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DETERMINING THE CONCENTRATION OF PROTEIN ON EGG ALBUMIN THROUGH LOWRY METHOD By: Alfath Agung Udayana (1013031004) Chemistry Department of Education, Faculty of Mathematic and Sciences, Ganesha University of Education, Singaraja ABSTRACT Proteins are substances needed by the body. In proteins there are about 20 kinds of standard amino acids. Determination of protein concentration can be performed using spectroscopy with Lowry method. Method of Lowry was used in the Folin-Ciocalteu reagent that can detect residue tyrosine (in proteins) due to the phenolic group in tyrosine to reduce reagent fosfotungstat and phosphomolybdate into tugstate and molybdenum blue. The sensitivity of the Folin-Ciocalteu reagent was experiencing a significant change when added to the ions Cu2+ (Biuret method). Complex of Cu-protein produced by the biuret reagents, will cause a reduction also in fosfotungstat and phosphomolybdate in the Folin-Ciocalteu reagent. This experiment aims to determine the protein concentration in unknown sample solution containing tyrosine with unknown concentration. At the time of determining the concentration of protein in the sample, should also be measured against some standard protein solution that has spanned a certain concentration to create a calibration curve so that by knowing and comparing absorbance of the sample size of egg whites.Spectroscopy experiment was carried out using the method of Lowry. Based on the results obtained by measuring the absorbance value of 0.13 for the unknown sample with concentration of protein is 0.24 ppm. Keyword : concentration of protein, spectroscopy, Lowry method Introduction Protein is a substance that is needed by the body, so the lack of protein will cause disruption in the body. Determination of protein concentration is a process routine in biochemical analysis. There are several methods used in order to determine the protein concentration, the biuret method, Lowry method, and so forth. Choice is good and proper method for a measurement depends on several factors: material or samples are available, the time required to perform measurements, and the tools available spectrophotometer (or UV Vis spectrophotometer) (Tika, 2007). Spectroscopy is one method of instrumental analysis is based on the interaction energy (electromagnetic radiation/ light) and matter (atom/ molecule). The electronic structure of a species or a molecule largely determines the light absorption by the species or molecule. The color of these compounds depends on the metal complexes involved and the number of its d orbital, which are associated with the oxidation state. However, there are several compounds of main group and transition class has no d orbital but the colored compound. Color is caused by electronic transitions involving the valence electrons to another. Spectrophotometer has very wide application in quantitative analysis. The results of quantitative measurements using this method have high accuracy, although not as accurate as atomic absorption or gamma rays. In studying the quantitative nature of the absorption of radiation, the radiation beam applied to the sample and then the intensity of transmitted radiation and the subsequent transmission was measured. Most of the job analysis of the solution, in which the substance to be

analyzed directly dissolved in the solvent or chemical treatment prior experience so as to absorb the radiation. Radiation absorbed by the sample can be determined by comparing the intensity of the radiation beam is passed. General steps in spectrophotometer, especially in the visible region are (1) The formation of molecules that can absorb light in the UV or visible region. If the compound is absorbed in the work area with sufficient intensity, then this step is not required, (2) Preparation and selection of the wavelength spectrum, (3) Preparation of calibration curve and (4) Measurement of the absorbance of samples. Formation of molecules that can absorb UV or visible light should be done when the initial compound (analyzed) did not absorb any area. Therefore, these compounds must be converted into other compounds that absorb UV area or converted into a colored compound. Formation of compounds with a longer conjugation chain and formation of complex compounds is often done for this purpose. The relationship between levels of a substance absorbing the radiation absorption is essentially defined by the Lambert-Beer (1989). Lambert-Beer law describes the exponential reduction of the transmitted radiation intensity due to increased levels of the arithmetic of absorbing substances, in order to obtain the equation: I 1 log o A log T log( ) I T Io/I called the optical density (OD) or absorbance (A), while the I / Io is called transmittance (T), which is the proportion of the transmitted radiation. If T is multiplied by 100% is called percent transmittance (%T). By using notations that are merging Bouguer law, the law can be written as follows A=bC

Where is the molar absorbtivity whose value depends on the wavelength and type of substance, b is a thick absorbing medium is usually expressed in centimeters, C is the molar concentration. At the time of determining the concentration of protein in the sample, measurements must be done well against some of the standard protein solution having a concentration range where the concentration of protein samples in the range. Protein was first inserted into a test tube, and then it is added water. The entire tube should have the same final volume. Complex-forming reagent being added last and usually takes finite time to complete the reaction. Protein standard solutions and samples was measured by spectrophotometer. The results of measurements made in a standard calibration curve obtained by measuring the absorbance of a series of standard solutions. For example, the calibration curves as follows: Absorbancy

