Lecture 3: Absorption - UV/Visible/IR and CD

Burning things to a crisp (as in the Bunsen Spectroscope)

can tell us about the atoms contained in a molecule
But this doesnt tell us anything about
how the molecule is put together
Fortunately, certain molecules absorb light in a characteristic
way. Over the years, this has helped us identify and quantify
biological molecules.
Absorption: Physical Basis
Absorption occurs when the energy contained in a photon is
absorbed by an electron resulting in a transition to an
excited state
http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/uvvisab1.htm
Since photon and electron energy levels are quantized, we
can only get specific allowed transitions
~ 115 nm
~ 200 400 nm
~ 150-250 nm
~ 400 - 700 nm
E=hv (h = 6.626*10
-34
J-s)
Absorption: Lineshape
t
hv
t
*

So, our absorption spectrum
should probably look like this:
But they dont
Absorption: Lineshape
This is because molecules are always rotating and vibrating.
Each rotational or vibrational state slightly changes the
energy of the transition.
Distrubtion of these states is a random walk.
So the lineshape of our
absorption spectra is
normally distributed
Absorption: Lineshape
Absorption is an additive property, that is, the spectrum is
the sum of the Gaussians associated with each transition
This can make looking at complex solutions quite hard,
especially if we are trying to say something about intensity
Absorption: Intensity
The absorption efficiency of an analyte is affected by:
The nature of the analyte
The number of available microstates
The solvent (sortof)
The absorption efficiency of an analyte generally not
affected by:
Other (low conc.) solutes
Concentration
This makes absorption spectroscopy one of the few
bioanalytical methods where the signal intensity is directly
proportional to the concentration
Temperature (within reason)
Absorption: The Beer-Lambert Law
The Beer-Lambert law sortof has the wrong name
Pierre Bouguer
(1698-1758)
Johan Lambert
(1728-1777)
Astronomer: Light
is diminished as it
passes through
the atmosphere.
Mathematician, first
to prove that t is
irrational. No
absorption coefficient.
August Beer (1825-1863): Added absorption co-
efficient and related to conc. in solution.
cl I I A c = = ) / log(
0 1
Extinction
coefficient
Concentration
Path length
Beer-Lambert Law Derivation (sortof)
The Beer-Lambert law is a result of the fact that a photon
has a constant probability of being absorbed as it travels
through the sample.
beam
particles
x
y
y
A P = ) (
x
y
Absorption
Total cross section of
particles concentration
Cross section of
beam
Populations with constant
probabilities of death
decline exponentially
cl
P
P
A
e
P
P
T
I
O
cl
I
O
o
o
=
|
|
.
|

