1. 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. Donaldson
Department 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
O
2
H2O
GOx
CAT
H2O2Glu
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
Acknowledgements
Special 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
GOx
CAT
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
0
100
200
300
400
500
600
700
800
900
nXGC XGC
nmolH2O2/mL
Max H2O2
Accumulation
nXGC
XGC
0
20
40
60
80
100
120
Time to
accumulate
(minutes)
Time to
decompose
(minutes)
Time(minutes)
H2O2 Accumulation
Decomposition Time
nXGC
XGC
0
5
10
15
20
25
30
35
40
0 50 100 150 200 250 300 350
nmolH2O2/mL
Time (minutes)
HRP Assay
nXGC
XGC
-400
-200
0
200
400
600
800
0 50 100 150 200 250 300 350
nmolH2O2/mL
Time (minutes)
5-Hour H2O2
Accumulation/Decomposition
nXGC
XGC
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
References
Liochev, 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.
X