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

Cyanide reaction with cytochrome c oxidase Wavelength (nm) 1 0.5 400 420 440 460 A 430 nm Time (m) +CN 0 1 10 100 1000 ACN=εCNcCNl A=εcl E-CN E

Time-resolved spectroscopy 0.4 0.2 -0.2 -0.4 ΔA 400 420 440 460 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

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

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

The timescales of fluorescence Excitation from ground state to excited state ~ 10-15 s Vibrational relaxation within excited state ~10-12 s Spontaneous emission 10-6-10-9 s Photochemistry – reactions from the excited state Phosphorescence (ms to minutes) Non-radiative relaxation to ground state Other processes

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

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

Some Intrinsic Fluorophores NA bases λex,max(nm) λem,max(nm) ΦF τF (ns) Adenine 260 321 2.6 x 10-4 0.02 Guanine 275 329 3.0 x 10-4 0.02 Cytosine 267 313 0.8 x 10-4 0.02 Uracil 260 308 0.4 x 10-4 0.02 Protein residues Trp 280 348 0.20 2.6 Tyr 274 303 0.14 3.6 Phe 257 282 0.04 6.4

Extrinsic Fluorescent probes Probe Use λex,max(nm) λem,max(nm) ΦF τF (ns) Dansyl chloride covalent adduct 330 510 0.1 13 via lys, cys 1,5-I-AEDANS “ “ 360 480 0.5 15 Fluorescein lys isothiocyanate (FITC) 495 516 0.3 4 8-Anilino-1- non-covalent 374 454 0.98 16 naphthalene protein binding sulfonate (ANS) Ethidium non-covalent 515 600 ~1 38 bromide nucleic acid complex

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

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

Formation of the GFP fluorophore

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

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

Scanning excitation and emission spectra

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 0.1 0.2 A Concentration (α A) Beware of changes to Aex + Aem