1. Chemical Modification
Variety
Oxidation
Nitrosylation
Dissociation
Effects
Folding
Controls
Environmental
Reactive

2. Protein structure - function
AA sequence
O-N pairing of backbone
Ionic/hydrophobic interaction of side chains
Chemical environment
Ionic strength
pH – ie: H+ ions
Reactive
Side chain modification

3. Chemistry A + B  AB With total A constant
AB increases with either A or B
Equilibrium constant Ka=[AB]/([A][B])
With total A constant
A/AB switch
A/AB indicator
Complex chemistry alters sensitivity

4. Multiple modifications
For fixed “A” the amount of product is
One “B”: AB
Two “B”: AB+AB2
More
Hill equation:
n: cooperativity
Kd: apparent dissociation constant ] [ 1 B K
A+AB AB + = Q 2 ] [ ' 1 ) ( B K + - = Q l å + = Q n B K a ] [ 1 n d x Kn + = Q

5. Chemical sensors 2-3 log dynamic range
Decreasing Kd increasing affinity, increases sensitivity
Increasing cooperativity increases gain

6. pH Charged amino acid side chains
H+ movement can dramatically alter molecular folding
pK=-log( )
[B-][H+]
[HB]

7. Bicarbonate buffers CO2 solubility ~0.03mM/Torr CO2 Hydration
Carbonic anhydrase
Henderson-Hasselbalch equation K=
pK= -log( )
pK = pH – log( )
pH = pK + log( ); pK=6.1
[HCO3-][H+]
[CO2]
[HCO3-][H+]
[CO2]
[HCO3-]
[CO2]
[HCO3-]
[CO2]

8. Bicarbonate buffers [HCO3-][H+] Ka = [CO2] pH 2 pH 7.2 5% CO2 pH 7.4

9. Open vs Closed Buffer Systems
Bicarbonate
Physiological
pCO2 = 40 mmHg
[CO2] = 1.2 mM
[CO2]+[HCO3]=31 mM
HEPES
Equivalent Buffer
[HEPES]+[HEPES-]=31 mM
[HEPES-]=14.7
[HEPES]=16.3

10. Open vs Closed Buffers Bicarb Immediate HEPES Immediate
Add 10 mM HCl
Immediate
[HCO3]=20 mM
[CO2]=11 mM
pH = 6.4 (400 nM)
HEPES
Add 10 mM HCl
Immediate
[HEPES-]=4.7 mM
[HEPES]=26 mM
pH=7.2 (63 nM)
Much better pH control near pKa

11. Open vs Closed Buffer System
Bicarb
CO2 solubility 1.2 mM
[HCO3]=20 mM-350 nM
[CO2] = 1.2mM+350 nM
pH=7.3 (50 nM)
Much better than 6.4 w/o exchange
HEPES
No mass exchange
pH =7.2
Could do bicarb in one step:

12. pH Control Cell membranes impermeable to H+ Compartmentalization of pH
Cytoplasm 7.15
Nucleus 7.2
Mitochondria 8.0
Golgi ~6.3
Lysosome 5.5
Transporters
H+/K+
HCO3-/Cl-

13. Dissociation of amino acid side chains
In cytoplasm, pH=7.15, so 80% of histidine is in base form (uncharged).
0.1% of lysine is in its base form.
In Golgi, pH=6.3, and 40% of histidine is in base form.

14. Block NHE Lower intracellular pH stabilize FAs
pH control demo
Talin-dependent adhesion/motility
Glycolysis
Block NHE Lower intracellular pH stabilize FAs
Talin structure
pH dependence of several glycolysis enzymes (Xie et al 2014)
Srivastava et al 2008

15. Reactive modification
NO S-nitrosylation
Cysteines in hydrophobic acid/base pockets
Hemoglobin
S-NO forms in oxidative environment
Allows NO release in low oxygen
Targets vasodilating NO to oxygen starved tissue
-S-H
-S-N=O

16. Reactive oxidation Partly reduced oxygen: O2·, H2O2, OH·
Protein modification
Cys, His, Phe, Tyr, Met
Sulfur
Ring structures
Chain break
Cross-linking
Chain reaction
DNA/Lipid modification

17. Reactive modification
Thymine
Thymine glycol
Amino acid modification changes local polarity
Crosslinking
Strand break
Thymine glycol distorts DNA structure
Kung & Bolton 1997

18. Electrochemistry Redox reactions describe electron transfer
Zn + CuSO4Cu + ZnSO4
Zn + Cu2+Cu + Zn2+
Zn Zn2+ + 2e- and Cu2+ + 2e- Cu
2 GSH + H2O2 GSSG + 2H2O
2 GSH GSSG + 2e- + 2H+ and H2O2 + 2e- + 2H+ 2H2O
Inorganic
Biological

19. Electrochemistry-free energy
Electrical
DG = -nFDE
Faraday constant C/mol
Concentration
DG = RT ln( Q)
Gas constant 8.31 J/K/mol
Whole reaction
DG = DG0 + RT ln(QProd) – RT ln(Qreac)
-nFDE = -nFDE0 + RT ln(Qprod/Qreac)
DE = DE0 - RT/nF ln(Qprod/Qreac)
Nernst Equation for redox reaction
Equilibrium at DG= DE = 0

20. Electrochemistry-half cells
Standard Reduction Potential E0
Metals (Daniell cell)
Zn Zn2+ + 2e- Zn2+ + 2e- Zn E0=-0.76V
Cu2+ + 2e- Cu E0=+0.34V
DE0=0.34-(-0.76) = 1.1V
Biological (glutathione)
2 GSH GSSG + 2e- + 2H+ GSSG + 2e- + 2H+  2 GSH E0=+0.18
H2O2 + 2e- + 2H+ 2H2O E0=+1.78
DE0=1.78-(0.18) = 1.6V

21. Cellular Redox State Biological Steady state trend
2 GSH + H2O2 GSSG + 2H2O
DE0= 1.6V
DG = -nF (1.6V) + RT ln( )
DE = 1.6V – RT/nF ln( )
Steady state trend
0 = 1.6 –(8.31*310)/(2*9.6e4) ln( )
= 1052
ie: Not a lot of free peroxide in a cell
Still needs a catalyst
Real cells have many potential half-cells
GSSG
GSH2 H2O2
GSSG
GSH2 H2O2
GSSG
GSH2 H2O2
GSSG
GSH2 H2O2

22. GSH:GSSG redox buffer GSH is abundant reducing agent
GSSG + 2e- + 2H+  2 GSH E0=+0.18
DE = 0.18 – RT/nF ln( )
DE = 0.18 – 0.03 log( )
GSH:GSSG ratio as marker of redox state
More GSH, more negative DE, more reducing
GSSG reduction appears as negative in whole reaction
Whole reaction more favorable with positive DE
More H+, more positive DE, more oxidizing
Neutral [H+]2 ~ 10-14
Many biological oxidations include H+
GSSG H2
GSH2
GSSG H2
GSH2

23. Cellular redox cascade
Oxygen radicals are not equivalent
ROS generation
Mitochondria
Photons (UV & ionizing radiation)
Inflammatory cells (NADPH oxidase)
Radical scavengers
O2•-H2O2 superoxide dismutase
H2O2H2O Catalase
H2O2 + GSH GSSG glutathione peroxidase
OH• hydroxyl (uncharged OH-)

24. Redox state Intracellular reductive Extracellular oxidative
Low free oxygen, relatively negative
Extracellular oxidative
High O2, relatively positive
Extracellular antioxidants
Extracellular signals that promote oxidative stress
Cytoplasmic oxidants
Cytoplasmic antioxidants