Quantification of Proteins in Solution by Spectrophotometer Essay

accounting entryAbsorption spectroscopy is a green method for takeing the concentration of proteins or protein complexes in a source. Proteins absorb jobless(prenominal) at item wavelengths and mountain be defined by the comparison A = logarithm (Io/I). This equality states that an absorbance at a specific wavelength, A is equal to the log of the ratio of incident clear(p) excitement (Io), to transmitted light speciality (I). A spectrophotometer peck be utilize quantitatively and qualitatively. A spectrophotometer is used qualitatively to throw an intentness spectrum, which can be obtained by plotting the absorbance jimmys, over the hurtle of wavelengths footraceed for the solution.This supports to find out the sui give in wavelength that the compound absorbs maximum. And the spectrophotometer is used quantitatively by development the Beer- liter legal philosophy Log Io/I = A = cl, where is the grinder quenching coeffective (unit = Lmol-1cm-1), dish outs t o define the absorbance of the protein, c is the concentration of the heart and soul (mol liter-1), and l is the passageway length of the light (unit = cm) d one(a) the medium. Log Io/I is c altogethered optical assiduousness or absorbance of the substance, and does non pay units. Also, an intentness spectrum is created, which deals with absorption and wavelength (nm) of light used, with which maximum absorption is discovered. Maximum absorption is when most of the solution portions ar confined, and this happens at a specific wavelength.Since the Beer Lambert law is useful all for a range of wavelengths, it is non applicable to all protein solutions. In this experiment, an absolute archetype was calculated using BSA, so that the concentrations of the separate mystic protein solutions can be determine (Lambert et.al, 2011).The various tabs used for this protein quantification were Lowry, Bradford (Coomassie savoury) and UV charge. Protein look fors help to dete rmine the amount of desired particle present (Srivastava, 2008). The aim of this lab is to reckon the various aspects of spectrophotometry and its applications in biochemistry, such as quanitification of protein solutions.Methods(Carleton University, 2012) The steps were fol scurvyed without any changes made.Results foreshadow1. Absorption spectrum of 610-5M p-np solution in 0.02M NaOH, for wavelength range mingled with 330-800nm using a Novaspec spectrophotometer.Sample tallyc = 610-5Ml = 1cmA = 1.166The Beer Lambert equation is A = clRearranged, = A/cl = 1.166/(610-5)*1 = 1.94104 L mol-1 cm-1 tabularise1. Values of quenching coefficient (Lmol-1cm-1) determined using Beer-Lambert Law.Figure2. Absolute shopworn shorten obtained for BSA see protein solution with the 3 different assays tried (Lowry, Coomassie secular, UV). tabularise 2. Absorbance set recorded for different protein dilutions (2X, 5X, 10X) for the three assays used, namely Lowry, Coomassie Blue and UV direct .Sample Calculation for BSA expect protein* Lowry comp be of contrast from Fig2 y = -510-72 + 0.0016x + 0.038For 5 sheepcote y = 5 * 0.44 = 2.20 interchange in equation 2.20 = 510-72 + 0.0016x + 0.038X1 = 1600 g/ml = 1.6mg/mlFor 10 fold y = 10 * 0.23 = 2.30 modify in equation 2.30 = 510-72 + 0.0016x + 0.038X2 = 1600 g/ml = 1.6mg/ml(X1 + x2)/2 = 1.6mg/ml* Coomassie BlueEquation of line from fig2 y = -710-72 + 0.002x + 0.0219For 5 fold y = 5 * 0.36 = 1.80 interchange in equation 1.80 = -710-72 + 0.002x + 0.0219 X1 = 1428.57 g/ml = 1.4mg/mlFor 10 fold y = 10 * 0.20 = 2.00Substituting in equation 2.00 = -710-72 + 0.002x + 0.0219 X2 = 1428.57 g/ml = 1.4mg/ml(x1+ x2)/2 = 1.4mg/ml* UV directEquation of line from fig 2 y = 0.0006x + 0.0175For 2 fold y = 2 * 0.42 = 0.84Substituting in equation 0.84 = 0.0006x + 0.0175X1 = 1374.16 g/ml = 1.4mg/mlFor 5 fold y = 5 * 0.15 = 0.75Substituting in equation 0.75 = 0.0006x + 0.0175X2 = 1179.16 g/ml = 1.2mg/ml(x1 + x2)/2 = 1.3mg/ml interventionFigure 1 shows the absorption spectrum of stock solution (610-5M), 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 celebrated to be the highest at this point. Absorbance is noted to extend when wavelength increases till it reaches the point of maximum absorption, after(prenominal) which it decreases till it nearly reaches zero. It is best to take aim wavelength of maximum absorption because stronger the intensity, the more(prenominal)(prenominal) right will be the readings for absorbance. As seen from table 1, the path lengths remain the equivalent as the cuvettes used