DETERMINATION OF PROTEIN CONTENT SPECTROPHOTOMETRICALLY

Name : Tsabit AlbananiNIM : 4301413003Major/Study Program : Chemistry/Chemistry EducationGroup : 7Date of lab work : 15th September 2015The lab work name : Determination of Protein Content Spectrophotometrically

A. Objection1. Understand the use of spectrophotometry as a tool to analyze the protein contents.2. Explain the basic principles of the use of spectrophotometryin the analysis of

protein content3. Skilfully use of spectrophotometry to determine of protein content.

B. Basic TheoryProtein is a high molecular organic compounds ranging from a few thousand to

millions. This protein is composed of atoms C, H, O and N, and other elements such as P and S which form the amino acid units. The sequences of the amino acid sequence with another amino acid, determining the biological properties of a protein. In nature found a wide 20-21 amino build proteins. Protein is a vital nutrient group. This compound is a protein found in the cytoplasm in living cells, both plant and animal. At the protein chemically is hetero-polymer of amino acids bound together by peptide bonds. Any protein and derived from any living creature also was only composed of 20 kinds of amino acids alone. Protein differences with each other due to the number and position of the amino acids that have common characteristics as configuration 1, which both have 1 COOH groups and one group CH2yang bound to the atom Ca (Lehinger, 2001).

When amino acids are linked together by acid–amide bonds, linear macromolecules (peptides) are produced. Those containing more than ca. 100 amino acid residues are described as proteins (polypeptides). Every organismcontains thousands of different proteins, which have a variety of functions. At a magnification of ca. 1.5 million, the semischematic illustration shows the structures of a few intra and extracellular proteins, giving an impression of their variety. The functions of proteins can be classified as follows (Koolman and Roehm, 2005).

The structural proteins give extracellular structures mechanical stability, and are involved in the structure of the cytoskeleton. Most of these proteins contain a high percentage of specific secondary structures. For this reason, the amino acid composition of many structural proteins is also characteristic.

Picture 1. Peptide bond and resonance in protein (Koolman and Roehm, 2005)

Measurement of protein content by Lowry method is the basic use of a spectrophotometer. This method can use a protein content of up to 5 Micrograms. The blue color that occurs by Folin reagent ciacalteu caused by the reaction between the protein with Cu in solution and reaction alkaline phosphotungstate salt and salt phosphomolybdat by tyrosine and tryptophan (Poedjiadi, 1994)

The wavelength controller (on the top of the instrument) adjusts the position of the diffraction grating so that different colors of light are selected by the slit. The zero % T control (on the left side of the front panel of the instrument) adjusts the electronics. With the sample compartment cover down and no sample in the cell holder, the zero control should be adjusted until the meter reads 0 for transmittance. This control adjusts for the so-called dark current, the current transmitted by the photocell in the dark. The 100%T control (on the right side of the front panel of the instrument) adjusts the amount of light output while the blank cuvette is in the cell holder.

A mechanical linkage is used to drive a wedge into the light beam for this adjustment. After inserting the blank cuvette into the cell holder, this knob should be adjusted until the transmittance is 100% (or the absorbance is zero). This allows you to correct for any stray absorbance resulting from the solvent alone.

A spectrophotometric method can be developed if the intensity of the coloration produced is essentially dependent upon the concentration of the desired constituent, and is stable long enough to permit measurement. The development of specific organic reagents and effective means of complexation have made possible the determination of single components found in complex systems. Furthermore, chemical phenomena which occur in solution, as complex formation, solvation, and equilibria, may be studied in situ by spectrophotometric methods. Absorption spectrophotometry thus provides a useful analytical and research tool which bridges the span between the data on atomic and ionic species supplied by emission spectroscopy and the information on molecular and crystalline states obtained from X-ray diffraction (Timma, 1952)

C. Equipment and MaterialsEquipment :1. Spectronic 202. Cuvette3. Pipette4. Test tube5. Erlenmeyer6. Pumpkin peck

7. Pipette volume8. Ball pipette9. Beaker glass10. Volumetrick Flask11. Test tube rack

Materials :1. Sample of Protein2. CuSO4.5H2O3. Sodium Potassium Tartrate4. Aquades5. NaOH 10%

6. Serum Albumin Pure7. Casein8. NaOH 3%

D. ProcedureExperiment 1: Making Biuret Reagent

Experiment 2. Preparation of Standarf Solution Protein

dissolve 1,5 grams of CuSO4 and 6,0 grams of sodium potassium tatrate (NaKC4O6.4H2O) in approximately 500 ml of distilled wate in a 1 liter

measuring flask

Add 300 ml of 10% NaOH with a whipped-cream

Add distilled water to the line

make a solution of pure serum albumin in distilled water

the yield of about 10 mg/ml to facilitate solubility add a few drops 3% of NaOH

Observe and note the product

Experiment 3. Preparation of Calibration Curve

Experiment 4. Measurement of Protin Samples

Prepare a standart solution with a concentration of 1 mg/ml protein, 3

mg/ml, 5 mg/ml, 7 mg/ml, and 9 mg/ml

insert standard solution into a test tube 5 pieces (eat 1 ml). add 4 ml

of biuret reagent

shake and let sand for 30 minutes at room temperature

enter into the cuvette, measure the absorbance using spectronic 20 at a

wavelength of 540 nm

use the blank solution containing a mixture of 1 ml of distilled water and 4 ml of biuret were silenced 30 mimutes at room

temperature

make a calibration curve based on spectronik 20 data

on a standard protein solution

Prepar the protein sample to be analyzed

Add 1 ml of the sample into a clean test tube, add 4 ml of

biuret reagent

shake and let stand for 30 minutes at room temperature

enter into the cuvette, measure the absorbance using spectronic

20 at wavelength 540 nm

use the blank solution as at the time of calibration curve

use the calibration curve obtained to calculate the protein content in the

sample

E. Result of ObservationTable 1. Result of observation

Name of Test tube

Concentration (mg/ml)

