Quantification of Proteins in Solution by Spectrophotometer
Lab bench# 1
Absorption spectroscopy is a common method for finding the concentration of proteins or protein complexes in a solution. Proteins absorb light at specific wavelengths and can be defined by the equation A = log (Io/I). This equation states that an absorbance at a specific wavelength, A is equal to the log of the ratio of incident light intensity (Io), to transmitted light intensity (I). A spectrophotometer can be used quantitatively and qualitatively. A spectrophotometer is used qualitatively to obtain an absorption spectrum, which can be obtained by plotting the absorbance values, over the range of wavelengths tested for the solution. This helps to find out the suitable wavelength that the compound absorbs maximum. And the spectrophotometer is used quantitatively by using the Beer-Lambert Law; Log [ Io/I] = A = ξcl, where ξ is the molar extinction coefficient (unit = Lmol-1cm-1), helps to define the absorbance of the protein, c is the concentration of the substance (mol liter-1), and l is the path length of the light (unit = cm) through the medium. Log [Io/I] is called optical density or absorbance of the substance, and does not have units. Also, an absorption spectrum is created, which deals with absorption and wavelength (nm) of light used, with which “maximum absorption” is observed. Maximum absorption is when most of the solution particles are absorbed, and this happens at a specific wavelength. Since the Beer Lambert law is useful only for a range of wavelengths, it is not applicable to all protein solutions. In this experiment, an absolute standard was calculated using BSA, so that the concentrations of the other unknown protein solutions can be determined (Lambert et.al, 2011).The different assays used for this protein quantification were Lowry, Bradford (Coomassie Blue) and UV direct. Protein assays help to determine the amount of desired particle present (Srivastava, 2008). The aim of this lab is to understand the various aspects of spectrophotometry and its applications in biochemistry, such as quanitification of protein solutions.
(Carleton University, 2012) The steps were followed without any changes made.
Figure1. Absorption spectrum of 6x10-5M p-np solution in 0.02M NaOH, for wavelength range between 330-800nm using a Novaspec spectrophotometer.
c = 6x10-5M
l = 1cm
A = 1.166
The Beer Lambert equation is A = Ɛcl
Rearranged, Ɛ = A/cl
Ɛ = 1.166/(6x10-5)*1
Ɛ = 1.94x104 L mol-1 cm-1
Table1. Values of extinction coefficient (Lmol-1cm-1) determined using Beer-Lambert Law.
Cuvette/Test tube| Pathlength (cm)| Concentration (M)| Absorbance| Extinction Coeffienct (L mol-1 cm -1)| 1| 1| 6.0x10-5| 1.166| 1.9x104|
2| 1| 3.0x10-5| 0.614| 2.1x104|
3| 1| 1.5x10-5| 0.265| 1.8x104|
4| 1| 0.6x10-5| 0.176| 2.9x104|
Wide| 2| 3.0x10-5| 1.09| 1.8x104|
Narrow| 1| 3.0x10-5| 0.529| 1.7x104|
Figure2. Absolute standard curve obtained for BSA test protein solution with the 3 different assays tested (Lowry, Coomassie Blue, UV).
Table 2. Absorbance values recorded for different protein dilutions (2X, 5X, 10X) for the three assays used, namely Lowry, Coomassie Blue and UV direct.
| Absorbance recorded by using the following methods|
Test Protein| Lowry| Coomassie Blue| UV|
| 5 fold| 10 fold| 2 fold| 5 fold| 10 fold| 2 fold| 5 fold| 10 fold| BSA| 0.44| 0.23| NA| 0.36| 0.20| 0.42| 0.15| NA|
Lysozyme| 0.66| 0.40| 0.49| 0.35| NA| NA| 0.71| 0.36| Hemoglobin| 0.45| 0.26| NA| 0.37| 0.23| NA| 0.43| 0.25| Ovalbumin| 0.33| 0.27| 0.58| 0.25| NA| 0.45| 0.18| NA| Gamma globulins| 0.63| 0.32| 0.38| 0.11| NA| NA| 0.30| 0.14| *NA – Not tested.
Sample Calculation for BSA stock protein:
Equation of line from Fig2; y = -5x10-7x2 + 0.0016x +...