|Topic:||Chemical Digestion of Proteins .|
|Details:|| The most commonly utilized chemical reagent that cleaves peptide bonds by recognition of specific amino acid residues is cyanogen bromide (CNBr). This reagent causes specific cleavage at the C-terminal side of M residues. The number of peptide fragments that result from CNBr cleavage is equivalent to one more than the number of M residues in a protein.
The most reliable chemical technique for C-terminal residue identification is hydrazinolysis. A peptide is treated with hydrazine, NH2–NH2, at high temperature (90°C) for an extended length of time (20-100hr). This treatment cleaves all of the peptide bonds yielding amino-acyl hydrazides of all the amino acids excluding the C-terminal residue which can be identified chromatographically compared to amino acid standards. Due to the high percentage of hydrazine induced side reactions this technique is only used on carboxypeptidase resistant peptides.
Size Exclusion Chromatography
This chromatographic technique is based upon the use of a porous gel in the form of insoluble beads placed into a column. As a solution of proteins is passed through the column, small proteins can penetrate into the pores of the beads and, therefore, are retarded in their rate of travel through the column. The larger proteins a protein is the less likely it will enter the pores. Different beads with different pore sizes can be used depending upon the desired protein size separation profile.
Ion Exchange Chromatography
Each individual protein exhibits a distinct overall net charge at a given pH. Some proteins will be negatively charged and some will be positively charged at the same pH. This property of proteins is the basis for ion exchange chromatography. Fine cellulose resins are used that are either negatively (cation exchanger) or positively (anion exchanger) charged. Proteins of opposite charge to the resin are retained as a solution of proteins is passed through the column. The bound proteins are then eluted by passing a solution of ions bearing a charge opposite to that of the column. By utilizing a gradient of increasing ionic strength, proteins with increasing affinity for the resin are progressively eluted.
Proteins have high affinities for their substrates or co-factors or prosthetic groups or receptors or antibodies raised against them. This affinity can be exploited in the purification of proteins. A column of beads bearing the high affinity compound can be prepared and a solution of protein passed through the column. The bound proteins are then eluted by passing a solution of unbound soluble high affinity compound through the column.
High Performance Liquid Chromatography (HPLC)
In column chromatography the smaller and more tightly packed a resin is the greater the separation capability of the column. In gravity flow columns the limitation column packing is the time it takes to pass the solution of proteins through the column. HPLC utilizes tightly packed fine diameter resins to impart increased resolution and overcomes the flow limitations by pumping the solution of proteins through the column under high pressure. Like standard column chromatography, HPLC columns can be used for size exclusion or charge separation. An additional separation technique commonly used with HPLC is to utilize hydrophobic resins to retard the movement of nonpolar proteins. The proteins are then eluted from the column with a gradient of increasing concentration of an organic solvent. This latter form of HPLC is termed reversed-phase HPLC.
Electrophoresis of Proteins
Proteins also can be characterized according to size and charge by separation in an electric current (electrophoresis) within solid sieving gels made from polymerized and cross-linked acrylamide. The most commonly used technique is termed SDS polyacrylamide gel electrophoresis (SDS-PAGE). The gel is a thin slab of acrylamide polymerized between two glass plates. This technique utilizes a negatively charged detergent (sodium dodecyl sulfate) to denature and solubilize proteins. SDS denatured proteins have a uniform negative charge such that all proteins will migrate through the gel in the electric field based solely upon size. The larger the protein the more slowly it will move through the matrix of the polyacrylamide. Following electrophoresis the migration distance of unknown proteins relative to known standard proteins is assessed by various staining or radiographic detection techniques.
The use of polyacrylamide gel electrophoresis also can be used to determine the isoelectric charge of proteins (pI). This technique is termed isoelectric focusing. Isoelectric focusing utilizes a thin tube of polyacrylamide made in the presence of a mixture of small positively and negatively charged molecules termed ampholytes. The ampholytes have a range of pIs that establish a pH gradient along the gel when current is applied. Proteins will, therefore, cease migration in the gel when they reach the point where the ampholytes have established a pH equal to the proteins pI.
Centrifugation of Proteins
Proteins will sediment through a solution in a centrifugal field dependent upon their mass. Analytical centrifugation measures the rate that proteins sediment through various density solutions. The most common solution utilized is a linear gradient of sucrose (generally from 5–20%). Proteins are layered atop the gradient in an ultracentrifuge tube then subjected to centrifugal fields in excess of 100,000 x g. The sizes of unknown proteins can then be determined by comparing their migration distance in the gradient with those of known standard proteins.
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