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 6×10-5M p-np solution in 0.02M NaOH, for wavelength range between 330-800nm using a Novaspec spectrophotometer.
c = 6×10-5M
l = 1cm
A = 1.166
The Beer Lambert equation is A = Ɛcl
Rearranged, Ɛ = A/cl
Ɛ = 1.166/(6×10-5)*1
Ɛ = 1.94×104 L mol-1 cm-1
Table1. Values of extinction coefficient (Lmol-1cm-1) determined using Beer-Lambert Law.
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.
Sample Calculation for BSA stock protein:
Equation of line from Fig2; y = -5×10-7×2 + 0.0016x + 0.038
For 5 fold; y = 5 * 0.44 = 2.20
Substituting in equation; 2.20 = 5×10-7×2 + 0.0016x + 0.038
X1 = 1600 µg/ml = 1.6mg/ml
For 10 fold; y = 10 * 0.23 = 2.30
Substituting in equation; 2.30 = 5×10-7×2 + 0.0016x + 0.038
X2 = 1600 µg/ml = 1.6mg/ml
(X1 + x2)/2 = 1.6mg/ml
* Coomassie Blue
Equation of line from fig2; y = -7×10-7×2 + 0.002x + 0.0219
For 5 fold; y = 5 * 0.36 = 1.80
Substituting in equation; 1.80 = -7×10-7×2 + 0.002x + 0.0219 X1 = 1428.57 µg/ml = 1.4mg/ml
For 10 fold; y = 10 * 0.20 = 2.00
Substituting in equation; 2.00 = -7×10-7×2 + 0.002x + 0.0219 X2 = 1428.57 µg/ml = 1.4mg/ml
(x1+ x2)/2 = 1.4mg/ml
* UV direct
Equation of line from fig 2; y = 0.0006x + 0.0175
For 2 fold; y = 2 * 0.42 = 0.84
Substituting in equation; 0.84 = 0.0006x + 0.0175
X1 = 1374.16 µg/ml = 1.4mg/ml
For 5 fold; y = 5 * 0.15 = 0.75
Substituting in equation; 0.75 = 0.0006x + 0.0175
X2 = 1179.16 µg/ml = 1.2mg/ml
(x1 + x2)/2 = 1.3mg/ml
Figure 1 shows the absorption spectrum of stock solution (6×10-5M), p-nitrophenol and 0.02M NaOH, and from the graph it can be inferred that 400nm is the wavelength of maximum absorption because absorption is noted to be the highest at this point. Absorbance is noted to increase when wavelength increases till it reaches the point of maximum absorption, after which it decreases till it nearly reaches zero. It is best to consider wavelength of maximum absorption because stronger the intensity, the more accurate will be the readings for absorbance. As seen from table 1, the path lengths remain the same as the cuvettes used were of the same size. The Beer-Lambert Law states that Abs = Ɛ.c.l, where Ɛ = molar extinction coefficient, c = concentration of protein solution, and l = path length of light through medium. Thus, it is noted that absorbance and path length share a directly proportional relationship, i.e. if path length increases, absorbance increases as well. It was clearly observed in the wide and narrow test-tubes, that as the path length was doubled, the absorbance value doubled too (Srivastava, 2008).
Also, from the same equation, it can be determined that absorbance and concentration share a directly proportional relationship meaning that as the concentration decreases, it directly affects the absorbance value obtained, and this value decreases too. Thus, as seen for the four cuvettes tested (in Table 1) as the concentration is halved in every cuvette, the absorbance value is halved correspondingly as well. It is known that the Beer-Lambert law says absorbance is proportional to number of absorbing molecules, and that this is valid for a variety of compounds over a wide range of concentrations.
But even as the molar extinction coefficient is seen to be attributed to wavelength, it is true only for monochromatic light (Lambert et.al, 2011). The relationship can be stated as “Ɛ is a measure of the amount of light absorbed per unit concentration”. Molar extinction coefficient is a constant for a particular substance, therefore according to the Beer-Lambert Law it is expected that if the concentration of the solution is halved so is the absorbance. A compound with a high molar extinction is very effective at absorbing light (of the appropriate wavelength), and hence low concentrations of a compound with a high molar extinction can be easily detected.
In the values determined (Table 1), the experimental values are in accordance with the theoretical statement except for one cuvette. The cuvette no.3 with Ɛ = 1.8×10-4 L mol-1cm-1 does not agree with the trend. Thus it can be deduced that due to experimental error, the Ɛ value is inaccurate. Also, from the equation it is understood that Ɛ and path-length are inversely proportional as well (i.e. Ɛ = Abs/cl) that means that as path-length increases, Ɛ decreases, assuming that the concentration is kept constant. But the experimental values do not agree with this statement, because it is seen that as the path-length increases so does the molar extinction coefficient, Ɛ. Biochemical methods are applied for to determine protein concentration in solutions. Many techniques are less used because they have limitations such as reduced sensitivity, time available for the assay, or they are highly specific about the amino acids in the protein solution being tested. But for every protein, the component amino acids are different, so there is no single assay that can be used for quantification of all proteins.
