UNIVERSITI TEKNOLOGI MARA FACULTY OF CHEMICAL ENGINEERING ENGINEERING CHEMISTRY LABORATORY (CBE 461) NAME STUDENT NO GROUP EXPERIMENT

DATE PERFORMED : SEMESTER PROGRAMME / CODE SUBMIT TO : MOHAMED FIRDAUS BIN NORDIN : 2012867046 : EH 222 2A :ESTIMATION OF PROTEIN : 15/5/2013 :2 : EH222 / CBE461 : MISS NUR SHAHIDAH

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Title Abstract/Summary Introduction Aims Theory Apparatus Methodology/Procedure Results Calculations Discussion Conclusion Recommendations Reference Appendix TOTAL MARKS

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TABLE OF CONTENT CONTENT Abstract / summary Introduction Aims / objectives Theory Material and apparatus Procedure Result Discussion Conclusion Recommendation References Appendices PAGE

ABSTRACT

The estimation of protein can be done in three methods which are Lowry method, Biuret method, and Bradford method. Lowry method: Lowry method is use for determining the total level of protein in solution. The color change of the sample solution in proportion to protein concentration is how the total protein concentration is exhibited, which can then be measured using colorimetric techniques. It is named for the biochemist Oliver Howe Lowry who invented the reagent in the 1940s. Biuret Method: The Biuret Method, the most widely used method for the total protein estimation, depend on the complexation of Cu 2+ by the function groups involved with the peptide bond. At least two peptide bonds is needed for the complexation to occur. Upon complexation, a violet color will appear. The absorbance of the Cu 2+ -protein complex is measured at 540 nm and then compared to a standard curve. Bradford method:

The Bradford method which use Bradford assay is very fast and uses about the same amount of protein as the Lowry assay. It is accurate and retest of samples that are out of range can be done within minutes. The Bradford method is recommended for general use, especially for determining protein content of cell fractions and assesing protein concentrations. the composition of the assay include color reagent, protein standard, and instruction booklet are available from Bio-Rad Corporation.

INTRODUCTION Protein is made up from simpler molecule called amino acid. Amino acid varies in 20 different types such as alanine, leucine, and lysine. Proteins have complex shapes and folding in proteins happens spontaneously. Chemical bonding between portions of the polypeptide chain help the proteins to hold together and giving it its shape. Globular proteins and fibrous proteins are two general classes of protein. Globular proteins are generally compact, soluble, and spherical in shape. Fibrous proteins are usually elongated and insoluble. Globular and fibrous proteins may exhibit one or more of four types of protein structure. These structure types are called primary, secondary, tertiary, and quaternary structure. Proteins are essential parts of organisms and involve in virtually every process within cells. Many proteins are enzymes that help catalyze biochemical reactions and are important to metabolism. Proteins also are important in cell signaling, immune responses, cell adhesion, and the cell cycle. Through the digestion process, animals break down ingested protein into free amino acids that are then used in metabolism. Proteins can be be purified from other components using many techniques such as precipitation.

OBJECTIVES To study the method of assay preparation in protein estimation and to determine the best assay in protein estimation.

THEORY In this experiment, two absorption spectroscopy assays which is a colorimetric assay and spectrophotometric assay were performed to calculate the concentration of protein in samples by mere estimation of spectral absorption. These analytical techniques are grouped under the chemical method of spectrophotometry. For the colorimetric assay, Biuret, Lowry, and Bradford assay was used. The reagent can be used either qualitatively or quantitatively. All these three assay share a common principle which is, under alkaline conditions the divalent copper ion forms a complex with peptide bonds in which it is reduced to a monovalent ion (causing change in colours). Spectrophotometric analysis relies on the interaction of electromagnetic radiation (light) with the matter of interest. Strictly speaking, every compound has a distinct absorption spectrum which allows its identification, in many cases, in the presence of other compounds. In addition to the identification of a compound, it is also possible to determine quantitatively the concentration of that compound. The principle behind the Lowry method of determining protein concentrations lies in the reactivity of the peptide nitrogen with the copper [II] ions under alkaline conditions and the subsequent reduction of the Folin-Ciocalteay phosphomolybdicphosphotungstic acid to heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic acids. The Lowry method is sensitive to pH changes and therefore the pH of assay solution should be maintained around 10 - 10.5. The Bradford assay is very fast and fairly accurate and samples that are out of range can be retested within minutes. The Bradford is recommended for general use, especially for determining protein content of cell fractions and assesing protein concentrations. The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change.

