Prabhakar Singh first sem biochem_ paper first _unit iv_ protein

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Peptide Cleavage

Ramachandran plot

ARamachandran plot(also known as aRamachandran diagramor a[,] plot), originally developed in 1963 byG. N. Ramachandran, C. Ramakrishnan, and V. Sasisekharan,[1]is a way to visualize backbonedihedral angles against ofamino acidresidues inprotein structureand identify sterically allowed regions for these angles.

The figure at left illustrates the definition of the and backbone dihedral angles[2](called and ' by Ramachandran). The angle at the peptide bond is normally 180, since the partial-double-bond character keeps thepeptideplanar.[3]The figure at top right shows the allowed , backbone conformational regions from the Ramachandran et al. 1963 and 1968 hard-sphere calculations: full radius in solid outline, reduced radius in dashed, and relaxed tau (N-Calpha-C) angle in dotted lines.[4]

Becausedihedral anglevalues are circular and 0 is the same as 360, the edges of the Ramachandran plot "wrap" right-to-left and bottom-to-top. For instance, the small strip of allowed values along the lower-left edge of the plot are a continuation of the large, extended-chain region at upper left.

Biuret Testlab procedure:mix albumin and 10% NaOH and add 0.5% CuSO4 drop by drop > dark violet colormix pentose and 10% NaOH and add 0.5% CuSO4 drop by drop > light violet colorexplanation:reacts with peptide bonds in proteinblue to violet colorTwo peptide bonds at leastXanthoproteic reactionlab procedure:mix albumin with conc. nitric acid and heat > yellow solutioncool the solution and add ammonium hydroxide > orange solutionexplanation:Nitric acid gives a color when heated with proteins containing tyrosine (yellow color) or tryptophan (orange color); the color is due to nitrationaromatic phenyl ring is nitrated to give yellow colored nitro-derivativesat alkaline pH, the color changes to orange due to the ionization of the phenolic group

Millons testlab procedure:mix albumin and millons rgt and heat> red flocculent pptexplanation:positive results with proteins containing the phenolic amino acid tyrosineGlyoxylic acid reactionlab procedure:mix albumin with hopkins-cole reagentincline the test tube and add conc. sulfuric acid >violet ringexplanation:Hopkins-Col reagent (magnesium salt of oxalic acid) gives positive results with proteins containing the essential amino acid tryptophan indicating a high nutritive valuespecific test for detecting tryptophanHellers Ring testlab procedure:mix 5 ml of conc. nitric acid with albumin by inclining the test tube >white ringexplanation:Nitric acid causes denaturation of proteins with the formation of a white pptused to test the presence of albumin in urine

Reduced Sulfur Testlab procedure:mix albumin with 40% NaOH and add 10 drops of lead acetate solution > black pptexplanation:Proteins containing sulfur (in Cysteine and cystine) give a black deposit of lead sulfide (PbS) when heated with lead acetate in alkaline mediumAdamkiewicz Reactionlab procedure:mix 3 drops of albumin with glacial acetic acidadd conc. sulfuric acid to the solution> violet ringexplanation:detect the presence of the amino acid tryptophan in proteinsred/purple colour

Thebiuret testis achemical testused for detecting the presence ofpeptide bonds. In the presence of peptides, acopper(II)ionformsviolet-coloredcoordination complexesin analkalinesolution.[1]Several variants on the test have been developed, such as the BCA test and the Modified Lowry test.[2]The biuret reaction can be used to assess theconcentrationof proteins because peptide bonds occur with the same frequency per amino acid in the peptide. The intensity of the color, and hence the absorption at 540nm, is directly proportional to the protein concentration, according to theBeer-Lambert law.Despite its name, the reagent does not in fact containbiuret((H2N-CO-)2NH). The test is so named because it also gives a positive reaction to the peptide-like bonds in the biuret molecule.

Procedure[edit]An aqueous sample is treated with an equal volume of 1% strong base (sodium or potassium hydroxide most often) followed by a few drops of aqueouscopper(II) sulfate. If the solution turns purple, protein is present. 5160mg/mLcan be determined. A peptide of a chain length of at least 3 amino acids is necessary for a significant, measurable color shift with these reagents.[3]

