PROTEIN EXPERIMENT

NIK NURUL FATEN ATIKAH BT NIK MUSTAFA D20071029526
NOR AINI BT ABDULLAH D20071029527
SYARIFAH NUR AMALINA BT SYED ZULFAKHAR D20071029552
MAAZIANA BT MUHAMAD D20071029558
AZILA BT MOHAMAD D20071029563



INTRODUCTION

This experiment is to determination of total protein content in gelatin solutions with the Lowry or Biuret Assay. Gelatins can be obtained from different sources and prepared using different processes, and the end product gelatin may vary in amino acid composition and molecular weight distribution. In the present study, the variation in "protein color" development among gelatins in colorimetric total protein content measurements was investigated at 540 nm using the Biuret assay and at 650 nm using the Lowry assay, with bovine serum albumin as the reference protein. In both the Biuret and Lowry assays, the color response varied significantly among gelatins. The difference in amino acid content was the major factor responsible for this variation, which probably influenced the gelatin helix → coil phase transition and resulted in the difference in gelatin associate state. Based on their "protein color" development abilities in both Biuret and Lowry, gelatins were classified into 2 major groups with the hierarchical cluster analysis: 1 group included all cold water fish gelatins, while the other included gelatins from warm water fish, avian, and mammalian species.

RESULT:

BIURET ASSAY







For biuret assay, the graph that we obtain is almost same with the theoretical. Which is, when the concentration of protein is increase, the absorbances also increase. This is because, the photometer is very sensitive. During experiment there might be finger print present outside of the cuvex, that affect the reading. Beside that, if a drop of water drop into the cuvex, the concentration of the sample will change, this will give not accurate reading, then the mixture of the sample does not mix well with buiret assay.



LOWRY ASSAY







DISCUSSIONS:

Lowry Assay

It offered a significant improvement over previous protein assays and his paper became one of the most cited references in life science literature for many years. The Modified Lowry Protein Assay uses a stable reagent that replaces two unstable reagents described by Lowry. Essentially, the assay is an enhanced biuret assay involving copper chelation chemistry.
Although the mechanism of color formation for the Lowry assay is similar to that of the BCA protein assay, there are several significant differences between the two. The exact mechanism of color formation in the Lowry assay remains poorly understood. The assay is performed in two distinct steps. First, protein is reacted with alkaline cupric sulfate in the presence of tartrate for 10 minutes at room temperature. During this incubation, a tetradentate copper complex forms from four peptide bonds and one atom of copper (this is the "biuret reaction"). Second, a phosphomolybdic-phosphotungstic acid solution is added. This compound (called Folin-phenol reagent) becomes reduced, producing an intense blue color. It is believed that the color enhancement occurs when the tetradentate copper complex transfers electrons to the phosphomolybdic-phosphotungstic acid complex. The blue color continues to intensify during a 30 minute room temperature incubation. It has been suggested that during the 30 minute incubation, a rearrangement of the initial unstable blue complex leads to the stable final blue colored complex which has higher absorbance.
The Lowry protein assay method combines the reactions of cupric ions with the peptide bonds under alkaline conditions (the Biuret test) with the oxidation of aromatic 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's reagent (a mixture of phosphotungstric acid and phosphomolybdic acid in phenol) in the Folin-Ciocalteu reaction. The reaction mechanism is not well understood, but involves reduction of the Folin reagent and oxidation of aromatic residues (mainly tryptophan, also tyrosine). The concentration of the reduced Folin reagent is measured by absorbance at 750 nm. As a result, the total concentration of protein in the sample can be deduced from the concentration of Trp and Tyr residues that reduce the Folin reagent.
The disadvantage of this method is the long incubation time and there are often interferences with commonly used buffers. This method is also subject to protein-to-protein variation due to the correlation of colour intensity dependent on the content of tyrosine and tryptophan in the protein.


Biuret Assay

Copper-based protein assays, including the BCA and Lowry methods, depend on the well-known "biuret reaction", whereby peptides containing three or more amino acid residues form a colored chelate complex with cupric ions (Cu2+) in an alkaline environment containing sodium potassium tartrate. This became known as the biuret reaction because it is chemically similar a complex that forms with the organic compound biuret (NH2-CO-NH-CO-NH2) and the cupric ion. Biuret, a product of excess urea and heat, reacts with copper to form a light blue tetradentate complex.


