UNIVERSITI PENDIDIKAN SULTAN IDRIS 35900 TANJONG MALIM, PERAK DARUL RIDZUAN FAKULTI : SAINS DAN TEKNOLOGI PRINCIPLE IN BIOCHEMISTRY (SBK3013) EXPERIMENT 2: PROTEIN ANALYSIS PREPARED BY :UMI ABIBAH BT SULAIMAN D20091034811SITI RAHAYU BT MOHAMED NOOR D20091034855AZMA AMIRA MOHAMAD D20091034859NUR AFIQAH BT MUHAMAD APANDI D20091034872AMEERA BT YAHYA D20091034814 SEMESTER 8 SESI 2009/2010 LAB SESSION THURSDAY(10.30 A.M. - 1.30 P.M.) LAB INSTRUCTOR DR ROSMILA MISNAN
PROTEIN EXPERIMENTExperiment 2: The determination of three protein samples using Biuret and Lowry Assay.Objective:1. To learn the principles of protein assays.2. To determine protein concentrations of protein samples using the Biuret Protein Assay and Lowry assay.Introduction: The determination of protein concentration is an essential technique in all aspects ofprotein studies.This lab activity is designed to teach students the principles behind a commonprotein estimation assay known as the BiuretProtein Assay and Lowry ProteinAssay.Although there are a wide variety of protein assays available none of the assays can beused without first consideringtheir suitability for the application. Each method has its ownadvantages and limitations and often it is necessary to obtainmore than one type of proteinassay for research applications. In the copper ion based protein assays, protein solutions are mixed with an alkalinesolution of copper salt, cupricions (Cu2+). The protein assay is based on the interaction ofcupric ions with protein in an alkaline solution and is commonlyreferred to as the Biuretassay. The interaction of cupric ions (Cu2+) with protein results in a purple color that can beread at 540nm. The amount of color produced is proportional to protein concentration. The Lowry protein assay method for protein concentration determination is one of themost venerable and widely-used protein assays. Hydrolysis is probably the most accuratemethod of determining protein concentration followed by amino acid analysis. Most othermethods are sensitive to the amino acid composition of the protein, and absoluteconcentrations cannot be obtained. The Lowry procedure is sensitive, and is moderatelyconstant from protein to protein. The Lowry protein estimation has been so widely used thatit is a completely acceptable alternative to a rigorous absolute determination in almost allcircumstances in which protein mixtures or crude extracts are involved.
Result:Biuret Assay Table 1.1: Biuret assay Concentration (mg/ml) Absorbance (nm) Blank 0.00 1 0.138 2 0.139 3 0.190 4 0.192 5 0.194 6 0.211 Table 1.2: Determination using Biuret assay Samples Absorbance Dilution Absorbance Concentration before after dilution (mg/ml) dilution (nm) (nm)Chicken egg 0.936 1mL of sample + 9 mL 0.106 2.0 x 10=20 of distilled water Quail egg 0.870 1mL of sample + 9 mL 0.090 1.6 x 10= 16 of distilled water Duck egg 0.764 1mL of sample + 9 mL 0.066 1.0 x 10= 10 of distilled waterChicken egg 1mL of sample + 9 mL 0.195 1.8 x 10= 18(ayam 0.230 of distilled waterkampung)Omega egg 0.256 1mL of sample + 9 mL 0.197 1.9 x 10= 19 of distilled water
Graph of absorbance value (nm) versus gelatin concentration (mg/ml) for Biuret assay 0.25 0.2absorbance value (nm) 0.15 Y-Values 0.1 0.05 0 0 1 2 3 4 5 6 7 Gelatin concentration (mg/ml)
Lowry Assay Table 2.1: Lowry assay Concentration Absorbance (nm) Blank 0.00 0.1mg/mL 0.053 0.2 mg/mL 0.133 0.3mg/mL 0.143 0.4 mg/mL 0.207 0.5mg/mL 0.185 0.6 mg/mL 0.166 Table 2.2: Determination using Lowry assay Samples Dilution Absorbance Concentration after dilution (mg/ml) (nm) Chicken egg 1mL of sample + 9 mL 0.072 0.16 x 10=1.6 of distilled water Quail egg 1mL of sample + 9 mL 0.134 0.3x 10= 3.0 of distilled water Duck egg 1mL of sample + 9 mL 0.185 0.56 x 10= 5.6 of distilled water Chicken egg 1mL of sample + 9 mL 0.173 0.41 x 10= 4.1 (ayam of distilled water kampung) Omega egg 1mL of sample + 9 mL 0.131 0.25 x 10= 2.5 of distilled water
Graph of absorbance value (nm) versus gelatin concentration (mg/ml) for Lowry assay 0.25 0.2Absorbance value (nm) 0.15 0.1 Y-Values 0.05 0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 Gelatin concerntration (mg/ml)
RESULT SUMMARY Table 3.1 Protein concentration obtained from Biuret assay and Lowry assay Type of sample Concentration (mg/ml) Biuret Assay Lowry Assay Chicken egg 20 1.6 Quail egg 16 3.0 Duck egg 10 5.6 Chicken egg 18 4.1 (ayam kampung) Omega egg 19 2.5Discussion: In this experiment, we had used Biuret assay and Lowry assay to determine proteinconcentration in five protein food samples which are chicken egg, quail egg, duck egg,chicken egg (ayam kampung) and omega egg. Biuret test indicate the presence of peptide bond between the amino group and thecarboxyl acid group on adjacent amino acids in a protein. The violet colour is a positive testfor the presence of protein. The greater the violet colour intense, the greater the number ofpeptide bonds that reacts. Biuret test also has its limitations and not very sensitive which are the Biuret only testwhether peptide bonds in protein are present in a sample, it will not determine how muchthere is unless we compare our sample of unknown concentration with a standard of knownprotein concentration. However biuret test will not detect free amino acids and the characteristic purplecolour will not show up if we only have small peptides, since the number of peptide bondsmay not be sufficient to yield colour. The Biuret assay is not much good for proteinconcentrations below about 5 mg/ml. By using the Folin‐Ciocalteu reagent to detect reducedcopper makes the Lowry assay nearly 100 times more sensitive than the Biuret reaction alone. Based on the result obtained we had plotted the standard curve by using Biuret assays,and we compare with the five type of albumin sample. We found that the absorbance value of
