Análisis de las técnicas de boxeo

 Ninhydrin- can detect microgram quantities of amino acids

–Forms Ruhemann’s purple colour with all amino acids EXCEPT Proline which gives yellow colour –Used to detect fingerprints

 Fluorescamine- detect picogram quantities of amino acids- Most sensitive method

Colour reactions of proteins and amino acids

Xanthoproteic test: Aromatic amino acids Millon’s test : Tyrosine, Phenylalanine

Aldehyde test: Tryptophan (Hopkins-Cole test) Acree-Rosenheim reaction : Tryptophan Sakaguchi’s test : Arginine

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Pauly’s test : Histidine, Tyrosine Sulphur test: Cysteine, Cystine

Biuret test: general test for proteins. At least 2 peptide bonds required

STRUCTURE OF PROTEIN - Different levels of protein structure Primary structure

Includes a.a sequence and position of disulfide bonds. Maintained by the covalent bonds of peptide linkages. Peptide bond- partial double bond character. Distance 1.32 Ao

Secondary structure

Relationship between residues which are 3-4 a. a apart H bonds

α helix, β pleated sheet, collagen helix

Alpha helix

Commonly right handed. Abundant in Hb and Mb

Absent in chymotrypsin

Pro and Hyp do not allow its formation Collagen: Triple helix

Glycine induces bends in the alpha helix.

β pleated sheet

Chains fully extended.Stabilised by H bonds.

Tertiary structure

-AA far apart. Bonds- Hydogen, Hydrophobic, Ionic, van der Waal’s

Quarternary – only when protein has 2 or more subunits

Stabilized by non-covalent interactions Domain- Compact globular functional unit of a protein

Amino acid residues which favor the secondary structure of proteins

Alpha helix Beta pleated sheet Turns Ala, Cys, Leu, Met, Glu, Gln

His, Lys, Arg

Ile, Val,

Phe, Tyr, Trp, Thr, Arg

Gly, Ser, Pro Asp, Asn Arg

Protein sequencing- detecting the amino acid sequence : Sanger Sanger’s reagent

Fluoro DinitroBenzene.Used for identification of N-terminal amino acid

End group analysis

N-terminal a.a identified by Sanger’s reagent C-terminal by Carboxypeptidase A and B

– Carboxypeptidase A will not act if C-terminal is Arg, Lys or Pro

– Carboxypeptidase B will act only if penultimate residue is Pro

Edman’s reagent

Phenyl isothiocyanate. Used for Sequential analysis of amino acids from N-terminal

Dansyl chloride

Combines with N-terminal amino acid. Used to assess the number of polypeptide chains

Cyanogen bromide

Hydrolyses peptide bonds following Mehionine

“Sequenator”: Automated instrument for protein sequencing

Ingram’s technique

Protein finger printing

Helps to identify qualitative abnormalities in proteins

Digestion by Trypsin followed by Chromatography and peptide mapping

51 Chaperones

Heat shock proteins which help in proper folding of proteins Different classes (hsp 70kDa, hsp 60 kDa)

Prevent misfolding Unfold misfolded regions

Keeps protein unfolded to pass through membranes Prevents inappropriate interactions with other proteins Chaperonins are hsp60 chaperones

Collagen

Most abundant protein in body

3 polypeptides (referred to as “α chains”) are wound around one another in a rope-like triple helix: held together by hydrogen bonds between the chains.

Variations in amino acids in the alpha chains forms different types of collagen Formed as procollagen

Every 3rd _{amino acid is glycine.}

Cross links are by lysine (hydroxy lysine)

Collagen diseases:

Ehlers-Danlos syndrome (EDS): result from a deficiency of collagen-processing enzymes (eg: lysyl hydroxylase

deficiency or procollagen peptidase deficiency), or from mutations in the amino acid sequences of collagen types I, III, or V. The most clinically important mutations are found in the gene for type III collagen

Vascular defects, fragile, stretchy skin and loose joints

Osteogenesis Imperfecta (Brittle bone syndrome): Brittle bones, delayed wound healing, humped back.

Type I : Osteogenesis imperfecta tarda. Due to decreased production of α1 and α2 chains. Presents in early infancy with pathologic fractures and may be suspected if prenatal ultrasound detects bowing or fractures of long bones. Type II : Osteogenesis imperfecta congenital; more severe, and patients die of pulmonary hypoplasia in utero or during the neonatal period.

Elastin

“Elastic” properties

Precursor- Tropoelastin (700 a.a)

Contains no hydroxylysine; Cross links by desmosine/isodesmosine

α1- Antitrypsin (α1-AT), produced primarily by the liver but also by tissues such as monocytes and alveolar

macrophages, prevents elastin degradation in the alveolar walls.

PROTEIN MISFOLDING

Misfolded proteins are usually tagged and degraded. If this system is not perfect, such abnormal protein are non functional, can form aggregates, cause cellular damage and result in diseases

1. Amyloidosis: Extracellular accumulation of β–pleated sheets forming long fibrils. If it occurs around neural cells, it is neurotoxic and is implicated in a neurodegenerative disease called Alzheimer’s disease The dominant component of amyloid protein in Alzheimer’s is amyloid β

Another protein accumulating in Alzheimer’s is abnormal tau (τ) protein as neurofibrillary tangles. Normal tau (τ) protein is required for microtubular assembly.

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2. Prion diseases: these are caused by purely protein particles called prions. Normal prion particle (PrPc) is coded by a gene situated on chromosome 20 in neuroglial cells. It has a structure of α–Helix. Misfolded prion particle (PrPsc) has a predominant structure of β–pleated sheets. Misfolded prion:-

a. Becomes infectious i.e. converts another PrP to infectious prion particle.

b. Gets precipitated around neurons and glial cells causing fatal neurodegenerative disorders called Transmissible spongiform encephalopathies (TSE)

i. Scrapie in sheep

ii. Bovine spongiform encephalopathy (Mad Cow Disease) in cattle iii. Creutzfeldt–Jacob disease in humans

iv. Kuru in cannibals

v. Gerstmann–Straussler–Scheinker Syndrome (GSS) vi. Familial Fatal Insomnia (FFI)

METABOLISM OF AMINO ACIDS