build a spectrophotometer Light source sample detector Measure I o 100 T Measure I T of sample Calculate A Change the monchromator to measure A versus wavelength Light source sample ID: 621179
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Slide1
Let’s build a spectrophotometer
Light source
sample
detector
Measure I
o
(100% T)
Measure I (T of sample)
Calculate ASlide2
Change the monchromator to measureA versus wavelength
Light source
sample
monochromator
detector
A
λ
(nm)
Scanning an absorbance spectrum
Resolution will be defined by dispersion
and slit width,
bandpass
≤ 2nm is
sufficientSlide3
Light source
sample
Measure all wavelengths at once
monochromator
Detector array
Diode array spectrometer
Sample receives broad irradiation (i.e., white light)
Dispersion element comes after the sample
Project spectrum onto an array of detectorsSlide4
1) Concentration Proteins
DNA2) Environmental
effects Effect of solvent
pH
Ligand or protein interactions3) Time – resolved measurements Measure time dependent changes TimescalesSome applications of spectrophotometrySlide5
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+ + AH2We 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) = 6.23Slide6
Measuring concentration (contd
)
A > 6 means % T < 0.0001 % , 99.9999 % of I
o
absorbedMost spectrophotometers are only linear up to A= 3 Stray lightHow can we fix the problem?Dilute the NADH. What about the pathlength?Measure off the peak.
A
[x] (M)
3
2
1Slide7
The spectrum of NADH
220 300 400
Wavelength (nm)
2.0
1.0
0
A
20
10
0
ε
(mM
-1
cm
-1
)
NAD
+
NADHSlide8
Environmental effects-Solvent perturbation
Trp
in aqueous buffer
b) + co-solvent
e.g., 10% DMSO260 280 300
2.0
1.0
0
A
Wavelength (nm)
E
1
E
2
Blue-shift to
Shorter
λ
Red- shift
Longer
λSlide9
Difference spectroscopy- small changes
Using
trp
in aqueous buffer
as reference, i.e., 100 % T260 280 300Wavelength (nm)
0.1
0
-0.1
Δ
A
0.05
-0.05Slide10
Sample
Trp
+ DMSO
Light source
monochromator
detectors
Reference
Trp
in buffer
Split-beam spectrophotometer
Beam splitter
mirrorSlide11
Light source
sample
monochromator
detector
Split- beam or split
cuvette
1
2
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 generatedSlide12
Ligand
binding
Cyanide binding to the respiratory enzyme-
cytochrome
c oxidaseWhat we know, cyanide is a potent poison of respiration CN inhibition to respiring mitochondria is instantaneous Blocks electron transfer to O2
[O
2
]
Time (min)
M
S
CNSlide13
Cyanide reaction with
cytochrome
c oxidase
A
Wavelength (nm)
1
0.5
0
400 420 440 460
A=
ε
cl
A
CN
=
ε
CN
cl
E
E-CN
A
430 nm
Time
+CN
0 1 10 100 1000Slide14
0.4
0.2
0
-0.2
-0.4
Δ
A
400 420 440 460
Wavelength (nm)
Time (m)
1000
700
400
100
1
Time-resolved spectroscopy
Isosbestic
points
E + CN
E-CN
3) Reactivity of the enzyme
in vitro
is different from
enzyme
in vivo
4)
Oxidase
exists in multiple states