Proteins are an important class of biological macromolecules that present in all organisms consist of amino acids which classified based on their physical size. The protein have four structure which is primary, secondary, tertiary and quarternary structure. The polymer of protein is also known as a polypeptide that consists of a sequence of 20 different L-α-amino acids, also referred to as residues. The protein able to perform their biological function by fold into one or more specific spatial conformations due to the presence of covalent bond, hydrogen bond, Van Der Waals forces, disulphide bond. To determine the functions of proteins at a molecular level, it is necessary to determine their three dimensional structure.
The primary structure is sequence of amino acid which is the primary structure of protein. The secondary structure is mostly the hydrogen-bonding interaction between adjacent amino acids residues and the polypeptide chain can arrange itself into characteristic helical or pleated segments. Tertiary structure is the polypeptide chains of protein molecules bend and fold to assume more compact three-dimensional shape and tertiary globular protein are roughly spherical in shape and the polypeptide chain is compactly folded so the hydrophobics amino acid side chainsare in the interior of the molecule and the hydrophilic side chains are on the outside exposed to the solvent. Quarternary structure is more than one protein is interacting polypeptide chains of characteristic tertiary structure.
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The α-amino acid consists of a backbone part which is present in all amino acid types, the side chain that is unique to each type of residue. An exception from this rule proline, because of the hydrogen atom is replaced by a bond to the side chain. Because the carbon atom is bound to four different groups called chiral carbon, however only one of the isomers occur in biological proteins. The 20 amino acids have different physical and chemical properties, including their electrostatic charge, pKa, hydrophobicity, size and specific functional groups which is the major role in molding protein structure. Because of the delocalization of the electrons from the double bond the peptide bond tend to be planar in shape. The rigid peptide dihedral angle, ω which is the bond between C1 and N) is always close to 180 degrees. The dihedral angles phi φ which is the bond between N and Cα) and psi ψ which is the bond between Cα and C1) can have a certain range of possible values. These angles are the internal degrees of freedom of a protein is important on controlling the protein's conformation. . When water molecules come in contact they are faced whichever way the water molecule face, it would appear that one or more of the four charges per molecule will have to point towards the inert solute molecule and thus to be lost in the hydrogen bond formation
Each of the amino acid has at least one amine and one acid functional group as the name implies. The different properties is cause by the variations in the structures of different R groups. The R group is always referred to as the amino acid side chain. Amino acids have special common names, however, a three letter abbreviation for the name is used most of the time. A second abbreviation , single letter, is used in long protein structures. There are basically four different classes of amino acids determined by different side chains which is non-polar and neutral, polar and neutral, acidic and polar, basic and polar. It involves several interactions between various amino acid side groups (R group) in the polypeptide chain. The R group can contain functional groups such as the sulfhydryl group, the hydroxyl group, or additional carboxyl or amino groups. Possible interactions of R groups that contribute to the tertiary protein structure such as hydrophobic interaction which amino acids with nonpolar side groups interact at the core of the protein, hydrogen bonds between side groups and ionic bonds between oppositely charged side groups.
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Native Gel electrophoresis
Native gel electrophoresis is known as a technique used in protein electrophoresis where the protein are not denatured and separated based on their charged-to-mass ratio. There are two types of native gel electrophoresis which are polyacrylamide gel and agarose gel. Native or non denaturing gel electrophoresis is run in the absence of SDS (sodium dodecyl sulfate polyacrylamide gel) because under native condition, separation of proteins depends on many factors including size, shape and native charge. One significant approach of native gel electrophoresis is to leave out the SDS and reducing agent from the standard SDS-PAGE since the gel electrophoresis solution is prepared without SDS. In native PAGE the mobility is depending on the both protein's charge and hydrodynamic size. The electric charge driving the electrophoresis is governed by the intrinsic charge on the protein at the pH of the running buffer. Since the protein retains its folded conformation, its hydrodynamic size and mobility on the gel also be vary with the nature of this conformation. Electrophoretic migration happens due to the proteins that carry the net negative charge in alkaline running buffer. The higher the negative charge density the faster protein will migrate. In order to maintain the integrity of proteins it is a must to keep apparatus cool and minimize the effect of denturation and proteolysis, where as the irreversible damages protein of interest such as denaturation or aggregation might occur if the extreme pH not avoided. The advantage of native gel is that is possible to recover the protein in their native form after the separation.Because it can be done by passive diffusion or electroelution.
