Enzyme Kinetics: Mitigating Oxidative Stress from Reactive Oxygen Species, Simulation of Peroxisomes by Enzyme Cross-Linking

James A. Randall, Katharine Wright, Dzhalal Agakishiev, Robert P. DonaldsonDepartment of Biology, The George Washington University, Washington, DC 20052

Introduction

• H2O2 is a reactive oxygen species (ROS) generated in μM concentrations during cellular respiration in peroxisomes. Accumulated ROS can lead to accelerating aging through damage to proteins, DNA, and lipids

• H2O2 is used as a chemical messenger at low concentrations

• From glucose (Glu), Glucose Oxidase (GOx) produces H2O2 (observable at 240nm in a spectrophotometer), which is consumed over time by Catalase (CAT) into water (H2O) and oxygen (O2)

• GOx serves as a good model for a peroxisomal oxidase

• This process takes time and can lead to H2O2 accumulation and ROS damage

• Hypothesis: If GOx and CAT are chemically cross-linked (XGC), there will be less detectable accumulation of H2O2 and a faster decomposition rate than the non-cross-linked enzymes (nXGC) since the enzymes will physically be in closer proximity

GOx

CAT

H2O2

H2O2

Glu

O2 H2O

GOx

CATH2O2Glu

O2 H2O

Experiment/Methods

Effective Cross-Linking

• Large XGC multimers were effectively produced• SDS gel electrophoresis shows the cross-linking procedure results in some

very large (850+ kD) multimers, smaller multimers of various size, and some monomers remain

• The large multimers were separated from the smaller ones via size exclusion chromatography

• The close physical proximity of the enzymes in the XGC system enables the H2O2 to quickly travel from GOx to CAT, where it is consumed faster than in the nXGC system

• Individual enzyme activities retained their activity after cross-linking• This diagram is a simplified representation of a peroxisome or large XGC multimer

Results• nXGC produced 800μM of H2O2 over 3 minutes followed by

nearly complete decomposition after 100 minutes• XGC produced 600μM H2O2 over 10 minutes followed by

nearly complete decomposition after 50 minutes• Less H2O2 was produced by the cross-linked

enzymes over a longer period of time and was degraded faster than nXGC

AcknowledgementsSpecial thanks to the Luther Rice Selection Committee, SURE Award Selection Committee, Dr. Robert Donaldson, and Dr. Michael Massiah

Conclusions• Cross-linking CAT with GOx can prevent H2O2 accumulation

and accelerate its decomposition• The XGC system could lead to enhanced antioxidant

capability and decrease the deleterious effects of ROS, such as aging and molecular damage

• This is a similar situation to peroxisomes where oxidases and catalase are together in a compartment

XGC

GOxCAT

850kD

250kD

75kD

50kD

• Enzymes were cross-linked using Disuccinimidyl Gluterate (DSG) and the excess DSG was removed using a desalting column

• Larger enzyme multimers were separated from smaller multimers and monomers via size exclusion chromatography

• H2O2 accumulation and decomposition rates of XGC were compared with the rates of accumulation and decomposition of H2O2 by nXGC with excess Glu and O2 over 5 hours in pH 7.4 KPO4 buffer

H2O2 Accumulation and Decomposition Rates

nXGC XGC0

100

200

300

400

500

600

700

800

900

Max H2O2 Accumu-lation

Max accumu-lation (nmol H2O2/mL)

nmol

H2O

2 /m

L

Time to

accumulate

(minutes)

Time to

decompose (m

inutes)0

20406080

100120

H2O2 Accumulation Decomposition Time

nXGCXGC

Tim

e (m

inut

es)

0 50 100 150 200 250 300 3500

5

10

15

20

25

30

35

40

HRP Assay

nXGCXGC

Time (minutes)

nmol

H2O

2 /m

L

0 50 100 150 200 250 300 350

-400

-200

0

200

400

600

800

1000

5-Hour H2O2 Accumulation/Decomposition

nXGCXGC

Time (minutes)

nmol

H2O

2 /m

L

XGC (red) and nXGC (blue) were incubated with excess Glu and O2 over 5 hours at 240nm in the spectrophotometer. nXGC increased to a higher concentration of H2O2 over a shorter period of time than XGC. And XGC decomposed the H2O2 at a faster rate than nXGC. XGC went into negative values likely due to uncertainty in the spectrophotometer at such low H2O2 levels

H2O2 levels were corroborated with horse radish peroxidase (HRP). O-dianisidine was added to samples of H2O2 taken from reacting XGC and nXGC solutions at 3, 10, 30, 60, and 300 minutes, which when combined with HRP turns orange. The intensity of the orange was captured using a spectrophotometer at 430nm. The peak H2O2 concentration varies in time; slope is important in the diagram

ReferencesLiochev, S. I. "Reactive oxygen species and the free radical theory of aging." Free Radical

Biology and Medicine 60 (2013): 1-4.Pye, Valerie E., et al. "Peroxisomal Plant 3-Ketoacyl-CoA Thiolase Structure and Activity

Are Regulated by a Sensitive Redox Switch." The Journal of Biological Chemistry 285 (2010): 24078-24088.Stone, James R. and Suping Yang. "Hydrogen Peroxide: A Signaling Messenger.” Antioxidants & Redox Signaling 8.3-4 (2006): 243-270.Tsuge, Haruhito, Osamu Natsuaki and Kazuji Ohashi. "Purification, Properties, and Molecular Features of Glucose Oxidase from Aspergilus niger." The Journal of Biochemistry 78.4 (1975): 835-843.

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