1. Ligand binding
Cyanide binding to the respiratory enzyme- cytochrome c oxidase
What we know,
cyanide is a potent poison of respiration
CN inhibition to respiring mitochondria is instantaneous
Blocks electron transfer to O2
[O2]
Time (min) M S CN

2. Cyanide reaction with cytochrome c oxidase
Wavelength (nm) 1
0.5
A 430 nm
Time (m)
+CN
ACN=εCNcCNl
A=εcl
E-CN E

3. Time-resolved spectroscopy
0.4
0.2
-0.2
-0.4 ΔA
Wavelength (nm)
Time (m)
1000
700
400
100 1
Isosbestic points
E + CN
E-CN
3) Reactivity of the enzyme
in vitro is different from
enzyme in vivo
4) Oxidized enzyme exists
in multiple states

4. Ligand binding-equilibrium case
[ligand] (mM)
KD= .011 mM
ΔA = .105

5. Emission spectroscopy
Fluorescence occurs when molecules emit light as they return to
The ground state following absorption E r hv
excitation
fluorescence I
Wavelength
(energy)
excitation
fluorescence
non-fluorescent
relaxation

6. The timescales of fluorescence
Excitation from ground state to excited state ~ s
Vibrational relaxation within excited state ~10-12 s
Spontaneous emission s
Photochemistry – reactions from the excited state
Phosphorescence (ms to minutes)
Non-radiative relaxation to ground state
Other processes

7. Fluorescence measurables
Excitation spectra (λmax,ex)
Emission spectra (λmax,em)
Intensity (Quantum yield)
Lifetime
Polarization

8. Lifetime, quantum yield and fluorescence intensity
Radiative decay to the ground state is a first order
process and lifetime is the inverse of decay rate,
τF = 1/kF
There are other processes (non-radiative) that
lead to depopulation of the excited state,
k = kF + ∑ki (overall lifetime τ=1/k)
The quantum yield (ΦF)is the fraction of molecules that return to
the ground state via a fluorescence pathway,
ΦF = kF / (kF + ∑ki) = τ / τF
where τ is the observed lifetime of the overall process
Fluorescence intensity (F) will then depend on the initial population
of the excited state (IA) and the quantum yield,
F = IA ΦF E r hv
excitation
fluorescence

9. Some Intrinsic Fluorophores
NA bases λex,max(nm) λem,max(nm) ΦF τF (ns)
Adenine x
Guanine x
Cytosine x
Uracil x
Protein residues
Trp
Tyr
Phe

10. Extrinsic Fluorescent probes
Probe Use λex,max(nm) λem,max(nm) ΦF τF (ns)
Dansyl chloride covalent adduct
via lys, cys
1,5-I-AEDANS “ “
Fluorescein lys
isothiocyanate (FITC)
8-Anilino non-covalent
naphthalene protein binding
sulfonate (ANS)
Ethidium non-covalent ~
bromide nucleic acid complex

11. Some structures 1,5-I-AEDANS Dansyl chloride Fluorescein isothiocyante
5-(dimethylamino)naphthalene
-1-sulfonyl chloride
5-[2-[(2-iodoacetyl)amino]ethylamino] naphthalene-1-sulfonic acid
Fluorescein
isothiocyante
ANS
Ethidium bromide

12. Another family of probes
The “fluorescent proteins” – Green fluorescent protein or GFP
-S65-Y66-G67- O2
“fused” chromophore
λex,max(nm) λem,max(nm)
T>30oC
398 nm H+
508 nm

13. Formation of the GFP fluorophore

14. Let’s build a fluorimeter
Light source
sample
detector
filter
slit

15. Scanning excitation and emission spectra
Light source
sample
detector
monochromators
Excitation (absorbance) and emission
Spectra
λmax,ex – excitation maximum
λmax,em – emission maximum

16. Scanning excitation and emission spectra

17. Fluorescence intensity and concentration
expected
As the concentration increases the signal strength does not increase proportionately
Inner filter effect I
observed
Correction factor= antilog
Aex + Aem 2
Aex + Aem < 0.1 A
Concentration (α A)
Beware of changes to Aex + Aem