Specific features of native electrophoresis techniques using dodecylmaltoside-solubilized membrane protein complexes.
SDS PAGE Electrophoresis
Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a technique to separate proteins based on its mobility electrophoretic or by determine the length of polypeptide chain or molecular weight. The binding of SDS results in fraction by size this is due to SDS gel electrophoresis of sample having identical charge per unit mass. The SDS page gel consist of resolving gel or separating gel which has a higher polyacrylamide content and it polymerized in a gel caster and to allowed polymerized a thin layer of butanol is added. After polymerized the thin layer of butanol is washed with distilled water then the loading gel or stacking gel is poured and the combed is placed to create the wells and the stacking gel is have large pores of polyacrylamide gel this condition provide an environment for Kohrausch reactions is determining molar conductivity and make SDS coated protein are concentrated to several fold and a thin starting zone of the order of 19μm is achieved in a few minutes . The electrophoresis is set up with running buffer covering the gel in the negative electrode chamber and in the lower positive chamber. Next the sample of denatured proteins added in the well using pipette finally the apparatus is supply with power source to separate the protein band. Because of the electric field is applied across the gel the negatively charge of protein is migrate across the gel toward positively charge electrode. The migration of protein is depend on their size, the shorter protein will move easily through the pore in the gel than the larger ones will have more difficulty. The smaller protein will have traveled farther down the gel and the larger protein will have remained closer to the origin. The sample of protein can be stained with coomasive brilliant blue or silver stain for allow visualization of the separated protein and different protein will appear as distinct bands within the gel.
Laemmli gels are composed of two different gels (stacker and running gel), each cast at a different pH. In addition, the gel buffer is at a third, different pH. The running gel is buffered with Tris by adjusting it to pH 8.8 with HCl. The stacking gel is also buffered with Tris but adjusted to pH 6.8 with HCl. The sample buffer is also buffered to pH 6.8 with Tris HCl (note all the chloride ions - they will become important in a minute). The electrode buffer is also Tris, but here the pH is adjusted to a few tenths of a unit below the running gel (in this case 8.3) using only glycine - nothing else. We run our gels at constant voltage.
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The migration of protein is depend on their size, the shorter protein will move easily through the pore in the gel than the larger ones will have more difficulty. The smaller protein will have traveled farther down the gel and the larger protein will have remained closer to the origin. The sample of protein can be stained with coomasive brilliant blue or silver stain for allow visualization of the separated protein and different protein will appear as distinct bands within the gel.
Lowry method, one of the method to quantitatively determine the concentration of protein in solution, is widely used due to its sensitivity is moderately constant from protein to protein. This method uses the characteristic of protein to produce stable complex with heavy metals such as copper. Under alkaline condition, copper ion will react with peptide bond of protein and produce Cu+. Cu+ reacts with Folin reagent and cause Folin Ciocalteau reaction, which the composition of Folin reagent, phosphomolybdotungstate is reduced to heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic amino acids. These reactions will result to the production of strong blue colour which depends on the tyrosine and tryptophan content. The blue color is then measured by absorbance at 750nm and the unknown protein concentration is determined by using graph of absorbance against the amount of standard protein. Lowry method sensitivity is about 0.01mg of protein/mL. The sensitivity can be improved up to 20% by vortex-mixing the two portions that have been added the Folin reagent, and improved up to 50% by add dithiothreitol 3 minutes after the addition of Folin reagent. Lowry method is very good for protein containing chromophore such as hemes and flavins. However, this method has some disadvantages. It is sensitive to pH changes, thus the pH of assay solution should be maintained at 10 to 10.5. It is also sensitive to variety of contaminants or compounds such as some amino acid derivatives, certain buffers, drugs, lipids, sugars, salts, nucleic acids and sulphydryl reagents.
Lowry reaction can also be interfered by Tris buffer, EDTA, nonionic and cationic detergent, zwitterionic buffers and thiol compounds. The effects of interference can be minimized by dilution.
Figure 4: Procedure of Lowry method