Concentration

Picture 1. Calibration curve With the calibration curve, the concentration of the sample solution can be easily determined or calculated by the regression equation. Based on the calibration curve, the equation can be obtained as follows: y = ax b in which: y = sample absorbantions a = tan x =samples concentration b = intercept

In determining the concentration, the detection reagent phenolic groups, such as Folin Ciocalteu reagent has been used in the determination of protein concentration by Lowry (1951) which became known as the Lowry method. In its simplest form Folin Ciocalteu reagent to detect the tyrosine residues (the protein) because of the phenolic group of tyrosine may reduce reagent and fosfomolibat fosfotungstat be a tungstate and molybdenum blue. Reagents and fosfomolibat fosfotungstat Ragen is a major constituent of the reduced Folin Ciocateu tungstate and molybdenum which produces shows a broad absorbance peak in the red and visible spectrum (600-800 nm). Folin Ciocalteu reagent sensitivity of these significant changes when its added to the ion-ion Cu2+ (biuret method). Cu-protein complex produced by the biuret reagent, will also lead to reduction in fosfotungstat and Folin Ciocalteu reagent fosfomolibat in. Approximately 75% of the reduction occurs by the system administrator due to the Cu-protein, and 25% reduced by the residues tyrosine and tryptophan. Folin Ciocalteu reagent is a complex composition obtained by heating under reflux of the Na-Na-tungstate and molibat with orthophosphoric acid. In addition of its, also included other components to improve the stability of the reagents in the normal condition of pale yellow. At the time of determining the concentration of protein in a sample, the measurement should be done well against some of the standard protein solution having a certain concentration range. Materials and Methods Materials used in this practicum were Na2CO3 solution 2% 25 mL, NaOH solution 0.1 N 25 mL, CuSO4.5H2O solution 0.5% 25 mL, sodium tartarat solution 25 mL, folin-ciocelteu reagen 25 mL, tirosin solution 100 mL, and aquades 500 mL,

The protein standard solution was mixed with distilled water to a final volume of 1.0 mL. The protein sample solution was also mixed with distilled water so that the final volume of 1.0 mL. Five mL of biuret reagent were prepared in each tube. It was incubated exactly 10 minutes at room temperature. After 10 minutes, 0.5 mL of Folin Ciocalteu reagent was added into each tube. It was shaked with stirring rod. This incubation time could be started after the addition and mixing of Folin Ciocalteu reagent into the tube last. The absorbance was read at a wavelength of 700 nm and the tube 1 as blank. Discussion In this experiment performed the analysis proetein levels in egg white samples using the method of Lowry. Lowry is the principle method for determining protein concentration in which there is amino acid-containing phenolic groups such as tyrosine and tryptophan. In this method used spectronic 20+ to analyze the absorbance of standard solutions and samples. In order to be absorbed radiation is the solution to be analyzed should show certain colors that can absorb visible light in the UV-visible region. For the purposes of Folin Ciocalteu reagent was used to detect the phenolic groups present in tyrosine and tryptophan residues (the protein). Phenolic groups present in amino acids can reduce fosfotungstat and fosfomolibat the Folin Ciocalteu reagent contained in a tungstate and molybdenum blue. The fofotungstat and fosfomolibat acts as a reducing agent phenolic groups present in the analyzed solution, wherein the phenolic group itself act as an oxidant to oxidize fosfotungstat and fosfomolibat be a tungstate and molybdenum blue complex ion. Tungstate and molybdenum produced in the reaction shows a broad absorption peak in the red region and the spectrum of visible light at wavelengths 600-800 nm.

The number of tyrosine residues in the reaction to oxidize slightly above the required constituents that may enhance the sensitivity of Folin Ciocalteu reagent. In this experiment the addition of biuret reagent. The addition of biuret reagent to tubes 1-7 (containing the standard solution) led to a colorless solution. The addition of biuret reagent to a solution aimed at increasing the sensitivity of Folin Ciocalteu reagent in which the addition of biuret reagent, Cu2+ complexes are formed by amino acid residues contained in the test solution Cu-protein. Cu-protein complex produced by the biuret reagent will cause a reduction of the Folin Ciocalteu reagent, where as many as 75% of the reduction that occurs due to the Cu-protein complexes, while the remaining 25% is reduced by the residues tyrosine and tryptophan. With the addition of the reagent is resulting color intensity will increase fourfold. Absorbance measurements of samples need to be making a calibration curve of standard solutions. Standard solutions used were Brovine Serum Albumin solution (BSA). Mother liquor is made by concentration 200g/mL. Mother liquor is then made in different concentrations through dilution. The purpose of diluting the standard solution is to make different concentrationsis to facilitate the making of the calibration curve. Next to the standard solution biuret reagent is added which serves to form the Cu-protein complexes leading to a reduction fosfotungstat and fosfomolibat tungstate and molybdenum of Folin Ciocalteu reagent. The results obtained after the standard solution in tube 1 to 7 Folin Ciocalteu reagent was added to a solution of clear blue of a concentration increase of the tube 1 to tube 7. The blue color indicates the formation of tungstate and molybdenum blue in the reaction.

