This document discusses spectrophotometry techniques for measuring light absorption by molecules. It covers the electromagnetic spectrum, Beer-Lambert law, applications of UV-vis spectroscopy like determining cell density and protein concentration, and methods for measuring absorbance of molecules like DNA, RNA, proteins, and other biological compounds. Key concepts explained include the relationship between absorbance, molar extinction coefficient, concentration, and path length in the Beer-Lambert law.
2. Objectives
• The electromagnetic spectrum
• The concept of transition between energy
levels
• The variety of spectroscopic techniques
available
• Applications of UV-Vis spectrophotometry
• Measurement of the concentration of a
solute in solution
4. The nature of light
• Light is a form of electromagnetic radiation
• Composed of electric and magnetic fields,
which are mutually perpendicular
• Energy content of the wave:
E = hc/l or E = hn,
where h is Planck’s constant, c is the speed of
light, l is the wavelength and n is frequency
10. Electromagnetic Spectrum Relevant to Physical
Biochemistry – Another View
-from Physical Biochemistry: Applications to Biochemistry and Molecular Biology by D. Freifelder, Freeman (1976)
11. Absorbance spectroscopy ...*
measures absorption of light in the
UV (200-340 nm) and visible (340-800 nm) ranges.
*Later, we will develop a crude “quantum mechanical
model” to describe the absorbance of light by
conjugated molecules
12. Basic Spectrophotometer
Tungsten lamp (340-650 nm) -- visible
Deuterium lamp (200-360 nm) -- UV
-from Biophysical Chemistry by C. Cantor and P. Schimmel, Freeman (1980)
13. Absorbance Measurement
Principle:
• want to relate I0 and I to the amount of absorber
in the sample
log I0/I = – log I/I0 = ecl = A = Absorbance
l = path length of sample cuvette (cm)
c = molar concentration of absorber
e = molar extinction coefficient or molar
absorptivity (M–1 cm–1)
What are I and Io?
14. Beer-Lambert Law
A = ecl
A is the absorbance at a particular wavelength.
e is the molar extinction coefficient (M–1 cm–1).
c is the concentration (M).
l is the path length of the sample cuvette (cm).
Note: A has no units!
Later in the class we will have more to say about
the above equation; for now, you need to know
it!
15. Applications of UV-Vis Spectroscopy
• Cell density
• Means of identification
• Monitor structural or chemical transitions
• Determination of concentration
• Measurement of enzyme activities
16. Cell Density
-from Physical Biochemistry: Applications to Biochemistry and Molecular Biology by D. Freifelder, Freeman (1976)
17. Absorption Spectrum for ATP
e for ATP at
pH 7 and 260 nm
is 15,400 M-1 cm-1
If A260 is 1.2, what
is the concentration
of ATP in a 1 cm
cuvette?
If 20 mL of a stock
solution of ATP had been combined with 980 mL of buffer to give
the above reading, what would be the millimolar concentration
of ATP in the stock solution?
http://www.bmglabtech.com/application-notes/absorbance/absorbance
-dna-quantitation-168.cfm
ÞVery Important: What is generally considered to be the
linear range of the spectrophotometer?
18. Two important and related concepts that you will
need to know and understand:
1) M1V1 = M2V2 or C1V1 = C2V2
2) If I combine 1 part of one solution and
4 parts of a second solution then I have
made a 1:5 dilution (1 part to 5 total parts)
and not a 1:4 dilution. It seems simple,
but it is easy to confuse when you are first
starting out in research.
19. UV Characteristics of dsDNA and ssRNA
pH 7.0
pH 1.0
pH 13.0
Fig. V-9 (Switzer & Garrity)
20. Absorbance Spectra for DNA and RNA
as a Function of Temperature
The shift in absorbance above the melting
temperature (Tm) is called the hyperchromic effect
-from Biochemistry: A Problems Approach by W. Wood, J. Wilson, R. Benbow, and L. Hood, Benjamin Cummings (1981)
21. Absorption spectra of NADH and NAD+
from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005
22. Methods for Determining Protein Concentration
• Chromogenic (color-forming) assays
- Biuret method
- Lowry Assay (Folin-Ciocalteu reagent)
- BCA method
- Bradford Assay (Coomassie Blue)*
• UV Absorbance at 280 nm*
*what you will do in lab
23. Biuret Method
Cupric complex
Figure 3-1 Ninfa & Ballou
• formation of a Cu(II) complex with 4 N atoms
from polypeptide backbone
• characteristic blue-purple color
• lmax = 550 nm for complex
24. Lowry Assay
• Biuret reaction followed by reduction of Folin-
Ciocalteu reagent, which consists of
phosphomolybdotungstate mixed acids:
(H2O)3(P2O5)(WO3)13(MoO3)5(H2O)10
characteristic blue-purple color development at
lmax ≈ 750 nm
25. BCA Method
• similar to Lowry except bicinchoninic acid (BCA)
is used in place of the Folin-Ciocalteu reagent
• purple-colored BCA-Cu(I)-protein complex has
lmax ≈ 562 nm
27. Using A280 readings to determine the concentration
of a protein…but first we have to get a good estimate
of the molar extinction coefficient of the protein. So…
How to estimate the molar extinction coefficient (e) of
a protein:
e280nm [M-1 cm-1] = 5500×nTrp + 1490×nTyr + 125×nCystines
Need sequence information to use this method, and
if you know if the protein has disulfides, the estimate
improves. (We will talk more about this in class.)
This empirical equation is taken from: Pace et al.
(1995) Protein Science 4, 2411
28. Oftentimes in the literature one might see the
following:
or
The first notation indicates that a 1% (w/v) solution
(i.e., 1 g/100 mL) would give an A280 of 4.1.
The second notation indicates that a 1 mg/mL
(or 1 g/L) would give an A280 of 0.74.
29. To five significant figures, calculate the extinction
coefficient for a protein that has 2 Trp, 3 Tyr, and
1 Cys. Assume the protein is cytosolic.
To two significant figures, calculate for the
above protein. The molecular weight of the protein
is 19,167.
30. To three significant figures, how many mg of xylitol
(Mr = 152) would you need to make 75.0 mL of a
35.0 mM solution?
___ mg = 1000 mg × 152 g × 0.035 mol × 0.075 L = 399mg
1 g mol L
31. A protein solution is prepared by dissolving 40.0 mg
of protein in 10.0 mL of water. A 0.500 mL sample of
this solution is diluted to a volume of 20.0 mL. To
one significant figure, how many mg of protein will
be in a 2.00 mL sample of the diluted solution?
M1V1 = M2V2 Þ M2 = M1V1/V2 Þ M2(2 mL) = __mg
M2 = (4.00 mg/mL)(0.500 mL) = 0.1 mg/mL
(20.0 mL)
(0.1 mg/mL)(2 mL) = 0.2 mg protein
