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ABSTRACT
Proteins are large molecules consisting amino acid chains which our bodies and the cells in our bodies need to be able to function properly. Our body structures, functions, the regulation of the body's cells, tissues and organs cannot exist without proteins. Our muscles, skin, bones and many other parts of the body contain significant amounts of protein. Protein accounts for about twenty percent of our total body weight. This part of the experiment was performed following the proper isolation of the protein myoglobin from the beef muscle by process of grinding. Myoglobin is important to oxygen storage in living organisms. It is especially found in muscles and it gives it its red colour. The mechanism used in the enzymatic hydrolysis of myoglobin is a naturally occurring enzyme which is usually used by the body to digest proteins in food. These enzymes are called proteases, peptidases, or proteolytic enzymes. They cleave the peptide bonds of proteins at specific amino acid residues, thereby separating and dividing the protein into smaller amino acid chains and some free amino acids. This experiment was performed with the use of a protein mixture, saturated protease solution, a phosphate buffer with pH 7.5, and incubation at 35-40C. The effect of the protease on the myoglobin was evident, as it turned the solution with cloudy brick-red residue into a clear solution with cloudy sediments at the bottom.
INTRODUCTION
Proteins are large molecules consisting amino acid chains which our bodies and the cells in our bodies need to be able to function properly. Our body structures, functions, the regulation of the body's cells, tissues and organs cannot exist without proteins. Our muscles, skin, bones and many other parts of the body contain significant amounts of protein. Protein accounts for about twenty percent of our total body weight. They are the most abundant organic molecules found in living cells. Myoglobin is a protein commonly found in muscle tissues. It is characterized by its cloudy reddish coloured residue and, functioning similarly to hemoglobin found in blood, it carries and stores oxygen for proper muscle function. It is the reason why muscle tissues appear to have a reddish tinge. Myoglobin is an unusual protein as it is made up almost exclusively of -helices joined by short loops. Most proteins have both -helices and pleated sheets.i Myoglobin can be isolated by ammonium sulfate precipitation from the buffered muscle extract.ii Meanwhile, the enzyme that was used to perform the enzymatic hydrolysis on the protein myoglobin is called protease. Proteases (also called peptidases or proteolytic enzymes) occur naturally in all organisms. Simply defined, protease catalyzes the hydrolytic breakdown of proteins.iii Protease refers to a group of enzymes whose catalytic function is to hydrolyze (breakdown) peptide bonds of proteins. They are also called proteolytic enzymes or proteinases. Proteases differ in their ability to hydrolyze various peptide bonds. Each type of protease has a specific kind of peptide bonds it breaks. Examples of proteases include: fungal protease, pepsin, trypsin, chymotrypsin, papain, bromelain, and subtilisin.iv
Myoglobin can be isolated from the meat by ammonium sulfate precipitation from the buffered muscle extract. The experimentation of enzymatic hydrolysis and the analysis of the products are done to determine and extract more information about their amino acid compositions. The twenty amino acids are commonly found as hydrolysis products of proteins because, as mentioned earlier, enzymes catalyze the breakdown of myoglobin. The product of this enzymatic hydrolysis of myoglobin was stored to be used for other experimental procedures. Examples of such tests are the following: the tests for qualitative color reactions, separation and identification of amino acids by thin layer chromatography, and protein assay using the Bradford Method. The qualitative color reactions involve several tests such as the Biuret test, the Ninhydrin test, the Xanthoproteic test, Millons Test, the Hopkins-Cole test, the Sakaguchi test, the Nitroprusside test, Fohls test, the test for amides, and the Pauly test. The separation and identification of amino acids by thin layer chromatography can be used for the qualitative analysis of the amino acid constituents of the acid, alkaline, and enzymatic protein hydrolysate. The separation in this type of chromatography is based on the polarity of the solutions. The Bradford Assay is commonly used to determine the total protein concentration of a sample. The method is based on the binding of Coomassie dye to proteins in acidic solution leading to an increased absorbance of the sample at 595 nm. The assay is sensitive to about 20 to 200 m protein.
