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Laboratory Hazards

Laboratory Hazards. Causes of injury. Direct contact ( skin , mouth by pipetting , stomach by swallowing) Inhalation ( leads to lung damage) Toxic systemic effects ( when absorbed and reached to liver, kidney, etc…..). Dangerous chemicals.

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Laboratory Hazards

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  1. Laboratory Hazards

  2. Causes of injury • Direct contact ( skin , mouth by pipetting, stomach by swallowing) • Inhalation ( leads to lung damage) • Toxic systemic effects ( when absorbed and reached to liver, kidney, etc…..)

  3. Dangerous chemicals • Corrosives (e.g : strong acids & alkali): treated with safety pipette. • Toxic (e.g : CN & Barbiturate) : discarded in flow of water. • Organic solvent (e.g: CHCL3, CCL4): toxic to BM & liver- treated in fuming cupboard • Flammable (e.g: Picric acid) : stored under water , explodes on percussion

  4. Units • Substance conc. : expressed by • Metric system : w/v ( gm/l – mg/dl) • Molarity : 1 mol is the molecular weight of sub. in grams 2. Enzyme activity : U/ml U ( international unit) is the activity which transforms 1 micromol of substrate per minute under definite conditions

  5. Proteins

  6. Nitrogenous organic compounds • High molecular weight • Composed of amino acid linked by peptide bonds • Subdivided into : a) simple: polypeptide chain only b) conjugated : pp chain with non protein part

  7. Chemical reactionS for identification • Classified into : • Reactions for peptide group • Reactions for free α-amino or α-carboxyl group • Reactions for specific side chain radicles • Used for identification of protein in biological media , specification of pp in chromatogrm

  8. Colour reactions

  9. Biuret reaction • For peptide groups • Principle: formation of violet complex with cupric ions in alkaline medium • Positive for peptides , non protein compounds (urea) that contain at least two peptide groups • Negative for free amino acids • Negative for glycylglycine (contains only one peptide bond)

  10. Ninhydrin reaction • For α-amino acid • Priciple : heating with ninhydrin leads to oxidative deamination of α-amino acids yielding violet blue complex of the reduced ninhydrin • Negative for β-amino acid (β-alanine)

  11. Xanthoproteic reaction • For aromatic amino acids • Principle: Aromatic ring react with conc. HNO3 to give dinitro derivative (yellow) quinoid str. in alkaline medium (orange). • Specific for phenylalanine,histidine,tyrosine,tryptophan

  12. Millon’s test • Specific for tyrosine • Principle: heating with millon’s reagent purple red colour (nitro tyrosine mercurial ) • Millon’s reagent : HNO3 + HgNO3

  13. Adamkiewiczs’s reaction • Specific for tryptophan • Principle : 2 tryp. + glacial acetic acid (contain glycoxylic acid) violet red compound • Conc. H2SO4 used as dehydrating agent

  14. Reactions for S- containINg amino acidS • E.g : cysteine , cystine , methionine • Principle: s-aa + heat in alk. medium Na2S a) fohl’s reaction: Na2S + sod. plumbitePbS ( black ppt) b) Nitroprusside reaction: Na2S + sod. nitroprusside violet red complex

  15. Protein denaturation • Denaturation : disruption of native structure of proteins ( 2ry, 3ry,4ry) resulting in alteration of its physicochemical properties ( solubility) and loss of its biological activity

  16. Practical application • Skin disinfection • Protein identification in urine • Treatment of poisoning by heavy metal • ppt of protein in biological media

  17. Denaturating agents • Chemical: acid , heavy metals • Physical : temp. , radiation • Biological : proteolytic enzymes ( trypsin)

  18. Chemical denaturating agents • Principle : destroy spatial structure of protein resulting in its ppt 1. Conc. mineral acids ( HNO3 ): by charge neutralization 2. Organic acid ( ACOH): by charge neutralization 3. Organic solvents ( ethanol): by destroying hydrophobic interaction in protein moiety 4. Heavy metal ( Hg): by binding to side chain of aa , excess heavy metal cause redissolution due to ion adsorption

  19. Determination of Isoelectric point (PI) • Practical application : Each protein has its IEP, by knowing it, we can: • Isolate certain protein by precipitation from a biological extract. • Purify protein preparation in pharmacy. • Protein carries +ve & -ve charges due to presence of acidic and basic amino acids, protein should be charged to stay in solution, the net charge of protein affected by the medium PH.

  20. Determination of Isoelectric point (PI) • Isoelectric point (PI) : It’s the PH at which the sum of +ve & -ve charges on the protein equal to zero (net charge = 0). • At the PI; the protein is unstable and prone to be precipitated; especially in presence of dehydrating agents (ethanol, acetone). • Each protein has its specific PI, which could be used for its isolation. • Milk souring is an example of PI for milk protein (Casein, PI=4.6), as it transit to unstable state, and form ppt at PH 4.6 (clouding of solution).

  21. Proteins salting out • Practical applications : Purification and fabrication of crytalline protein preparations. Salting Out: Process of protein precipitation by alkaline earth metal salts (Na2SO4, NaCl, KCl, MgSO4); and neutral salts (NH4)2SO4 • These salts neutralize the charge on protein moiety leading to its precipitation. Salting out is a REVERSIBLE process: the ppt protein when redissolved in H2O, it restores its physical, chemical and biological properties. • Proteins could be separated by different concentrations of salt solutions, depending on their charges and hydrophilic properties.

  22. Proteins salting out • Practical applications : Salting Out of Globulin and Albumin. The method depends on that (NH4)2SO4 neutralizes charges on protein molecules ( compete with it for H2O molecules) and induces their dehydration resulting in protein ppt (SALTING OUT). Half Saturation ppt of globulin As it’s less hydrophilic and has larger molecular mass; as compared with albumin. Complete saturation ppt of albumin.

  23. Conjugated proteins • Hemoproteins e.g:Hb , Myoglobin, cytochrome , catalase • It’s protein part + heme part ( tetra pyroleheterocycle bound to Fe) • Heme has peroxidase like reaction due to structure similarity SH2 + H2O2 S + 2 H2O • Unlike peroxidase , heme retain its catalytic properties after boiling

  24. HbexperImentvery sensitive and used in trace analysis of blood

  25. 2. Phosphoproteins • E.g : casein, vitellin, phosphorylase • H3PO4 linked by ester bond to –OH of serine or threonine of protein moiety • Principle: Protein part tested by biuret reaction H3PO4 tested by amm. Molybdateamm. Phosphomolybdatereducing agent molybdenum blue

  26. Nucloprotein • Abundant in nucleated cell such as liver, spleen • Not found in RBC’s • Classfied into RNP , DNP

  27. DNA isolation Spleen cells grind broken cells CTAB weakened cells Hypotonic saline spilled cytoplasm DNP CTAB detergant : solubilize cell membrane , inactivate nucleases EDTA ( chelating agent ) : chelate necessary cations for nucleases activity so inhibit DNA degradation

  28. DNP identification • Protein part : biuret reaction • H3PO4 part : molybdenum reaction • Deoxy ribose : di-phenylamine test ( blue colour) • Purines : amm. AgNO3 test ( pale brown ppt)

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