\
|
=
= =

ln
UV/Visible Spectroscopy: Instrumentation
In absorption spectroscopy, we measure c as a function of
wavelength
The instrument we use to do this is called a UV/visible
Spectrophotometer
The Major Components Are:
A light source
A monochromator
A detector
A Sample Compartment
UV/Visible Spectroscopy: Light Sources
Xenon, Mercury/Xenon
Flash Arc-Lamps light
generated from Xe plasma
Pure Xenon has very wide emission spectrum ~200 1200 nm
Xenon/Mercury is blue shifted for more power in the UV
region, used more often for sterilization
UV/Visible Spectroscopy: Light Sources
Deuterium
D
2
gas is discharged by contact with a high voltage tungsten
cathode
Continuous spectrum from ~150 nm - ~370 nm
Usually used in conjunction with a tungsten/halogen source,
which handles the visible spectrum
Monochromators
The light sources we use produce continuous emission
spectra. But we need single wavelengths, so
This is called the Czerny-
Turner setup
D is either a prism or a
diffraction grating (almost
always the latter these days
B and C set up a beam that
is at infinite focus
E refocuses one wavelength on slit F
Different wavelengths can be focused on F by rotating D or
E (its almost always D).
Monochromators: Defraction Gratings
Diffraction gratings are a surface with closely spaced parallel
lines
These can be semi-transparent gratings or ridged mirrors
After passing though a slit (or bouncing off a ridge)
the angle at which the light leaves is given by
u n a = ) sin(
wavelength!
distance between slits
diffraction order
Sample Compartments/Holders
These days, sample compartments are designed to accept
accessories
The sample itself is held in a cuvette, usually plastic or
quartz:
The detector
Silicon diode:
Basically a solar cell light ionizes n-doped (phosphate)
silicon, placing the electrons in the conduction band (i.e.
having a voltage).
Wide wavelength range, less sensitivity
Photomultiplier
In photomultipliers, light hits a photocathode, releasing a small
number of electrons, which are then made to collide with a
series of dynodes, each more positive than the last
Each collision produces more and more activated electrons
Sensitive, but noisy. Pretty much needed for low energy (IR)
photons
The Whole Instrument
Light Source
Czerny-Turner
Sample
Detector
UV/Visible - Applications
UV/visible absorption spectroscopy may not be a new and
sexy method, but it has one advantage: The signal is
proportional to the concentration.
Consequently, it is most commonly used for concentration
measurements or validation:
Protein concentration with dyes (Bradford) and without
(A
280
- Tryptophan)
Purity of protein or nucleic acid preps (A
260
/A
280
)
c = 5,579 M
-1
cm
-1
at 278 nm
UV/visible: Applications
UV/visible is still used in current research,
especially for heme-containing proteins, which have
absorbance in the Soret region that is sensitive to
the state of the protein
3d shell of Fe
2+
has 6 electrons
High Spin Low Spin
UV/Visible Applications
This paper looks at iron-sulfur clusters in a native and a
mutant protein
C196S Mutant lacks broad
absorption band between 400-
600 nm which is diagnostic of an
2Fe-2S cluster
JBC (1998)Vol. 273, No. 35;28 pp. 2231122316
Time-resolved UV/visible
The main protein signal (at 280 nm) doesnt change much
with protein folding/activity
But Soret region absorption does
(cytochrome P450
cam
)
Spolitak T, Dawson JH, Ballou DP (2005) J. Biol.
Chem. 280 (21): 20300-20309 2005
Circular Dichroism (CD)
So far, weve gotten our light down to a single wavelength,
but its not polarized
Plane polarized (i.e.
a laser)
Circularly polarized
CD and Chirality
Chiral molecules absorb left and right circularly polarized
light differently
L R L R L R L
L R L
R L R L
This difference, which can be expressed as
R L
c c c = A
which is the circular dichroism
Usually expressed as molar elipticity: c | A = 2 . 298 , 3
CD and Proteins
In proteins there are a number of circularly dichroistic
electronic transitions that are of use:
nt
*
(220 nm)
tt
*
(190 nm)
dipole orientation
of F, Y, C and W
(250-300 nm)
But these are all weakly dichroistic. In proteins Ac ~ .0001
CD Spectra of Proteins
In CD Spectra, we measure Ac as a function of wavelength:
-ve Peaks at 208 and -222 nm
= o-helix
-ve, broad Peak at -218 nm
= |-sheet
+ve at 212, -ve at 190 nm =
|-turn
Problem: Overlap!!!
CD Data Analysis:
CD data can provide very specific information about
secondary structure.
We should be able to convert the intensity of the peak at
222nm, for example, into % alpha helix. But what if theres
overlap with the | sheet peak?
The most common solution is to use a basis set of archetypal
spectra, adding them together in a way that most closely
matches the observed spectrum.
Helix 35%
Sheet 22%
Turn 10%
CD is Noisy!
Not only is CD a very small signal, but in the far UV region,
there are lots of sources of noise
One of these is the production ozone (O
3
) in the sample
compartment. This is why most CD instruments are flushed
with N
2

H
T
C

Noise/Signal
(effectively)
CD Instrumentation
So we need most of the same
things as for a UV/visible:
Lamp (Xe or Xe/Hg)
Monochromator (Czerny-
Turner)
Detector (photomultiplier)
But we also need:
Polarizer
Electro-optic modulator
Sample compartment
Applications
CD compliments other more general methods of monitoring
(un)folding.
Babu KR, Douglas DJ, (2000) Biochemistry
39 (47): 14702-14710
Tertiary structure
Secondary Structure
Time-resolved CD
CD compliments other more general methods of monitoring
(un)folding.
Dartigalongue T, Hache F, Chirality 18
(4): 273-278 MAY 5 2006
Kaushik JK, Ogasahara K, Yutani K, J.
Mol. Biol., 316 (4): 991-1003 MAR 1 2002
Time-resolved CD
CD compliments other more general methods of monitoring
(un)folding.
J. Mol. Biol., 372 (1): 236-253 2007