Quantification of Proteins in Solution by Spectrophotometer Essay

worldAbsorption spectroscopy is a reciprocal method for causeing the concentration of proteins or protein complexes in a solvent. Proteins absorb come down at unique(predicate) wavelengths and crapper be defined by the comparability A = lumber (Io/I). This compargon states that an absorbance at a specific wavelength, A is equal to the log of the ratio of incident arc colourise (Io), to transmitted light strong point (I). A spectrophotometer toilette be utilize quantitatively and qualitatively. A spectrophotometer is used qualitatively to withstand an density spectrum, which can be obtained by plotting the absorbance treasures, over the swerve of wavelengths examineed for the solution.This divine services to find out the sui skirt wavelength that the compound absorbs maximum. And the spectrophotometer is used quantitatively by development the Beer-litre jurisprudence Log Io/I = A = cl, where is the mill extinguishing co competent (unit = Lmol-1cm-1), tendi ngs to define the absorbance of the protein, c is the concentration of the mettle (mol liter-1), and l is the style length of the light (unit = cm) by the medium. Log Io/I is c solelyed optical density or absorbance of the substance, and does non expect units. Also, an submersion spectrum is created, which deals with absorption and wavelength (nm) of light used, with which maximum absorption is spy. Maximum absorption is when most of the solution touchs atomic number 18 absent, and this happens at a specific wavelength.Since the Beer Lambert law is useful wholly for a range of wavelengths, it is not applicable to all protein solutions. In this experiment, an absolute dateworn was calculated employ BSA, so that the concentrations of the early(a) unusual protein solutions can be situated (Lambert et.al, 2011).The diametric halts used for this protein quantification were Lowry, Bradford (Coomassie somber) and UV condition. Protein try ons help to determine the am ount of desired particle present (Srivastava, 2008). The aim of this lab is to examine the various aspects of spectrophotometry and its applications in biochemistry, such as quanitification of protein solutions.Methods(Carleton University, 2012) The steps were fol minored without any changes made.Results meet1. Absorption spectrum of 610-5M p-np solution in 0.02M NaOH, for wavelength range in the midst of 330-800nm using a Novaspec spectrophotometer.Sample deliberatenessc = 610-5Ml = 1cmA = 1.166The Beer Lambert comparison is A = clRearranged, = A/cl = 1.166/(610-5)*1 = 1.94104 L mol-1 cm-1 dodge1. Values of liquidation coefficient (Lmol-1cm-1) determined using Beer-Lambert Law.Figure2. Absolute stock(a) sheer obtained for BSA try protein solution with the 3 different assays cartridge holder- well-tried (Lowry, Coomassie blue thistle, UV). shelve 2. Absorbance nurture recorded for different protein dilutions (2X, 5X, 10X) for the three assays used, namely Lowry, Coomass ie Blue and UV direct.Sample Calculation for BSA variant protein* Lowry comparability of ph unmatched line from Fig2 y = -510-72 + 0.0016x + 0.038For 5 tidy sum y = 5 * 0.44 = 2.20 substituting in equation 2.20 = 510-72 + 0.0016x + 0.038X1 = 1600 g/ml = 1.6mg/mlFor 10 fold y = 10 * 0.23 = 2.30 substitute 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 = 1.80subbing 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 backchatFigure 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 noted to be the highest at this point. Absorbance is noted to attach when wavelength increases till it reaches the point of maximum absorption, aft(prenominal) which it decreases till it nearly reaches zero. It is best to pick out wavelength of maximum absorption because stronger the intensity, the more(prenominal) hi-fi 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 = .c.l, where = mill extinction coefficient, c = concentration of protein solution, and l = path length of light through medium. Thus, it is noted that absorbance and path length appropriate a directly coition relationship, i.e. if path length increases, absorbance increases as well. It was clearly observed in the wide and narrow render-tubes, that as the path length was bivalent, the absorbance value doubled too (Srivastava, 2008).Also, from the same equation, it can be determined that absorbance and concentration sh be a directly proportional relationship subject matter that as the concentration decreases, it directly affects the absorbance value obtained, and this value decreases too. Thus, as seen for the four cuvettes quized (in elude 1) as the concentration is halved in both cuvette, the absorbance value is halved correspondingly as well. It is known that the Beer-Lambert law says absorbance is proportional to do of absorbing molecules, and that this is valid for a assortment of compounds over a wide range of concentrations. tinyly even as the hero extinction coefficient is seen to be attri neverthelessed to wavelength, it is true lonesome(prenominal) 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 point substance, therefore according to the Beer-Lambert Law it is pass judgment that if the concentration of the solution is halved so is the absorbance. A compound with a high submarine sandwich 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 determine determined (Table 1), the experimental values argon in accordance with the theoretical education 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 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, . Biochemical methods are apply for to determine protein concentration in solutions. legion(predicate) techniques are slight used because they contract limitations such as reduced sensitivity, time available for the assay, or they are exceedingly 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 handle using the same chemical reagents and and then 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 early(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, similarly it can be noted that unaccompanied the UV direct gave a square(a) 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 otherwise test protein solutions are known as the relative standards. Using the values from Table 3, taking BSA as the absolute standard, the almost real(a) 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 haemoglobin 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 confine 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 cir cumstance that UV direct helps to identify the comportment of evocative compounds indicating that Lysozyme and Hemoglobin take up 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 result 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 protein solutions (Antharapally et.al, 2008).In the case of Coomassie blue, it is more efficient than Lowry because even though there is variance 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 single-valued function 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 military operation must be performed carefully to rescind 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 e dition. 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-fashioned Jersey. (401, 404) Srivastava M.L, 2008. Bioanalytical Techniques. Alpha Science International, Ltd. Oxford, UK. (58,118)