APPARATUS 1. Spectrophotometer 2. Pipette 3. Beaker 4. Cuvettes

MATERIALS 1. Biuret reagent 2. Lowry reagent 3. Bradford reagent 4. sodium carbonate 5. 0.4% NaOH 6. Folin-Ciocalteu phenol (folin) reagent 7. Distilled water 8. Protein:bovine serum albumin (BSA) and gelatin

PROCEDURE Estimation of protein 1) Lowry (reagent 1) 1 volume of reagent B is mixed with reagent A (50 volume) Reagent A 1) prepared 500ml 2) 2% NaCO2 (10g) is mixed with 0.4% NaOH (2g) 2) Lowry (reagent 2) 5ml folin is mixed with 5ml of distilled water

Analysis 1) Biuret - 1ml of protein is mixed with 5ml of biuret reagent (blank sample). It is measured 2) at 540nm.

Lowry - 0.5ml of protein is mixed with lowry reagent 1. After 10 minutes, 0.5ml lowry reagent 2 is added and mixed well. After 30 minutes, it is measured at 750nm.

3)

Branford - 0.5ml of protein is mixed with 5ml of Bradford reagent. It is measured at 595nm.

RESULT Table shows the absorbance of gelatin using 3 reagents; Biuret, Lowry and Bradfort.

Absorbance Biuret (540 nm) 0.00 0.08 0.09 0.10 0.20 0.30 0.40 0.00 0.106 0.317 0.314 0.327 0.338 Lowry (750nm) 0.00 1.464 1.515 1.372 1.704 2.037 Bradfort (595 nm) 0.00 1.716 0.979 0.61

Graph of the each of the absorbance of gelatin using 3 reagents; Biuret, Lowry and Bradfort.

Standart Curve of Gelatin for Biuret Reagent

0.45 0.4 0.35 absorbance (540nm) 0.3 0.25 0.2 0.15 0.1 0.05 0 0 0.1 0.2 concentration (mg/mL) 0.3 0.4 gelatin Linear (gelatin) y = 0.9929x + 0.1062 R = 0.5284

Standart Curve of Gelatin for Lowry Reagent

2.5 y = 2.6838x + 1.2051 R = 0.9313 2

Axis Title

1.5 Lowry Linear () Linear (Lowry)

0.5

0 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 Axis Title

Graph of Absorbance versus Concentration of Gelatin in Bradford reagent

2 1.8 1.6 1.4

Absorbance, nm

1.2 1 0.8 0.6 0.4 0.2 0 0 0.1 0.2 0.3 0.4 0.5 0.6 concentration of gelatin, g/mL Series1 y = 0.1808x + 0.7856 R = 0.0031 Series2 Linear (Series1) Linear (Series2)

Table below shows the absorbance of BSA using 3 reagents; Biuret, Lowry and Bradfort

Absorbance Biuret (540 nm) 0.00 0.08 0.09 0.10 0.20 0.30 0.40 0.50 0.00 0.046 0.232 0.261 0.301 0.316 Lowry (750nm) 0.00 1.933 2.912 2.935 3.067 3.163 Bradfort (595 nm) 0.00 2.037 2.054 2.218 2.917 2.96 3.11

Graph of the each of the absorbance of BSA using 3 reagents; Biuret, Lowry and Bradfort.

