Isolation of Proteins: Myoglobin from muscle Michelle Nhat Ly Reyes, Kristiana Margarette Romina, Cyla Mariel Salvadora, Joanna

Gee San Pedro, Gabriel Santos Group 6 2F-Pharmacy Biochemistry Laboratory ABSTRACT
Myoglobin is a vital protein for oxygen transport in vertebrates. It is present in large concentration in muscle and is responsible for the red color of the organ. Myoglobin is isolated by ammonium sulfate precipitation from the buffered muscle extract and hydrolyzed by acid, alkaline and enzymatic and subjected to different qualitative tests. Paper chromatography had been done to analyze the different amino acid components of the protein.

Introduction
Proteins are a class of organic compounds which are present in and vital to every living cell. Proteins hold together, protect, and provide structure to the body of a multi-celled organism; they catalyze, regulate, and protect the body chemistry and in the form of hemoglobin, myoglobin and various lipoproteins, they affect the transport of oxygen and other substances within an organism. Myoglobin is a singlechain globular protein of 153 amino acids, containing a heme which is an ironcontaining porphyrin prosthetic group in the center around which the remaining apoprotein folds. Biologically, active proteins like myoglobin, are made up of polymers consisting of amino acids linked by covalent peptide bonds. The following are considered in the isolation of an intact protein from its source: Three dimensional (3D) structure of protein (fibrous or globular) Interactions that keep the native conformation of the protein functional (electrostatic, covalent, hydrophobic, H-Bonding, and van der Waals) Acid-base property; and Solubility of protein in different solvents. The solubility of proteins can be altered by changing the pH of their environment. Proteins are insoluble at their isoelectric pH, the pH at which the net charge is equal to zero. This will cause denaturation of the protein meaning that the proteins conformation is altered by the breaking of peptide bonds. This results to a solution containing amino acid fragments which is then called the hydrolysate. Denaturation alters protein function, demonstrating a relationship between structure and function. Hydrolysis of protein and analysis of products are done to obtain information about their composition. The three most common types of hydrolysis are acid, basic and enzymatic. Acid hydrolysis implies a chemical mechanism of hydrolysis catalyzed by a Bronsted or Arrhenius acid. The type of hydrolysis carried out with a basic medium is termed basic hydrolysis. An enzymatic hydrolysis is by the addition of proteolytic enzymes which refer to a group of enzymes each to hydrolyse specific peptide bonds of protein. The following experiments aim to isolate the intact protein from Myoglobin by precipitation (isoeletric and salt-induced) and by difference in solubility (selective dissolution). The isolated proteins will be

characterized qualitatively by colorimetric reactions and paper chromatography.

Express the dark-red extract into a new beaker using a cheese cloth. Centrifuge the extract at 13,000 x g got 5 minutes. Transfer 1.5 mL of the supernatant into another empty centrifuge tube.

EXPERIMENTAL
A. Reagents and materials Minced beef muscle (or steak) (NH4)2SO4 crystals 70% buffer-diluted (NH4)2SO4 solution, pH 7.5 Centrifuge

B. Procedure

Place 6.0 g minced beef heart (or steak) and 6 mL 70% (NH4)2SO4 solution in a small beaker. Gently stir the mixture for 1 minute to release the myoglobin. Figure 2. centrifuge. Supernatant liquid after

Add ~0.30-0.35 g of (NH4)2SO4 crystals ground to fine powder. Mix gently until the solid dissolves. Avoid frothing.

Figure 1. extract)

Myoglobin

extract

(dark-red Figure 3. (NH4)2SO4 crystals ground to fine powder.

Centrifuge the sample again for at least 5 minutes. Decant off the supernatant.

REFERENCES: Books: [1] Campbell, Mary; Farell, Shawn. (2008). Biochemistry (6th ed.). Canada: Brooks/Crole. [2] The Biochemistry Department. (2008). Laboratory manual in General Biochemistry. Quezon city, Philippines: C & E Publishing Inc. Websites: Isolation of protein. Retrieved December 18, 2013 from http://www.chemguide.co.uk/organicprops/ aminoacids/isolationofproteins.html

Figure 4. Purified myoglobin residue.

http://www.kmu.lt/nsc/biochemija/Laborat ory_manual_PART%20I.pdf

Results and Discussion

Protein isolated: Myoglobin The pure myoglobin that was extracted from the meat that was mixed with (NH4)2S04 crystal solution was dark-red solution with residue at the bottom. The meat color is a result of the concentration of the heme pigments, reactions of the pigments with gaseous elements or compounds, and the structural properties of the muscle protein. The most important meat pigment is myoglobin, color is determined by state of the heme group in the pigment. (NH4)2S04 solution significantly decreased the heat stability of myoglobin under all temperature conditions, and the unfolding of polypeptide chains could expose the heme group to aqueous solution thus explaining the dark-red color of the solution.

http://www.public.iastate.edu/~duahn/publicati on/pdf%20files/2.%20PS%20681218%20Effects%20of%20Sodium%20Chloride.p df