Absorbance

1 1 0,31

2 3 0,16

3 5 0,25

4 7 0,34

5 9 0,41

Blank - 0,00

Sample of Protein 0,40

0 1 2 3 4 5 6 7 8 9 100

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

f(x) = 0.0326301369863014 x + 0.109041095890411R² = 0.601121317102628

calibration curve

times

Abso

rban

ce

Graph 1. Relation between concentation protein and absorbance

F. Data Analysis and ExplanationIn this experiment, purposed to determine the level of a protein in a solution by

using biuret reagent. In this experiment used a standard protein sample and random

sample solutions. The protein that used in these experiments are Albumin is by

dissolving 1 gram of albumin into 100 mL of distilled water. In this experiment used a

protein solution with a concentration that is different, that of 1, 3, 5, 7 and 9 mg / ml.

After each standard solution was put into a test tube and add biuret reagent

solutions then is left for 20 minutes. It is intended that the process of formation of

colored complex compounds can take place with absolutely perfect. After the colored

complex compounds are formed, then measured it with the UV spectrometer.

Measurements were taken at a wavelength of 540 nm. In the spectrophotometer,

protein solution adsorbs light that given to it. This is an appearance of the interaction of

an atom with light. In which the electromagnetic energy is transferred to the atoms or

molecules so that the particles in protein promoted from a lower energy level to a

higher energy level, namely the level of excited. From the results of the identification

of the spectrophotometer, obtains of price absorbance at each concentration. The

greater the concentration, the more protein is absorbed, so the price the greater the

absorbance obtained as well.

Complex compounds is seen immediately after the addition of reagent biuret

with formation of a purple color in the solution. To get the color, then the protein

solution is treated with copper elements in the Biuret reagent. So we get a purple

protein solution at each concentration. The purple color is formed by the reaction

between Cu2+ in biuret reagent by peptide bonds of proteins in solution casein earlier,

precisely a bond with -NH of proteins as in the equation.

From the results of the identification of the spectrophotometer, the price

obtained absorbance at each concentration as shown in the table. The greater the

concentration, the more protein is absorbed or absorbed, so the price the greater the

absorbance obtained as well.

Calculation:R2 = 0,6011y = 0,0326x +0,109concentration of sample protein: y = 0,0326x +0,109

0,40 = 0,0326x +0,109x = 8,926 (the correct answer is 8)

Percentage of error = result of observation−the correct answer

the correct answer x 100%

= 8,926−8,000

8,000 x 100%

= 11,5 %

Prior to testing samples of proteins, first attempted to create a calibration curve.

Measurement calibration curve was made by measuring levels of a protein in the

albumin with various concentrations is 1 mg/ml, 3 mg/ml, 5 mg/ml, 7 mg/mL and 9

mg/ml. Then measured absorbance at a wavelength of 540 nm which is the wavelength

of maximum. Absorbance value is directly proportional to the concentration. The

higher the absorbance value, the higher the concentration.

Based on the calibration curve, obtained by the equation y = 0,0326x +0,109.

Once the sample is obtained absorbance of the sample is measured so that the protein

content was obtained is 8,926 mg/ml. This result protein content of the sample is 8

mg/ml. This experiment can conclusion that this experiment is correct, because

experiment error is 11,5 %.

G. Conclusion and SuggestionConclusion

1. The higher the concentration of protein contained, more concentrated solution also produced a purple color complex.

2. Absorbance of the solution is directly proportional to the concentration, so that the greater the concentration used, the greater the absorbance used.

3. Concentration of random sample obtained is 8.926 mg/ml.

Suggestion1. Try to understand the material before doing the lab work2. We should use safety equipment when we are doing experiment.

3. Careful in use spectronic 20.4. Don’t forget to give label on test tube, so the test not will get accidently exchanged.

H. Refferences Gilbert, Hiram F. 2000. Basic Concepts In Biochemistry. Texas: Baylor College of

MedicineKoolman, J and Roehm K.H. 2005. Color Atlas of Biochemistry. Germany: Philipps

University Marburg and Institute of Physiologic ChemistryLehninger, Albert. 1995. Dasar-dasar Biokimia Jilid 1. Jakarta: Erlangga.Poedjiadi, A. 1994. Dasar-dasar Biokimia. Jakarta: UI-Press.Timma, D.L. 1952. Absorption Spectrophotometry. Columbus: Ohio Journal of Science

Semarang, 18th October 2015Lab Work Lecturer Student

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Samuel Budi W.K M.Si, M.Sc Tsabit AlbananiNIP 198204182006041002 NIM 4301413003

Question1. Explain the legal basic of the analysis using spectrophotometry tool2. Describe the work of spectrophotometry instrument. Describe the work of visible

spectrophotometric instruments3. Explain the advantaged and disadvantages of the use of the biuret method with

analysis spectronic 20 for protein content4. Why the reaction in this experiment is called biuret reaction ?

Answer1. The underlying law is the law of Lambert-beer:

"Measuring the intensity of monochromatic light through a colored solution that lasts exponentially and depends on the wavelength of the light passed through the solution and the concentration of substances in solution"

A = log II o

= - KCL

Description:I = intensity of the lightIo = the intensity of the light emittedK = concentration based on the nature of the substance in solutionC = concentration of soluteL = length of light through which the solution

2. S D L be continued

Description:S = light sourceD = sample/solutionL = prism

3. Pros: Reagents biuret more durable in storage, showed a positive reaction to a bully biuret compounds.Disadvantages: the bully biuret compounds

4. The reaction of this experiment called biuret reaction because it gives a positive reaction to biuret.