Friday, July 23, 2010

43 - Ninhydrin test or Ninhydrin reagent

Ninhydrin (2,2-Dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or primary and secondary amines. When reacting with these free amines, a deep blue or purple color known as Ruhemann's purple is produced. Ninhydrin is most commonly used to detect fingerprints, as the terminal amines or lysine residues in peptides and proteins sloughed off in fingerprints react with ninhydrin.

Ninhydrin can also be used to monitor deprotection in solid phase peptide synthesis (Kaiser Test). The chain is linked via its C-terminus to the solid support, with the N-terminus extending off it. When that nitrogen is deprotected, a ninhydrin test yields blue. Amino-acid residues are attached with their N-terminus protected, so if the next residue has been successfully coupled onto the chain, the test gives a colorless or yellow result.

Ninhydrin is also used in amino acid analysis of proteins: Most of the amino acids are hydrolyzed and reacted with ninhydrin except proline; Also, certain amino acid chains are degraded. Therefore, separate analysis is required for identifying such amino acids that either react differently or don't react at all with ninhydrin. The rest of the amino acids are then quantified colorimetrically after separation by chromatography.

A solution suspected of containing the ammonium ion can be tested by ninhydrin by dotting it onto a solid support (such as silica gel); treatment with ninhydrin should result in a dramatic purple color if the solution contains this species. In the analysis of a chemical reaction by thin layer chromatography (TLC), the reagent can also be used. It will detect, on the TLC plate, virtually all amines, carbamates and also, after vigorous heating, amides.

When ninhydrin reacts with amino acids, the reaction also releases CO2. The carbon in this CO2 originates from the carboxyl carbon of the amino acid. This reaction has been used to release the carboxyl carbons of bone collagen from ancient bones for stable isotope analysis in order to help reconstruct the palaeodiet of cave bears.

A ninhydrin solution is commonly used by forensic investigators in the analysis of latent fingerprints on porous surfaces such as paper. Amino acid containing fingermarks, formed by minute sweat secretions which gather on the finger's unique ridges, are treated with the ninhydrin solution which turns the amino acid finger ridge patterns purple and therefore visible.

The carbon atom of a carbonyl bears a partial positive charge enhanced by neighboring electron withdrawing groups like carbonyl itself. So the central carbon of a 1,2,3-tricarbonyl compound is much more electrophilic than one in a simple ketone. Thus indane-1,2,3-trione reacts readily with nucleophiles, including water. Whereas for most carbonyl compounds, a carbonyl form is more stable than a product of water addition (hydrate), ninhydrin forms a stable hydrate of the central carbon because of the destabilizing effect of the adjacent carbonyl groups.

Note that in order to generate the ninhydrin chromophore, the amine is condensed with a molecule of ninhydrin to give a Schiff base. Thus only ammonia and primary amines can proceed past this step. At this step, there must also be an alpha proton (H* in the diagram) for Schiff base transfer, so an amine adjacent to a tertiary carbon cannot be detected by the ninhydrin test. The reaction of ninhydrin with secondary amines gives an iminium salt, which is also coloured, and this is generally yellow-orange in color.

42 - Biuret test or Biuret reagent

The biuret test is a chemical test used for detecting the presence of peptide bonds. In the presence of peptides, a copper(II) ion forms a violet-colored complex in an alkaline solution. Several variants on the test have been developed.
The Biuret reaction can be used to assay the concentration of 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 540 nm, is directly proportional to the protein concentration, according to the Beer-Lambert law.
In spite of its name, the reagent does not in fact contain biuret ((H2N-CO-)2NH). The test is so named because it also gives a positive reaction to the peptide bonds in the biuret molecule.

Procedure :

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 aqueous copper(II) sulfate. If the solution turns purple, protein is present. 5–160 mg/mL can be determined.

Biuret reagent :

The biuret reagent is made of potassium hydroxide (KOH) and hydrated copper (II) sulfate, together with potassium sodium tartrate. The reagent turns from blue to violet in the presence of proteins, blue to pink when combined with short-chain polypeptides.
Not all biuret tests require the biuret reagent. The reagent is commonly used in a biuret protein assay, a colorimetric assay used to determine protein concentration—such as UV-VIS at wavelength 540 nm (to detect the Cu2+ ion).