these five types of albumin sample is higher than the standard curve. Then the five must dothe dilution to lower the reading from the standard curve. For dilution, the ratio used is 1 mlof protein samples to 9.0 ml of distilled water. After dilution, we get the results which are theprotein concentration in chicken egg is the highest which is 20 mg/mL, the second higher ofprotein concentration by using Biuret assays is omega egg which is 19 mg/mL and the thirdhigher protein concentration using Biuret assays is chicken egg(ayam kampung) which is18mg/mL. Quail egg show the protein concentration 16mg/mL and the lowest proteinconcentration by using Biuret assays is duck egg which is 10 mg/mL. Next we had carried out Lowry assay to determine the protein concentration in threeprotein samples. The Lowry method is more sensitive since it combines the reactions ofcopper ions with the peptide bonds under alkaline conditions with the oxidation ofaromatic protein residues. The Lowry method is best used with protein concentrations of 0.01-1.0 mg/mL and is based on the reaction of Cu+, produced by the oxidation of peptide bonds,with Folin-Ciocalteu reagent. The following substances are known to interfere with the Lowry assay are detergents,carbohydrates, glycerol, potassium compounds, sulfhydryl compounds, most phenols, uricacid, guanine, and calcium. Many of these interfering substances are commonly used inbuffers for preparing proteins or in cell extracts. From the result of Lawry assays we also had been plotted standard curve graph, andwe compare with the five type of albumin sample. For dilution, the ratio used is 1 ml ofprotein samples to 9.0 ml of distilled water. After dilution, we get the results which are theprotein concentration in duck egg is the highest among the other four which is 5.6 mg/mL, thesecond higher of protein concentration by using Lowry assays is chicken egg (ayamkampung) egg which is 4.1 mg/mL and the third highest protein concentration using Lowryassays is quail egg which is 3.0 mg/mL. Omega egg shows the protein concentration2.5mg/mL and the lowest protein concentration by using Lowry assays is chicken egg whichis 1.6 mg/mL. When compare the result of Lowry assay with biuret essay, there is some differences.This is might be because of some errors that occur while we conduct our experiment. Forinstance we just estimate the volume of the sample when we poured into the vial. It should an
actual volume that is same for all protein. Second, there might be error when we dilute theprotein, caused by parallax error when reading the measuring cylinder. As the conclusion, Lowry assays is best method in determining the proteinconcentration because it is best used with protein concentrations of 0.01-1.0 mg/mL which issuitable when test with 0.25 mL of protein sample of albumin of chicken, duck, quail, ayamkampong and omega which is in the range of 0.01-1.0 mg/mL. Meanwhile, Biuret assay is notvery efficient in determining the protein concentration as it is not much good for proteinconcentrations below about 5 mg/ml. Thus, albumin of duck has the highest proteinconcentration, albumin of ayam kampong is the second highest in protein concentration,albumin of quail has the third highest in protein concentration, omega chicken has the fourthhighest in protein concentration and the last one is albumin of chicken. Instead of these two method, there are several method can be used in determiningprotein concentration. First is The Bradford assay, a colorimetric protein assay, is based onan absorbance shift of the dye Coomassie Brilliant Blue G-250 in which under acidic conditions thered form of the dye is converted into its bluer form to bind to the protein being assayed. It also hasdisadvantages which is the Bradford assay is linear over a short range, typically from 0 µg/ml to2000 µg/ml, often making dilutions of a sample necessary before analysis. It is also inhibited by thepresence of detergents.
Conclusion 1. Protein concentration can be determined using Biuret protein assay and Lowry protein assay. 2. The protein concentration in chicken egg is 20mg/mL , omega egg is 19 mg/mL, chicken egg(ayam kampong) is 18mg/mL, quail egg is 16mg/mL and duck egg is 10mg/mL based on Biuret method. 3. The protein concentration in duck egg is 5.6mg/mL , chicken egg(ayam kampong) is 4.1 mg/mL, quail egg is 3.0mg/mL, omega egg is 2.5mg/mL and chicken egg is 1.6mg/mL based on Lowry method. 4. Protein concentration can be found out from the standard curve.References1. www.gbiosciences.com/.../633453707995878750.pdf2. wolfson.huji.ac.il/purification/PDF/Protein.../PIERCE_BIURET.pdf3. biochemistry.musc.edu/.../Lowry%20Protein%20Assay... - United States4. www.molecularstation.com/protein/lowry-protein-assay/