Transcript Slide 1

Let’s build a spectrophotometer
Light source
sample
detector
1) Measure Io (100% T)
2) Measure I (T of sample)
3) Calculate A
Scanning an absorbance spectrum
Light source
sample
monochromator
detector
A
Change the monchromator to measure
A versus wavelength
Resolution will be defined by dispersion
and slit width, bandpass ≤ 2nm is
sufficient
λ (nm)
Measure all wavelengths at once
Light source
Detector array
sample
monochromator
Diode array spectrometer
1) Sample receives broad irradiation (i.e., white light)
2) Dispersion element comes after the sample
3) Project spectrum onto an array of detectors
Some applications of spectrophotometry
1) Concentration
Proteins
DNA
2) Environmental effects
Effect of solvent
pH
Ligand or protein interactions
3) Time – resolved measurements
Measure time dependent changes
Timescales
Measuring concentration
Suppose we are studying an enzyme that consumes NADH as part of
its catalytic reaction, we can use the long wavelength absorbance band
of the pyridine chromophore to measure NADH concentration,
and thereby activity.
A + H+ + NADH → NAD+ + AH2
We know the KM of the enzyme for NADH is 100 μM (and for A, KM =5μM)
We wish to measure the total activity present in a series of extracts.
Measure Vmax at saturating S, (i.e., NADH & A)
[NADH] = 1 mM (10-fold> KM)
Measure A at 340 nm, A = ε c l
A= 6.23 x 103 M-1 cm-1 (1 x10-3 M) = 6.23
Measuring concentration (contd)
A > 6 means % T < 0.0001 % , 99.9999 % of Io absorbed
Most spectrophotometers are only linear up to A= 3
Stray light
How can we fix the problem?
3
A
Dilute the NADH.
2
What about the pathlength?
1
Measure off the peak.
[x] (M)
The spectrum of NADH
2.0
20
NAD+
ε (mM-1cm-1)
A
1.0
NADH
0
10
0
220
300
400
Wavelength (nm)
Environmental effects-Solvent perturbation
2.0
a) Trp in aqueous buffer
A
b) + co-solvent
e.g., 10% DMSO
1.0
E2
Red- shift
Longer λ
0
260
280
Wavelength (nm)
300
E1
Blue-shift to
Shorter λ
Difference spectroscopy- small changes
ΔA
Using trp in aqueous buffer
as reference, i.e., 100 % T
0.1
0.05
0
-0.05
-0.1
260
280
Wavelength (nm)
300
Split-beam spectrophotometer
mirror
Reference
Trp in buffer
Beam splitter
Sample
Light source
Trp + DMSO
monochromator
detectors
Split- beam or split cuvette
In the two compartments (1 and 2)we have two proteins A and B
that we suppose form a complex,
A+B
AB
Place equal volumes of the two protein solutions in the two sides,
Measure this as reference, i.e., 100 %T, then mix and record again,
The difference spectrum is generated
1 2
Light source
sample
monochromator
detector
Ligand binding
Cyanide binding to the respiratory enzyme- cytochrome c oxidase
What we know,
cyanide is a potent poison of respiration
CN inhibition to respiring mitochondria is instantaneous
Blocks electron transfer to O2
S
[O2]
CN
M
Time (min)
Cyanide reaction with cytochrome c oxidase
A
ACN=εCNcl
1
A=εcl
0.5
A 430 nm
E-CN
E
+CN
0
400 420 440 460
Wavelength (nm)
0
1 10 100
Time
1000
Time-resolved spectroscopy
Time (m)
1000
ΔA
1) Isosbestic points
700
0.4
400
0.2
2) E + CN
100
1
0
E-CN
3) Reactivity of the enzyme
in vitro is different from
enzyme in vivo
4) Oxidase exists in multiple states
-0.2
-0.4
400
420
440
Wavelength (nm)
460