biomimetic enzyme nanocomplexes and their use as antidotes and preventative measures for alcohol...
TRANSCRIPT
Presentation Outline:
Primer on enzymes
Enzymatic function in biological systems
Formation of enzyme nanocomplexes
Characterization of form and function
Conclusion: Antidote for alcohol intoxication
Enzymes mediate chemical reactions in biological systems
http://classes.midlandstech.edu/carterp/courses/bio225/chap05/Slide2.GIF
Lowers activation energy and permits unfavorable reactions
Enzymes are polymeric amino acids that assemble into 3-D structures of the lowest free energy
http://www.ebi.ac.uk/training/online/sites/ebi.ac.uk.training.online/files/user/84/images/figure1.png
Structure formation via electrical and hydrophobic interactions
Many possible conformations but only one likely structure (lowest free energy)
REPRODUCIBILITYhttp://www.indiana.edu/~oso/lessons/prot/folding1_files/image002.jpg
Substrates bind to enzyme resulting in conformation change and the breaking/formation of substrate molecular bonds
http://upload.wikimedia.org/wikipedia/commons/thumb/2/24/Induced_fit_diagram.svg/648px-Induced_fit_diagram.svg.png
SubstrateBinding Site
Conformation Change
Bond Reorganization
Increased [H2O2] induces immune activation1 and cell death2
Model of biological enzymatic process: oxidation of glucose
Glucose Oxidase (GOx) oxidizes carboalkoxy group
H2O2 IS A TOXIC INTERMEDIATE
Glucose + 02D-glucono-δ-lactone + H202
Glucose Oxidase (GOx)
OXIDATION
1. Los, M., Droge, W., Stricker, K., Baeuerle, P., and Schulze-Osthoff, K. (2005). Hydrogen peroxide as a potent activator of T-lymphocyte functions. Eur. J. Immun. 25(1):159-165.2. Gardner, A.M., Xu, F., Fady, C., Jacoby, F.J., Duffey, D.C., Tu, Y., and Lichtenstein, A. (1997). Apoptotic vs. nonapoptotic cytotoxicity induced by hydrogen peroxide. Free Rad. Biol. Med. 22(1-2):73-83.
Peroxidases: Nature’s solution to the toxic intermediate problem
Peroxidases employ H2O2 to mediate reduction reactions
Substrate + H202 Product + H20
Horseradish Peroxidase (HRP)
REDUCTION
GOx + HRP = concerted process (reaction + H2O2 disposal)
Mimic concerted process to solve toxic intermediate problem
Mimic cellular membrane to ensure proximity of complimentary enzymes
Perform multi-enzyme-mediated reactions in the extracellular compartment
Design of nanocomplexes accomplished by mimicking nature
Biomimetic: mimic biology
Synthesis of Enzyme Nanocomplexes
1.) HRP & GOx tethered in proximity with inhibitor DNA complex
3.) Inhibitor DNA scaffolds removed
4.) Purification of nanocomplexes: Dialysis with PBS Size-exclusion chromatography
2.) Tethered enzymes encapsulated within polymer network
Monomer:acrylamide
Crosslinker:Bis-methylacrylamide
Transmission Electron Micrograph (TEM) of complexes in solution
Nanocomplex diameter = 30±8nm TEM of nanocomplexes after GOx and HRP labelled with 1.4nm gold particles
Measuring proximity by Förster Resonance Energy Transfer (FRET)
Fluoroisothiocyanate (FITC): @ 450nm excitation, emits at 520nm
Rhodamine B (RhB): @ 450nm excitation, no emission@ 520nm excitation (from FITC emission) emits at 585nm
http://www.molecular-beacons.org/toto/images/FRET.jpg
Fluorosceins:
Requires close proximity (<10nm) for energy transfer
520nm 585nm
(450nm)
<10nm
Spectroscopic determination of enzyme identity within complexes
HRP labelled with RhB
GOx labelled with FITC
Free enzymes(A): no FRET
Indicates that HRP and GOx are within 10nm proximity and that complexes contain both enzymes
Nanocomplexes(B): FRET observed
Polymer encasement not only acts as confining structure but also as protective barrier
Free enzymes and complexes incubated at 65°C (denaturation)
At t=60min, free enzymes lost all function whereas 75% of nanocomplex enzymes maintained functionality
Functional Denatured
HEAT
http://www.ib.bioninja.com.au/_Media/denaturation_med.jpeg
Rate of hydrogen peroxide elimination of Dual-enzyme vs. mono-enzyme analog
Dual-enzyme complex exhibits >900% efficiency of H2O2 elimination over mono-enzyme analog
B
AGOx
+Cat
vsCatGOx +
BADual-enzyme:
Mono-enzyme Analog:
2H2O2
Catalase (Cat)2H2O + O2
Summary of nanocomplex form and function
Average 30nm diameter
Enhanced stability of enzymes
Enhanced function of complimentary enzymes
Excellent prospect as antidote for alcohol intoxication: halt intoxication via enzymatic elimination of ethanol in vivo
Enzyme nanocomplex as antidote for alcohol intoxication
2H2O2
Catalase (Cat)2H2O + O2
CH3CH2OH + 02
Alcohol Oxidase (AOx)
CH3CHO + H202
Employing Alcohol Oxidase:
…and Catalase:
…to eliminate ethanol in vivo
Toxic Intermediate
Loss of intoxicant function
Nanocomplexes exhibit enhanced elimination of ethanol in vivo
Measure of mice BAC at 45min intervals after intoxication with ethanol and administration of placebo (PBS) or enzyme nanocomplex antidote n(AOx-Cat).