Differential protein precipitation
EXERCISE 3: DIFFERENTIAL PROTEIN PRECIPITATION
Vanessa Andrea Estepa
University of Mindanao, College of Science and Mathematics
ABSTRACT: Denaturation is the disruption in the original conformation of the protein wherein the secondary, tertiary and quaternary structures are all affected. Denaturation is brought about by various kinds of physical and chemical means; this includes the addition of strong acids, heavy metal cations, alkaloidal reagents, salting out and addition of organic solvents. Precipitation is commonly observed when a protein is denatured. The experiment was done to explain the principles behind each method of denaturing proteins. In addition of strong acids to protein solution, a precipitate formed due to the disruption of hydrogen bonds in the protein. In addition of heavy metal, a precipitate also formed due to some binding activities and disruption of disulfide linkages. In addition of alkaloidal reagents, precipitate formed because of some neutralization of the charges. Organic solvents interfere with H-bonds and protein molecules to cause precipitation. And in the salting out, when ammonium sulfate was added to the protein solution, the shell of hydration is removed and the solubility changed that’s why the protein precipitated. Only a small percent recovery was obtained.
INTRODUCTION
Proteins are polymers of amino acids. A typical protein may be composed of hundreds of amino acids. They are held in their native or natural confirmations by a combination of forces: hydrogen bonds, salt bridges (also called ionic interactions), disulfide bridges, and hydrophobic interactions. Changing the conformation of a protein by disrupting these forces is called denaturation (Willbrand, n.d.). According to Ophardt (2003), denaturation of proteins involves the disruption and destruction of the bonding interactions responsible for the secondary and tertiary structure. A variety of reagents and conditions can cause denaturation. In this experiment,
Vanessa Andrea Estepa
University of Mindanao, College of Science and Mathematics
ABSTRACT: Denaturation is the disruption in the original conformation of the protein wherein the secondary, tertiary and quaternary structures are all affected. Denaturation is brought about by various kinds of physical and chemical means; this includes the addition of strong acids, heavy metal cations, alkaloidal reagents, salting out and addition of organic solvents. Precipitation is commonly observed when a protein is denatured. The experiment was done to explain the principles behind each method of denaturing proteins. In addition of strong acids to protein solution, a precipitate formed due to the disruption of hydrogen bonds in the protein. In addition of heavy metal, a precipitate also formed due to some binding activities and disruption of disulfide linkages. In addition of alkaloidal reagents, precipitate formed because of some neutralization of the charges. Organic solvents interfere with H-bonds and protein molecules to cause precipitation. And in the salting out, when ammonium sulfate was added to the protein solution, the shell of hydration is removed and the solubility changed that’s why the protein precipitated. Only a small percent recovery was obtained.
INTRODUCTION
Proteins are polymers of amino acids. A typical protein may be composed of hundreds of amino acids. They are held in their native or natural confirmations by a combination of forces: hydrogen bonds, salt bridges (also called ionic interactions), disulfide bridges, and hydrophobic interactions. Changing the conformation of a protein by disrupting these forces is called denaturation (Willbrand, n.d.). According to Ophardt (2003), denaturation of proteins involves the disruption and destruction of the bonding interactions responsible for the secondary and tertiary structure. A variety of reagents and conditions can cause denaturation. In this experiment,
References: Baldwin, M.M. 1996. Salting out. Carpio, Paulo T. n.d. Isolationand characterization of egg white proteins. Elearnbiotech. Education Portal. Life Science. Precipitation of proteins and nucleic acids. Moore, Ryan. 2009 Protein Analysis. Ophardt, Charles E. 2003. Virtual Chembook. El mhurst College. Shankara. 2008. Laboratory Manual for Practical Biochemistry. Willbrand, Ann. N.d. Proteins and Denaturing Agents. Young, Derek R. 1994. Introduction to Biochemical Engineering Term Project