Download Rick`s UV-Vis Absorption Lecture

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.