Place 6.0 g minced meat beef heart (or steak) and 6 mL 70% (NH4)2SO4 solution in a small beaker. Gently stir the mixture for one minute to release the myoglobin. Express the dark-red extract into a new beaker using cheesecloth. Centrifuge the extract at 13,000 x g for 5 minutes. Transfer 1.5 mL of the supernatant into another empty centrifuge tube. Add 0.30-0.35 g (NH4)2SO4 crystals ground to fine powder. Mix gently until the solid dissolves. Avoid frothing. Centrifuge sample again for at least five minutes. Decant off supernatant, describe the appearance of purified myoglobin residue.
Before undertaking this dilution method, one must first know how to compute for dilutions. The protein is diluted by adding one part of protein to 9 parts of distilled water, giving it a 9:1 ratio, involving taking one part of the sample and addition it to 9 parts of distilled water. In this case the dilution factor is 10. This procedure is continued until the ratio of 1:1000 is achieved. Several test tubes, a graduated cylinder, and a pipette were used by the group to perform this dilution.
Assay
Using
Bradford
Bradford reagent Bovine serum albumin (BSA) standard (10 mg/mL) Evaporated milk sample
4. Incubate the hard glass tube in a water bath maintained at 35-40C for 60 minutes
In this part of the procedure, the water bath to be used depends on the source of the enzyme.
* Alternatively, the digestion of myoglobin may be carried out overnight at room temperature 5. Allow the mixture to cool off before using it in the procedures for qualitative color reactions and for the separation and identification of amino acids by thin layer chromatography.
Identification of by Paper
Draw the origin as pencil line across the paper with a 1.5 cm margin from the bottom of the longer edge. Mark thirteen equidistant points for spotting. Apply standards 5 times and samples 10 times. Place the paper inside a pre-equilibrated chamber. The lever of the solvent should be below the origin. Cover the chamber. It is important that the solvent be absorbed by the paper undisturbed. Air-dry chromatogram and spray lightly with 1% ninhydrin reagent. Place chromatogram inside an oven (group used hot plate istead) and amino acids will appear as blue, purple or yellow spots.
Assay
Using
Bradford
Prepare a series of test tubes as follows: mL Standard 0 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 mL H20 1.5 1.40 1.35 1.30 1.25 1.20 1.15 1.10 1.05
Dilute the milk sample 1:500, 1:1000, and 1:2000 with distilled water. Take 1.5 mL of diluted and label them as numbers 10, 11, and 12. Add 1.5 mL Bradford reagent to each test tube. Mix well. Let it stand for 5 minutes. Read the absorbance at 595 nm within the hour. Use tube 1 as the blank.
Isolation of the protein myoglobin resulted in a milky yellow curd. Enzymatic hydrolysis of the isolated protein yielded to a clear solution with sediments.
Due to the lack of time, the group was not able to finish the paper chromatography, thus the ascension of the solvent only reached half way.
Table 1. Results of Qualitative Color Reactions with Enzyme Hydrolyzed Samples Test Biuret Test Ninhydrin Test Xanthoproteic Test Millons Test Hopkins-Cole Test Sakaguchi Test Nitroprusside Test Fohls Test Test for Amides Pauly Test Reaction (+) Blue upper layer (+) Violet solution (+) Turbid solution (+) Turbid solution (+) Yellow layer (+) Colorless solution (+) Yellow solution (+) Yellow solution, brown sediments (+) Yellow layer, litmus paper is red to red (+) Pale yellow
References
i
The biuret test is used to detect the presence of peptide bonds while the Ninhydrin test is a typical test for an -amino acid. The Xanthoproteic test detects side chains of aromatic amino acids while the Millons and Hopkins-Cole tests determine tyrosine and tryptophan residues, respectively. The Nitroprusside test is used to find out if sulfur-containing amino acids are present; test for amides it used to detect R-groups of asparagine and glutamine.
Figure 2. Paper Chromatography of Acidic, Basic, and Enzymatic Hydrolyzed Samples and Amino Acids
Acidic
Basic
Enzymatic
Amino Acids