Concentration of each tube is known from the following calculation: Test tube 1 : The concentration in test tube 1 was 0 ppm because it wasnt added tyrosin standard solution. In test tube 1 was added aquades, biuret reagent and phenol reagent. Test tube 2 : V1M1 = V2M2 x mL.10 ppm = 5 mL. 1 ppm x = 0.5 mL So, to make tyrosin 1 ppm from 10 ppm, it should be added aquades 4.5 mL until the volume 5 mL. Test tube 3 : V1M1 = V2M2 x mL.1 ppm = 5 mL. 0.2 ppm x = 1 mL So, to make tyrosin 0.2 ppm from 1 ppm, it should be added aquades 4 mL until the volume 5 mL. Test tube 4 : V1M1 = V2M2 x mL.1 ppm = 5 mL. 0.4 ppm x = 2 mL So, to make tyrosin 0.4 ppm from 1 ppm, it should be added aquades 3 mL until the volume 5 mL. Test tube 5 : V1M1 = V2M2 x mL.1 ppm = 5 mL. 0.6 ppm x = 3 mL So, to make tyrosin 0.6 ppm from 1 ppm, it should be added aquades 2 mL until the volume 5 mL. Test tube 6 : V1M1 = V2M2 x mL.1 ppm = 5 mL. 0.8 ppm x = 4 mL So, to make tyrosin 0.8 ppm from 1 ppm, it should be added aquades 1 mL until the volume 5 mL. After that, it was added a solution of 1.0 mL after biuret and Folin Ciocalteu reagent volume to 6.5 mL finally. The addition of this volume is calculated considering the volume of biuret reagent and Folin Ciocalteu reagent were added to each tube has the same volume.

After the addition of biuret reagent and Folin Ciocalteu reagent, obtained by the color of the solution in each tube to increase the thick of the tube 1 to tube 7. On the tube 8, contains a sample of protein (egg albumin), the resulting color is the color between the standard solution.

Based on the equation that obtained, it can be calculated tyrosine concentrations present in the sample by the following formula. A = mC + b where, A = absorbance of solution C = concentration of solution (g/mL) b = interscept in y axis The slope (m) from curve can calculated as follow:
m 0.30 0.07 A m m 0.255 C 1 .0 0 .1 A mC b

Picture 2a. After Picture 2b. After adding Buret adding Phenol reagent and reagent and incubated 10 incubated 30 minutes minutes In the manufacture of calibration curves, to obtain more accurate data then used the data %T of the measurement results of each standard solution. The absorbance value from the practicum are as follows:
Concentration (ppm) 1 0.8 0.6 0.4 0.2 0.1 Absorbance 0.3 0.21 0.2 0.17 0.09 0.07

0.07 0.255 ( 0.1) b b 2.74 From the observation results, the sample solution has 0.13 of absorbance (A). Thus the concentration of the sample solution can be calculated using the equation above, namely: A = mC + b A = 0.255 x C + 0.07 0.13 = 0.255x C + 0.07 0.06 = 0.255x C C = 0.24 ppm C = 0.24 ppm = 0.24 mg/L Based on the above calculation can be detected the concentration in unknown samples of tyrosine is 0.24 mg/L.

So, the curve is follow:

Picture 3. Calibration curve of BSA

Conclusion Based on the results and discussion above, it can conclude that the protein contained in the unknown sample solution is 0.24 mg/L. Acknowledgment The authors thank to Dr. I Nyoman Tika,M.Si, lecture of biochemistry in chemistry department, Ganesha University of education The authors thank to Dr. Hj. Siti Maryam M.Si, lecture of biochemistry in chemistry department, Ganesha University of education The author thank to I Wayan Gde Oka Prabawa and Made Eka Guna Tresna, assistants of biochemistry in chemistry department, Ganesha University of education The author thank all of students in Pioneering chemistry department, Ganesha University of education References Anwar, Chairil, dkk. 1996. Pengantar Praktikum Kimia Organik. Yogyakarta : Dedikbud Muderawan, I Wayan. 2010. Analisis Instrumen. Singaraja: Universitas Pendidikan Ganesha Poedjadi, Anna dan Titin Supriyanti. 1994. Dasar-Dasar Biokimia. Jakarta: Universitas Indonesia. Redhana, I Wayan. 2004. Penuntun Praktikum Biokimia. Yogyakarta: Universitas Negeri Yogyakarta Tika, I Nyoman. 2010. Penuntun Praktikum Biokimia. Singaraja: Universitas Pendidikan Ganesha Wirahadikusuma, Muhamad. 1989. Biokimia: Protein, Enzim, dan Asam Nukleat. Bandung: Penerbit ITB

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