Standart Curve of BSA for Biuret Reagent

0.4 0.35 0.3 absorbance (540nm) 0.25 0.2 0.15 0.1 0.05 0 0 0.1 0.2 0.3 0.4 0.5 0.6 concentration (g/mL) Series1 Linear (Series1) y = 0.6783x + 0.0231 R = 0.8771

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Standart Curve of BSA for Lowry Reagent

4 3.5 3 absorbance (750nm) 2.5 2 1.5 1 0.5 0 0 0.1 0.2 0.3 0.4 0.5 0.6 concentration (g/mL) y = 5.4971x + 0.9607 R = 0.7025

Series1 Linear (Series1)

Standard Curve BSA for Bradford Reagent

3.5 3 2.5 Axis Title 2 1.5 1 0.5 0 0 0.2 Axis Title 0.4 0.6 Linear (Absorbence Value (nm) Bradford) Absorbence Value (nm) Bradford Linear () y = 2.6412x + 1.9903 R = 0.7659

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DISCUSSION This experiment is conducted by using three different assays which is Biuret, Bradford and Lowry assays to estimate two types of proteins used which are Bovine Serum Albumin (BSA) and gelatin. The proteins used is in different concentrations which are 0.08, 0.09, 0.1, 0.2, 0.3, 0..4 mg/ml for the gelatin and 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5 mg/ml for BSA.

For Biuret assays, after its reacts with the gelatin and BSA sample, the absorbance for the different concentrations is determine using spectrophotometer set at 540nm and the data is tabulated and the graph is plotted. The steps were also carried out for Lowry assay and Bradford assay where the setting of spectrophotometer is 750nm and 595nm respectively. Based on the result obtained, the R2 obtain for the gelatin in Biuret assay, Lowry assay and Bradford assay are 0.5842, 0.9313 and 0.0031 respectively. Based from the theoretical, the experiments results are seem to be valid as it approaching 1 which is the theoretical value except for the last which is the Bradford assay. This undesired value may due to the impurities in the apparatus used. The proper cleaning of apparatus must be done before it is being used.

While for BSA sample, the R2 obtained are 0.8771 for Biuret assay, 0.7025 for the Lowry assay, and 0.7659 for the Bradford assay. Since the values approaching the value of the theoretical which is 1, the results obtain are all valid for BSA. Lowry assays is very sensitive to the tyrosine present in the protein. The reaction involves reduction of the Folin reagent and oxidation of aromatic residues mainly tryptophan and also tyrosine. Both BSA and gelatine contain the tyrosine in their composition.

Based on the theoretical results, the Bradford assay is said to be the best assay, which is should be showing the highest of the R2. However, due to the error while handling the experiment, the result obtained is far differ to the theoretical value.

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CONCLUSION Overall, the results obtained for the BSA sample and the gelatin sample by the Biuret assay, Lowry assay and the Bradford assay are valid based on theoretical which show the value approaching 1. The assay that should give the highest value based on theoretical is Bradford assay, but in the gelatin sample, the Lowry assay has the highest R2 value which is opposite to the theoretical one. For the BSA sample, the same situations occur but the different is that the highest R2 value is for Biuret reagent. Based on experiment conducted, Lowry is the easiest assays to be prepared among all. From the theory, Bradford assays is the best in estimation of protein but based on experimentally observation, the best assays to used in this experiments is Lowry assays. As the conclusion, due to the criteria discuss based on the experimentally results, the best assays for protein estimation in this experiment is Lowry assays.

RECOMMENDATION 1. During the experiment, proper attire such as gloves, goggles and lab coat must be wear. 2. When doing experiment on gelatin, the procedure need to be done fast and very carefully because the gelatin will became more viscous. 3. Weigh the gelatin accurately because when it is dilute with water is became viscous and hard to detect the absorbance. 4. The value of absorbance must be taken repeatedly to ensure the consistency of reading. 5. Follow the laboratory regulation and rules throughout the experiment period. 6. Make sure all the apparatus clean and safe before using it.

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REFERENCE 1. http://www.gbiosciences.com/ResearchProducts/Protein-Research/ProteinQuantificationAssays.aspx

2. http://www.vivo.colostate.edu/hbooks/genetics/biotech/basics/prostruct.html

APPENDICES

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