SpectrophotometryReading:

Ninfa & Ballou, Ch. 3

Quantification of Protein ConcentrationReading: Ninfa & Ballou, Ch. 4

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

Spectrophotometric techniques …

monitor the interaction of light with matter.

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/λ or E = hν,

where h is Planck’s constant, c is the speed of light, λ is the wavelength and ν is frequency

R O Y G. B I V

Absorption and Emission of Light

From: Molecular Probes

R

O

Y

G

B

V

I

700 nm

400 nm

Electromagnetic Spectrum Relevant to Physical Biochemistry

Table 3-1 (Ninfa & Ballou)

Table 3-1

Electromagnetic Spectrum Relevant to Physical Biochemistry – Another View

-from Physical Biochemistry: Applications to Biochemistry and Molecular Biology by D. Freifelder, Freeman (1976)

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 mechanicalmodel” to describe the absorbance of light by conjugated molecules

Basic Spectrophotometer

Tungsten lamp (340-650 nm) -- visibleDeuterium lamp (200-360 nm) -- UV

-from Biophysical Chemistry by C. Cantor and P. Schimmel, Freeman (1980)

Absorbance Measurement

Principle:• want to relate I0 and I to the amount of absorber in the sample

log I0/I = – log I/I0 = εcl = A = Absorbance

l = path length of sample cuvette (cm)c = molar concentration of absorberε = molar extinction coefficient or molar absorptivity (M–1 cm–1)

What are I and Io?

Beer-Lambert Law

A = εcl

A is the absorbance at a particular wavelength.ε 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!

Applications of UV-Vis Spectroscopy

• Cell density

• Means of identification

• Monitor structural or chemical transitions

• Determination of concentration

• Measurement of enzyme activities

Cell Density

-from Physical Biochemistry: Applications to Biochemistry and Molecular Biology by D. Freifelder, Freeman (1976)

Absorption Spectrum for ATP

ε for ATP atpH 7 and 260 nmis 15,400 M−1 cm−1

If A260 is 1.2, what is the concentration of ATP in a 1 cm cuvette?

If 20 μL of a stock solution of ATP had been combined with 980 μL 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

⇒Very Important: What is generally considered to be thelinear range of the spectrophotometer?

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.

pH 7.0

pH 1.0

pH 13.0 Fig. V-9 (Switzer & Garrity)

UV Characteristics of dsDNA and ssRNA

Absorbance Spectra for DNA and RNAas a Function of Temperature

-from Biochemistry: A Problems Approach by W. Wood, J. Wilson, R. Benbow, and L. Hood, Benjamin Cummings (1981)

The shift in absorbance above the melting temperature (Tm) is called the hyperchromic effect

Absorption spectra of NADH and NAD+

from Lehninger’s Biochemistry by Michael M. Cox and David L. Nelson, W.H. Freeman, 2005

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

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• λmax = 550 nm for complex

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 λmax ≈ 750 nm

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 λmax ≈ 562 nm

Bradford Assay

Figure 3-3 Ninfa & Ballou

Coomassie Brilliant Blue G-250

How to estimate the molar extinction coefficient (ε) of a protein:

ε280nm [M−1 cm−1] = 5500⋅nTrp + 1490⋅nTyr + 125⋅nCystines

Need sequence information to use this method, andif 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

Using A280 readings to determine the concentrationof a protein…but first we have to get a good estimateof the molar extinction coefficient of the protein. So…

Oftentimes in the literature one might see thefollowing:

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.

To two significant figures, calculate for the above protein. The molecular weight of the protein is 19,167.

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 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

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 ⇒ M2 = M1V1/V2 ⇒ 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