Protein and Amino Acid Chemistry

Reactions of Proteins

• The colloidal nature of proteins can be observed through heat coagulation and salting out.
• Acidity and basicity of a protein refer to its reaction with a reagent, leading to precipitation by mineral acids, salts of heavy metals, and alkaloidal reagents.
• The presence of specific chemical groups determines protein reactions.

Amino Acids

• Amino acids include glycine (Gly, G), alanine (Ala, A), serine (Ser, S), threonine (Thr, T), and cysteine (Cys, C).
• Valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), methionine (Met, M), and proline (Pro, P) belong to amino acids family.
• Phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan (Trp, W), aspartic acid (Asp, D), glutamic acid (Glu, E) are also types of amino acids.
• Asparagine (Asn, N), glutamine (Gln, Q), histidine (His, H), lysine (Lys, K), and arginine (Arg, R) are amino acids as well.

Ninhydrin Reaction

• Free amino acids, small peptides, and proteins react with ninhydrin, producing a purple color.
• Ninhydrin forms hydrindantin with amino acids, which then reacts with ammonia and another ninhydrin molecule to create Ruhemann's purple.
• Imino acids yield a yellow color due to the absence of alpha amino acids.

Biuret Test

• Copper from copper sulfate forms a coordination complex with peptide bonds in an alkaline medium.
• Compounds containing two or more peptide bonds react with cupric ions in a basic solution, resulting in a violet-pink complex.
• A blue solution indicates the tested compound is an amino acid, a dipeptide, or neither

Xanthoproteic Test

• Aromatic amino acids like phenylalanine, tyrosine, and tryptophan respond positively.
• Phenylalanine shows a weak positive result.
• Concentrated nitric acid causes the aromatic phenyl ring to undergo nitration, producing yellow colored nitro-derivatives.
• At alkaline pH, the color shifts to orange because of the phenolic group's ionization.

Millon-Nasse Test

• Specifically tests for the phenolic group of tyrosine; the hydroxy benzene group reacts with Millon's reagent, forming a red colored complex.

Hopkins-Cole Reaction

• Presence of amino acid containing indole group is indicated by the test.
• Glyoxylic acid functions as the oxidizing agent, oxidizing the indole nucleus of tryptophan.
• The oxidized indole ring reacts with formaldehyde, giving a violet colored ring where two liquids connect.

Bromine Water Test

• This test identifies free tryptophan.
• Adding bromine water and n-amyl alcohol to a sample with tryptophan leads to the formation of a pinkish lavender or violet color in the alcohol layer.

Pauly Reaction

• Specifically identifies tyrosine or histidine.
• The reagent contains sulphanilic acid dissolved in hydrochloric acid.
• Upon diazotization, sulphanilic acid, in the presence of sodium nitrite and hydrochloric acid, forms a diazonium salt.
• The diazonium salt couples with either tyrosine or histidine, in an alkaline medium, the product is a red coloured chromogen (azo dye).

Lead Acetate Reaction

• Boiling a protein solution with an alkali splits the sulfur of cysteine and cystine, forming sodium sulfide.
• Black lead sulfate is formed when this combines with lead acetate.
• Methionine does not answer this test as sulfur is in a thioether linkage and isn't split through boiling with strong alkali.

Sakaguchi Reaction

• Under alkaline conditions, α-naphthol (1-hydroxy naphthalene) reacts with Arginine.
• When treated with hypobromite or hypochlorite, it forms a red color indicating a positive result.
• Arginine, whether free or combined, tests positive.

Nucleic Acids

• Nucleic Acids are macrobiopolymers, high molecular weight, with mononucleotide as the repeating unit.
• The two structural types are DNA and RNA, made of nitrogenous bases, sugar, and a phosphate group.
• Nitrogenous bases can be pyrimidines (cytosine, thymine, uracil) or purines (adenine & guanine).
• Deoxyribonucleic Acid carries genetic information

DNA Extraction Process

• Lysis involves breaking open cells to release the nucleus, and then breaking the nucleus to release DNA.
• DNA extraction buffer components include H₂O and detergent, which contains surfactants to dissolve the cell membrane (lipid bilayer).
• Sodium chloride removes proteins and carbohydrates.
• Borax buffers the pH, and ethanol precipitates the dissolved DNA.
• Cold ethanol precipitates DNA; it's less soluble in alcohol
• Low temperatures protects by slowing enzyme activity, which prevents nucleases from breaking it down .
• Purification removes remaining cellular debris and unwanted material and is done by alcohol rinsing.
• Hydrolysis of nucleic acid by either chemical or enzymatic methods, yields purine and pyrimidine bases, oligonucleotides which may contain upto 20 residues linked together, nucleosides, ribose or deoxyribose and phosphates.

Interactions of Nucleic Acids

• Phosphodiester bonds join nucleotides in each chain.
• Hydrogen bonds join the bases.
• Van der Waal's forces between stacked bases

Qualitative Tests for Nucleic Acids

• Benedict's test (test for reducing sugars)
• Orcinol Test (test for pentoses)
• Test for purine bases
• Test for inorganic phosphate

Benedict's Test

• Test for reducing sugars where aldehydes and ketones are oxidized in the presence of an alkaline alkaline copper solution
• The cupric ions are reduced to cuprous and brick-red Cu₂O creating a precipitate

Orcinol Test

• Test for pentoses where pentoses decomposes after being heated with concentrated hydrochloric acid, resulting in the formation of furfural
• The furfural them condenses with orcinol (3,5-dihydrotoluene) to give blue colored compounds

Test for Purine Bases

• Hydrolysate + NH4 + AgNO3 creates a whitish precipitate
• Unhydrolyzed +NH4 + AgNO3 creates a Grayish-white precipitate

Test for Inorganic Phosphate

• Presence of inorganic phosphate can be detected though the the formation of a yellow precipitate after specific binding.