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Transcript
UV-Electronic Transitions In Proteins Same rationale as formaldehye but the nitrogen takes on sp2 hydbridization (not sp3) as in NH3! Electronic Transitions: π → π* 2pz - 2pz 2pz + 2pz The π → π* transition involves orbitals that have significant overlap, and the probability is near 1.0 as they are “symmetry allowed”. Electronic Transitions: n → π* The n-orbitals do not overlap at all well with the π* orbital, so the probability of this excitation is small. The ε of the n→π* transition is about 103 times smaller than ε for the π→π* transition as it is “symmetry forbidden”. Relating the MO transitions to the Jablonski Diagram ΔS≠0 The Jablonski Diagram Back To The Farm UV-visible Absorption of Amino Acids & Proteins n to π* π to π* amide absorbance of amino acids Strong absorbance by both at 280 nm Crude estimate of protein concentration but varies with amounts of each amino acid in protein. Amide linkages absorbances of various amino acids UV-visible By Amino Acids Sidechains UV-visible Absorption of Amino Acids & Proteins UV spectrum of Bovine Serum Albumin (BSA) • Contains information on conformation and concentration •π to π* transition • Most proteins are colorless in the visible region. •Absorption maxima at 190-200 nm (large) ✓ π to π* transtion amide backbone •Absorption maxima at 280 nm (smaller) ✓ π to π* transtion in F,W,Y aromatics Secondary Structure Affects Absorption Properties •π to π* transition in amide group • Changes in abs properties reflect changes in “electronic environment by: • local charge re-distribution of chromophore • conformational changes altering the π to π* transition amide backbone • bonding interactions •PROVIDES INFORMATION! • Circular dichromism is more sensitive then UV for conformational changes UV spectrum of DNA with enzymatic digestion and thermal “melting” (right) Increase in absorbance as DNA duplex “melts” by increasing temperature (or digestion as shown above) exposing more bases and increasing absorption = hyperchromicity UV Absorption of Nucleic Acid Bases Beer’s Law and Multiple Absorption 1. Multi-component systems obey Beer’s Law A = ΣAi = Σ!i Ci l -For N-component mixture we can measure Ci for all N by measuring Atotal at N different wavelengths 2. Isosbetic Point --is the wavelength were the molar absorptivity of two different absorbing species is equal. Two or more isosbestic points in a spectra of a series of solutions of the same total concentration demonstrates the presence of two and only two components absorbing in that spectra region.