Biuret reagent[edit]TheBiuret reagentis made ofsodium hydroxide(NaOH) and hydrated copper(II) sulfate, together withpotassium sodium tartrate.[4]Potassium sodium tartrate[5]is added to complex to stabilize the cupric ions. The reaction of the cupric ions with the nitrogen atoms involved in peptide bonds leads to the displacement of the peptide hydrogen atoms under the alkaline conditions. A tri or tetra dentate chelate of with the peptide nitrogen produces the "buret" color. This is found with dipeptides (Datta,S.P., Leberman,R., and Rabin,B.R., Trans.Farad.Soc. (1959), 55, 2141.)The reagent is commonly used in the biuretproteinassay, acolorimetrictest used to determine proteinconcentrationbyUV/VIS spectroscopyat wavelength 565nm.High sensitivity variants of the biuret test[edit]Two major modifications of the biuret test are commonly applied in modern colorimetric analysis of peptides: the bicinchoninic acid (BCA) assay and the Lowry assay. In these tests, the Cu+formed during the biuret reaction reacts further with other reagents, leading to a deeper color.In theBCA test, Cu+forms a deep purple complex withbicinchoninic acid(BCA),[6]which absorbs around 562nm, producing the signature violet color. The water-soluble BCA/copper complex absorbs much more strongly than the peptide/copper complex, increasing the sensitivity of the biuret test by a factor of around 100: the BCA assay allows to detect proteins in the range of 0.0005 to 2mg/mL). Additionally, the BCA protein assay gives the important benefit of compatibility with substances like up to 5% surfactants in protein samples.In the Lowry protein assay Cu+is oxidized back to Cu2+by MoVIinFolin-Ciocalteu's reagent, which formsmolybdenum blue(MoIV). Tyrosine residues in the protein also form molybdenum blue under these circumstances. In this way, proteins can be detected in concentrations between 0.005 and 2mg/mL.[7]Molybdenum blue in turn can bind certain organic dyes such asmalachite greenandAuramin O, resulting in further amplification of the signal.[8]

Theory:Amino acids are building blocks of all proteins, and are linked in series by peptide bond (-CONH-) to form the primary structure of a protein. Amino acids possess an amine group, a carboxylic acid group and a varying side chain that differs between different amino acids.There are 20 naturally occurring amino acids, which vary from one another with respect to their side chains. Their melting points are extremely high (usually exceeding 200C), and at their pI, they exist as zwitterions, rather than as unionized molecules.Amino acids respond to all typical chemical reactions associated with compounds that contain carboxylic acid and amino groups, usually under conditions where the zwitter ions form is present in only small quantities. All amino acids (except glycine) exhibit optical activity due to the presence of an asymmetric Carbon atom. Amino acids with an L configuration are present in all naturally occurring proteins, whereas those with D forms are found in antibiotics and in bacterial cell walls.

Protein foldingProtein foldingis the process by which aproteinstructure assumes its functional shape or conformation. It is the physical process by which apolypeptidefolds into its characteristic and functionalthree-dimensional structureAmino acids interact with each other to produce a well-defined three-dimensional structure, the folded protein (the right hand side of the figure), known as thenative state. The resulting three-dimensional structure is determined by the amino acid sequence (Anfinsen's dogma).[2]Experiments[3]beginning in the 1980s indicate the codon for an amino acid can also influence protein structure.The correct three-dimensional structure is essential to function, although some parts of functional proteinsmay remain unfolded,[4]so thatprotein dynamicsis important. Failure to fold into native structure generally produces inactive proteins, but in some instances misfolded proteins have modified or toxic functionality.Relationship between folding and amino acid sequenceThe amino-acid sequence of a protein determines its native conformation.[7]A protein molecule folds spontaneously during or afterbiosynthesis. While thesemacromoleculesmay be regarded as "folding themselves", the process also depends on thesolvent(waterorlipid bilayer),[8]the concentration ofsalts, thepH, thetemperature, the possible presence of cofactors and of molecularchaperones.Minimizing the number of hydrophobic side-chains exposed to water is an important driving force behind the folding process.[9]Formation of intramolecular hydrogen bonds provides another important contribution to protein stability.[10]The strength of hydrogen bonds depends on their environment, thus H-bonds enveloped in a hydrophobic core contribute more than H-bonds exposed to the aqueous environment to the stability of the native state.[11]

The process of folding often beginsco-translationally, so that theN-terminusof the protein begins to fold while theC-terminalportion of the protein is still beingsynthesizedby theribosome. Specialized proteins calledchaperonesassist in the folding of other proteins.[12]ChaperoninChaperoninsare proteins that provide favourable conditions for the correct folding of other proteins, thus preventing aggregation. Newly made proteins usually mustfoldfrom a linear chain of amino acids into a three-dimensional form. Chaperonins belong to a large class of molecules that assist protein folding, calledmolecular chap