, the activity of the original AP was

determined (Vmax = 41.7 uM/min).• [AP] = 250 nM for each• [TCEP] = 5, 10, 15, 20, 50, 100, 500 mM• The samples were heated at 45 °C for 30 minutes, and enzyme

activity tests were performed to ensure no activity remained.• 10 mM TCEP was the lowest concentration of TCEP where no

activity remained, while any concentration above 50 mM TCEP lowered the pH of the solution appreciably.

• 5 mM TCEP still gave ~ 4 % of the original activity after 30 minutes (Vmax = 1.5 uM/min), but no activity persisted after an hour.

Methods• 35 uL of TCEP/AP (5 mM/ 250 nM) samples were then

incubated with a slight ( 5 % ) molar excess of K3[Fe(CN)6] (stock solution = 40 mM) at room temperature, 45 °C, and 0 °C, to give a final volume of 50 uL.

• These samples were then subjected to enzyme activity after set intervals of time (30 minutes, 60 minutes, 90 minutes, 120 minutes) to see if activity was regained.

• A calibration curve was also prepared for the Ellman’s test, using beta-mercaptoethanol as a standard.

• 1 mL of the 10 mM TCEP/ 250 nM AP was added to 300 uL of K3[Fe(CN)6] and allowed to react for 30 minutes at 45 °C, which was then reacted with Ellman’s reagent to determine the thiol (and subsequently, disulfide) concentration.

Results

A B C 5 mM 50 mM 100mM 500mM

Figure 2: Sample A contains fully reduced AP using 5 mM TCEP, B contains pure AP enzyme, and C is AP post oxidation with K3[Fe(CN)6]). A pH strip post-reduction with TCEP is depicted.

Results

Results

Conclusions

• Oxidation with potassium ferricyanide returns some activity, but not full activity. Also, the oxidation does not occur at 0 °C, and there is little discernible difference between oxidation at room temperature and at 45 °C.

• The Ellman’s test confirmed that 40 % of the disulfide bonds had been oxidized, however only 20 % of the activity remained. Discrepancies could be due to unequal oxidation between Cys-286/Cys-336 and Cys-168/Cys-178.

Future Work

• Perform experiments to determine which pair of disulfide bonds are preferentially oxidized, and if other experimental conditions influence the oxidation/enyzmatic activity of AP.

References• M. Sone, S. Kishigami, T. Yoshihisa, and K. Ito(1997) J. Biol. Chem. 272, 6174 - 6178.

Roles of Disulfide Bonds in Bacterial Alkaline Phosphatase.

• Murphy, J.E., Tibbitts, T.T., Kantrowitz, E.R. (1995) J.Mol. Biol. 253: 604-617. Mutations at positions 153 and 328 in Escherichia coli alkaline phosphatase provide insight towards the structure and function of mammalian and yeast alkaline phosphatases.

• Sedlak J, Lindsay RH (1968) Anal. Biochem. 25:192-205. Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent.

• Chen L,Annis I, Barany G. Current Protocols in Protein Science Current Protocols in Protein Science (2001) 18.6.1-18.6.19. Disulfide Bond Formation in Peptides

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