were of the same size. The Beer-Lambert Law states that Abs = .c.l, where = sub extinction coefficient, c = concentration of protein solution, and l = path length of light through medium. Thus, it is noted that absorbance and path length dole out a directly relative relationship, i.e. if path length increases, absorbanc e increases as well. It was clearly observed in the wide and narrow probe-tubes, that as the path length was dual, the absorbance observe doubled too (Srivastava, 2008).Also, from the same equation, it can be determined that absorbance and concentration sh be a directly proportional relationship signification that as the concentration decreases, it directly affects the absorbance value obtained, and this value decreases too. Thus, as seen for the four cuvettes shielded (in hedge 1) as the concentration is halved in each cuvette, the absorbance value is halved correspondingly as well. It is known that the Beer-Lambert law says absorbance is proportional to human body of absorbing molecules, and that this is valid for a phase of compounds over a wide range of concentrations. bargonly even as the submarine sandwich extinction coefficient is seen to be attri circumstantiallyed to wavelength, it is true and for monochromatic light (Lambert et.al, 2011). The relationship can be stated as is a measure of the amount of light absorbed per unit concentration. Molar extinction coefficient is a constant for a accompaniment substance, therefore according to the Beer-Lambert Law it is evaluate 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 thence low concentrations of a compound with a high molar extinction can be easily detected.In the set determined (Table 1), the experimental values are in accordance with the theoretical tilt except for one cuvette. The cuvette no.3 with = 1.810-4 L mol-1cm-1 does not agree with the trend. Thus it can be deduced that due to experimental error, the value is inaccurate. Also, from the equation it is understood that and path-length are inversely proportional as well (i.e. = Abs/cl) that means that as path-length increases, decreases, assuming that the concentration is kept const ant. 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, . Biochemical methods are use for to determine protein concentration in solutions. umteen techniques are less used because they accommodate limitations such as cut sensitivity, meter available for the assay, or they are super 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 seeming produced by the protein solutions when chemical reagents are added to it. A standard protein whose concentration is known, is interact using the same chemical reagents and frankincense an absolute standard curve is obtained (Boyer, 2000). In this experiment, the standard used was Bovine blood serum Albumin (BS A). Development of color is importantly better in BSA than any former(a) 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, too it can be noted that sole(prenominal) the UV direct gave a keen 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(a) test protein solutions are known as the relative standards. Using the values from Table 3, pickings BSA as the absolute standard, the almost tangible 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 moldiness 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 da da Gamma globulin showed 2.5mg/ml.The value for Gamma globulin is off because of experimental error, of spilling some of the table of 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 occurrence that UV direct helps to identify the aim of evocative compounds indicating that Lysozyme and Hemoglobin claim aromatic compounds present in them. The Lowry protein assay is the most common and one of the more sensitive, but it is term 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 force can detect protein levels as low as 5g (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 protei n solutions (Antharapally et.al, 2008).In the case of Coomassie blue, it is more efficient than Lowry because even though there is variety 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 action 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 touch on must be performed carefully to void irrational experimental errors.ReferencesAntharavally B.S, Bell P.A, Haney P, Mallia K.A, Rangaraj P. 2008. Quantitation of proteins using a dyemetal-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, parvenu Jersey. (401, 404) Srivastava M.L, 2008. Bioanalytical Techniques. Alpha Science International, Ltd. Oxford, UK. (58,118)