Increasing the sensitivity of the biuret test :

Cu+ is a strong reducing agent which can react for example with Mo(VI) in Folin-Ciocalteu's reagent to form molybdenum blue. In this way, proteins can be detected in concentrations between 0.005 and 2 mg/mL. Molybdenum blue in turn can bind certain organic dyes (malachite green, Auramin O), resulting in further amplification of the of the signal.
Cu+ forms a deep purple complex with bicinchoninic acid (BCA), which allows proteins in the range of 0.0005 to 2 mg/mL to be determined. This assay is often referred to as "Pierce assay" after the manufacturer of a reagent kit.

41 - Millon's test or Millon's reagent

Millon's reagent is an analytical reagent used to detect the presence of soluble proteins.

A few drops of the reagent are added to the test solution, which is then heated gently.

A reddish-brown coloration or precipitate indicates the presence of tyrosine residues which occur in nearly all proteins.

Millon's test is not specific for proteins (it actually detects phenolic compounds), and so must be confirmed by other tests for proteins such as the biuret test and the ninhydrin reaction.

The reagent is made by dissolving metallic mercury in nitric acid and diluting with water.

The test was developed by the French chemist Auguste Millon (1812–67).

Monday, February 22, 2010

40 - Tetrahydrobiopterin (THB)

*Tetrahydrobiopterin (THB, BH4; Kuvan) or sapropterin, is a naturally occurring nutrient and essential cofactor of the three aromatic amino acid hydroxylase enzymes, used in the biosynthesis of the neurotransmitters serotonin (5-hydroxytryptamine (5-HT)), melatonin, dopamine, norepinephrine (noradrenaline), epinephrine (adrenaline), and nitric oxide (NO).

*THB was discovered to play a role as an enzymatic cofactor by Seymour Kaufman. The first enzyme found to use THB is phenylalanine hydroxylase (PAH).

*THB is biosynthesized from guanosine triphosphate (GTP) by three chemical reactions, those of which are mediated by the enzymes (GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS), and sepiapterin reductase (SPR).

*THB has the following responsibilities as a cofactor:

- Tryptophan hydroxylase (TPH) for the conversion of L-tryptophan (TRP) to 5-hydroxytryptophan (5-HTP)
- Phenylalanine hydroxylase (PAH) for conversion of L-phenylalanine (PHE) to L-tyrosine (TYR)
- Tyrosine hydroxylase (TH) for the conversion of L-tyrosine to L-DOPA (DOPA)
- Nitric oxide synthase (NOS) for conversion of a guanidino nitrogen of L-arginine (L-Arg) to nitric oxide (NO)
- Glyceryl ether monooxygenase (GEMO) for the conversion of 1-alkyl-sn-glycerol to 1-hydroxyalkyl-sn-glycerol.

*A deficit in THB biosynthesis and/or regeneration results in phenylketonuria (PKU) from excess L-phenylalanine concentrations or hyperphenylalaninemia (HPA), as well as monoamine and nitric oxide neurotransmitter deficiency or chemical imbalance. The chronic presence of PKU can result in severe brain damage, including symptoms of mental retardation, microcephaly, speech impediments such as stuttering, slurring, and lisps, seizures or convulsions, and behavioral abnormalities, among other effects.

*THB, developed by BioMarin under the brand name Kuvan and approved by the United States (U.S.) Food and Drug Administration (FDA) on December 13, 2007, is a synthetic preparation of the dihydrochloride salt of the substance, used in the treatment of PKU. HPA due to THB deficiency-mediated PKU.

*According to researchers at the University of Illinois at Chicago, THB may be of benefit in the management and treatment of intractable systolic heart failure based on research in mice. This is especially significant given that THB has already been tested and determined to be safe in human patients using it in the management of phenylketonuria, and also because there are far fewer ways to manage systolic heart failure than for diastolic heart failure.

39 - Type I and Type II Skeletal Muscle Fibers

*A Sprinter Uses Creatine Phosphate and Anaerobic Glycolysis to Make ATP, Whereas a Marathon Runner Uses Oxidative Phosphorylation.

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