Structures of urea, biuret and peptide. Because polypeptides have a structure similar to biuret, they are able to complex with copper by the biuret reaction.
Single amino acids and dipeptides do not give the biuret reaction, but tripeptides and larger polypeptides or proteins will react to produce the light blue to violet complex that absorbs light at 540 nm. One cupric ion forms a colored coordination complex with four to six nearby peptides bonds. The intensity of the color produced is proportional to the number of peptide bonds participating in the reaction. Thus, the biuret reaction is the basis for a simple and rapid colorimetric reagent of the same name for quantitatively determining total protein concentration. The working range for the biuret assay is 5-160 mg/ml, which is adequate for some types of industrial applications but not nearly sensitive enough for most protein research needs.


Which one gave more accurate value to determine protein contained in eggs?




For the Biuret method, is the most linear because its color depends on a direct complex between the peptide bonds of the protein and Copper(II) ion. It is not highly sensitive since the complex does not have a high extinction coefficient. Its sensitivity range is just about 1 to 10 mg of protein.
The Lowry assay is dependent on the presence of aromatic amino acids in the protein. First, a cupric/peptide bond complex is formed and then this is enhanced by a phosphomolybodate complex with the aromatic amino acids. Overall, about 10 to 50 times more sensitive than the Biuret method.

What is an ‘appropriate blank’ and why?
The last sample in the list with no protein added is blank that should be used to zero the spectrophotometer. Blanks are useful when there are other substances in the experimental tube besides the substance you are trying to measure. Since those other substances are not the chemical that you are trying to measure, they often interfere with the absorbance reading of the chemical of interest. A much more suitable way to deal with this problem is to exclude these other substances from our spectrophotometer reading without removing them from the experimental tube. The way to do that is to use a blank. A blank contains all the substances (or substance) in the experimental tube except the substance that is being measured. Then, before reading your experimental tube, you place the blank tube in the spectrophotometer.


MAKING A PROTEIN GLUE

OBSERVATIONS OF PROTEIN GLUE
By adding vinegar to milk we produce a solution containing white solids (precipitation), and by filtering the milk we separate the solution into two substances (called curds and whey). The curds can be dried with a paper towel to produce a cheese-like substance.
The baking soda, when added to the curds, causes them to become a sticky glue. We noted bubbles coming from the solid when the baking soda was added. The glue dries to become a plastic-like substance. It has different physical properties than the original milk and vinegar individually. Milk contains a variety of substances suspended in water. Much of the protein found in milk is in the form of casein. Casein consists of long chains of protein molecules that are grouped together into small droplets called micelles.


DISCUSSION OF PROTEIN GLUE

Full fat milk also contains fat globule. Although most of the fat globules have been removed from fat free skim milk, it still contains most of the casein, which is why it works well for this experiment. As a side note, these micelles and fat globules are approximately 1 micrometer in size, which is close enough to the wavelength of visible light to cause scattering. This is the reason milk is white.
Altering the acidity of the milk causes the structure of the proteins in the casein to change. Casein is a protein that is found in milk and used independently in many foods as a binding agent. Technically, it is part of a group called phosphoproteins, collections of proteins bound to something containing phosphoric acid. Casein may also be called caseinogen, particularly in European foods. Before the acid is added, the proteins are in the form of tiny micelles. After adding the acid, the proteins form larger structures.
By straining the solids (called curds) through the coffee filter, you can remove a majority of the liquids. Next, by drying the casein with a paper towel you can remove most of the remaining liquid. Adding a base (sodium bicarbonate) to neutralize the acid (and produce water) causes the casein to become soluble again, except now the concentration is much higher. What makes this experiment so cool is that it demonstrates several fundamental chemical processes. An acid is used to precipitate a solid, which is then extracted and purified. Next, a base is then used to reverse the reaction leaving behind a highly concentrated form of the original material.
Non-food products also use casein, some examples being adhesives, plastics and cosmetics. Finally, we examine the residual glue after it has dried a few days. It should dry into plastic like material. Below is picture of the result that we get.