VL 13 (Frank Bier) Flashcards
What is Spectroscopy?
Spectroscopy is the Method of Analysis of the Interaction of electromagnetic radiation with Matter
to be used for:
* determination of concentration
* measurement of enzyme activity
* localize molecules
* determine and quantify interactions of molecules
* observe conformational changes
* observe movement and flows (e.g. within a cell
What is electromagentic radiation?
E = h * v = (h * c) / lambda
UV/Vis-spectroscopy: e- transition from occupied to non occupied molecular e- orbitals
Spectroscopy usage
c-determination
* protein- nucleic acid content of cell extracts
* correction for contribution of light scattering
* c(protein) after Gill & von Hippel
measure enzyme activity, c(S)
* NADH-reduction: enzyme activity of dehydrogenases
* NAD-oxidation: lactate determination
measure ligand binding
* CN- binding to catalase
In situ
* mitochondrial respiratory chain
observe conformational changes
* Tm of DNA-helices
What are molecular energy levels?
- molecule in E0 can occupy different energy states between E0 – E1 because it can have vibrational energy (e.g. E0v1)
- transition:
–> E0v0→E1v1: middle
–> E0v0→E1v2:left
–> conclusion: high energy = low λ
What is the line spectra?
Heterogeneity of molecular environment + limited instrument resolution
Explain how a spectrophotometer is build
- light hits monochromator→diffracted into its spectrum
- one wavelength transmitted through slit→hits sample
- detector measures emerging light intensity
What is the labert-Beer law?
- exponential decay of light intensity with increasing layer thickness (cuvette)
- number of absorbing photons/time unit (= decay of light intensity dI) on path dL in cuvette is proportional to
–> number of incident photons/time unit
(= light intensity I)
–> number of absorbable molecules
(= product from c and path length dL)
extinction, absorption, absorbance, transmission:
* absorption designates (bezeichnet) a molecular process
* extinction resp. absorbance: observed decay of light intensity: E = log10
* light scattering can also contribute to extinction
* transmission describes medium permeability for waves: T = I/I0
extinction coefficient:
* describes how much radiation is absorbed by substance at 1 cm path length in medium
* dependent on: path length in media (cuvette thickness), c
Biochemically important chromophores
Typical spectrums of Proteins and DNA
Spectroscopy with polarised light ORD and CD Spectroscopy
ORD (Optical Rotation Dispersion)
* also visible beside of absorption bands
* because of the different propagation of L- and R-circular polar. light
* Needs high concentrations and long optical paths (thickness of cuvette)
CD (Circular Dichroism)
* Caused by different absorption of L- and R-circular polar. light
* less material needed (compared to ORD)
* Applications:
–> Protein structure estimate from Far-UV-CD-Spektra
–> Near-UV-CD diagnostic use for complete (native) proteins with
preserved tertiary structure
–> structural changes in proteins and nucleic acid
Fluorescence - Excitation and Emission
Some biochemically relevant fluorophores
Natural Fluorophores:
* aromatic amino acids (Tryptophan, Tyrosin, Phenylalanin)
* NAD(P)H, FAD, FMN
* Retinoide, Porphyrine (e.g.. Chlorophylle)
* rare bases in tRNA, Steroid hormones
NOT fluorescent:
* nucleotides and nucleic acids, lipides, carbohydrates,
* non-aromatic amino acids, primary metabolites and intermediates,
* usual metal ions
Fluorescence – Quantum yield
and the impact of the environment