The absorbance assays use the method of testing the intensity of the color produced by the protein solutions when chemical reagents are added to it. A “standard protein” whose concentration is known, is treated using the same chemical reagents and thus an absolute standard curve is obtained (Boyer, 2000). In this experiment, the standard used was Bovine Serum Albumin (BSA). Development of color is significantly better in BSA than any other protein, and this makes it one of the most preferred test solutions for quantification of proteins (Antharavally et.al, 2008). Hence figure 2 is obtained by performing the three suitable assays on BSA to produce a standard curve, also it can be noted that only the UV direct gave a straight line passing through zero, whereas the Lowry and Coomassie Blue gave curved lines, passing through zero. Table 2 shows the absorbance values recorded, for different dilutions of the test protein in three different assays.
With the help of the values obtained in Table 2, and with the equations obtained from Figure 2, the concentration of protein (mg/ml) was calculated and presented in Table 3. Since all the values in Table 3 were deduced from the equation of standard curve BSA, it is considered as the absolute standard, and the other test protein solutions are known as the relative standards. Using the values from Table 3, taking BSA as the absolute standard, the almost actual concentration of the protein (mg/ml) can be concluded, and they are 1.6 (mg/ml) for Lowry assay, 1.4 (mg/ml) for Coomassie Blue and 1.3 (mg/ml) for UV direct. For Lowry assay, the concentration value for all test proteins was 1.6 mg/ml, which must mean that the value obtained is accurate. For Coomassie blue, BSA and Hemoglobin were the same (1.4mg/ml), Ovalbumin and Lysozyme had similar values of 1.9mg/ml, and 1.8mg/ml respectively, whereas Gamma globulin showed 2.5mg/ml.
The value for Gamma globulin is off because of experimental error, of spilling some of the contents from the cuvette while transferring it to the spectrophotometer for calibration. For UV direct, BSA and Ovalbumin have similar readings (1.3mg/ml and 1.5mg/ml respectively), Gamma globulin is 2.5mg/ml, but Lysozyme is 5.9mg/ml and Hemoglobin is 3.8mg/ml. The reason for this could be due to the fact that UV direct helps to identify the presence of aromatic compounds indicating that Lysozyme and Hemoglobin contain aromatic compounds present in them. The Lowry protein assay is the most common and one of the more sensitive, but it is time consuming, on the other hand Coomassie blue (the Bradford assay) is much more sensitive as compared to Lowry, and requires less time too.
They both show change of color with proteins. As for UV direct method, it is one of the faster methods too, and it is helpful to identify aromatic compounds because aromatic residues absorb 280nm light (Boyer, 2000). The Lowry procedure can detect protein levels as low as 5µg (Boyer, 2000). It depends on the color development by the reagent Folin-Ciocalteu. Peptide bonds are formed under alkaline Cu2+ conditions and reduced from Folin-Ciocalteu phosphomolybdate-phosphotungsten by aromatic amino acids (tyrosine and tryptophan) to heteropolymolybdenum blue. The standard curve obtained with BSA helps to determine concentration of unknown protein solutions (Antharapally et.al, 2008).
In the case of Coomassie blue, it is more efficient than Lowry because even though there is variation with different proteins, there is very less interference by non protein components (Borley, 2000). Therefore, according to literature, Coomassie Blue is the most preferred protein assay but this contrasts the experimental inferences, because through experimental procedure it was seen that Lowry method gave the most accurate and precise results. With this experiment, the method to quantify unknown protein concentrations has been understood. Also, that this process must be performed carefully to avoid irrational experimental errors.
Antharavally B.S, Bell P.A, Haney P, Mallia K.A, Rangaraj P. 2008. Quantitation of proteins using a dye–metal-based colorimetric protein assay. Analytical Biochemistry. 385; 342-245. Boyer R, 2000. Modern Experimental Biochemistry, third edition. Addison-Wesley Longman, Inc. USA. (41-45). Lambert J.B, Gronert S, Lightner D.A, Shurvell H.F, 2011. Organic Structural Spectroscopy, second edition. Pearson Education, Inc, New Jersey. (401, 404) Srivastava M.L, 2008. Bioanalytical Techniques. Alpha Science International, Ltd. Oxford, UK. (58,118)