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*

The π → π* transition involves orbitals that have significant overlap, and the probability is near 1.0 as they are “symmetry allowed”.

2pz + 2pz

2pz - 2pz

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”.

ΔS≠0

Relating the MO transitions to the Jablonski Diagram

The Jablonski Diagram

Back To The Farm

UV-visible Absorption of Amino Acids & Proteins

Amide linkages absorbances of various amino acids

Strong absorbance by both at 280 nm

Crude estimate of protein concentration but varies with amounts

of each amino acid in protein.

n to π* π to π* amide absorbance of amino acids

UV-visible By Amino Acids Sidechains

UV-visible Absorption of Amino Acids & Proteins

UV spectrum of Bovine Serum Albumin (BSA)

• 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

•π to π* transition

• Contains information on conformation and concentration

Secondary Structure Affects Absorption Properties• 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

•π to π* transition in amide group

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 = !Ai = !!iCil

-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.