Enzymology namrata

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Enzymology namrata

  1. 1. A SEMINARONPROTEIN SEQUENCING1GUIDED BY –DR. K.L. TIWARI (DIRECTOR)DR. SEN (PRINCIPAL)MR. AJAY SINGH (ASST. PROF.)PRESENTED BY –NAMRATA KAHARM.Sc. 2nd SEM (Biotech)G. D. RUNGTA COLLEGE OF SCIENCE AND TECHNOLOGYKOHKA- KURUD ROAD, BHILAI, 490023
  2. 2. PROTEIN SEQUENCING Introduction History Determination of amino acid sequences by-HydrolysisSeparationQuantitative Analysis Mechanism of protein sequencing by–Chemical methodPhysical method Application of protein sequencing Summary Conclusion References2SYNOPSIS
  3. 3. PROTEIN SEQUENCING• Protein sequencing is a technique to determine the aminoacid sequence of a protein.• It is a method to understand the structure and function ofproteins in living organism.• Amino acid sequence determines the eventual threedimentional structure of the protein.• All proteins are polymers of amino acid.3INTRODUTION
  4. 4. PROTEIN SEQUENCING• Pehr Edman (1947) -He found the method to decode the aminoacid sequence of a protein usingchemicals.• Fredrick Sanger (1955) -He was able to present the completesequence of insulin.4HISTORY
  5. 5. PROTEIN SEQUENCINGDetermination of amino acid sequences -5DETERMINATIONS
  6. 6. PROTEIN SEQUENCING•HydrolysisThe peptide is hydrolyzed into its constituent aminoacids by heating it with 6N HCL at 110º c for 24hours.•SeparationSeparation of protein is done by chromatography,dialysis, fractionation etc.6DETERMINATIONS
  7. 7. PROTEIN SEQUENCING•Separation by gel chromatography -7DETERMINATIONSFig . No.- 1. Gel chromatography
  8. 8. PROTEIN SEQUENCING•Quantitative AnalysisThe amino acid residue of peptides reacts quantitatively withninhydrin.On heating, an α-amino acids reacts with two molecules ofninhydrin to yield an intensely coloured product.Purple colour is given in this test by all amino acids andpeptide having a free α -amino group where as prolinegives yellow colour.Amino acid + 2 ninhydrin CO2 + aldehyde + finalcomplex (yellow/purple) + 3H2O8DETERMINATIONS
  9. 9. PROTEIN SEQUENCING9DETERMINATIONSFig . No. 2. Ninhydrin reactionNinhydrin reagent Amino acidPurple /yellow colour complexReduced ninhydrinNinhydrin reagentAldehyde
  10. 10. PROTEIN SEQUENCINGPROTEIN SEQUENCING BYCHEMICAL METHODSanger’s methodFredrick Sanger wasdevelop first this methodEdman’s methodPehr Edman firstdeveloped this methodin (1950)10MECHANISM
  11. 11. PROTEIN SEQUENCING Sanger showed for the first time that amino acids arecovalently together by α-amino and α carboxyl group. He suggested stepwise release and identification ofamino acid starting from N-terminal. He used a reagent fluro dinitro benzene (FDB)which iscommonly called Sanger’s reagent. The FDB reacts with free NH2 group of N-terminus. Upon hydrolysis a yellow coloured dinitrophenol (DNP)derivative of N-terminal amino acid is produced. The DNP amino acid is identified comparing it with aknown standard DNP-amino acid by using gelchromatography.11MECHANISMSanger’s method
  12. 12. PROTEIN SEQUENCING12MECHANISMFig . No. 3. Sanger’s method
  13. 13. PROTEIN SEQUENCINGDansyl Chloride• The Dansyl chloride is commonly used because it formsan intensively coloured derivatives.• That can be detected with high sensitivity that thedinitrophenyl compound .• It reacts with an uncharged C and N terminal to form asulfonamide derivatives that is the stable undercondition that by hydrolyser peptide bonds.• Although the densyl method for determining the aminoterminal residue is sensitive and powerful.13MECHANISM
  14. 14. PROTEIN SEQUENCING14MECHANISMFig . No. 4. Dansyl methodDANSYL CHLORIDEDANSYL AMINO ACIDDANSYL PEPTIDEPEPTIDEFREE AMINO ACIDS-HCL
  15. 15. PROTEIN SEQUENCINGIt cannot be used repeatedly on the same peptidebecause the peptide is totally degraded in the acidhydrolysis step.The Sanger’s method is more sensitive and efficientprocedure.The dansyl chloride is 100 times more sensitive thanthe Sanger’s method.The Sanger’s method is only useful for identificationof N-terminal residue of a peptide.15DISADVANTAGE
  16. 16. PROTEIN SEQUENCING It involves sequential identification of amino acids from N to Cterminals. Phenyl isothiocynate (PTC) reagent is used for the Edmandegradation. The amino terminal (N-terminal) residue of a protein can beidentifies by reaction the protein with the PTC that forms a stablecovalent link with the free α amino group prior to hydrolysis with6M HCl Phenyl isothiocynate reacts with the uncharged terminal aminogroup of the peptide to form a phenyl thiocarbonyl derivation.16MECHANISMEdman’s method
  17. 17. PROTEIN SEQUENCING17MECHANISMThe labeled N-terminal amino acid can be identified by comparisonof its chromatographic properties with stanrdard fluorodinitrobenzene and dansyl chloride.Then, under mild acidic the cyclic Phenylthio hydantoin (PTH) of theterminal amino acid is librated which leaves an intact peptide shortedby one amino acid.The released PTH amino acid is identified by high performanceliquid chromatography.The sequencing technique has been automated and refined so thatupwards of 50 residues from the N-termines of a protein can besequenced from picomolecule quantities of material.
  18. 18. PROTEIN SEQUENCING18MECHANISMFig . No. 5. Edman methodPhenyl thiocarbamoylpeptide
  19. 19. PROTEIN SEQUENCING• The great advantage of the Edman method is that the rest ofthe peptide chain after removal of the N-terminal amino acid isleft intact for further cycles of this procedure, thus the Edmanmethod can be used in a sequential fashion to identify severalor many consecutive amino acid residues starting from the N-terminal end.19ADVANTAGE
  20. 20. PROTEIN SEQUENCING•The Edman degradation proceeds from the N-terminus of theprotein it will not work it the N-terminal amino acid has beenchemically modified.•It also required the use of either guess work or a separate-procedure to determine the position of disulfide bridges.20LIMITATIONS
  21. 21. PROTEIN SEQUENCINGProtein sequencing by MassSpectrometry21PHYSICALMETHOD•Mass spectrometry is an important emerging method for thecharacterization of proteins.• The two primary methods for ionization of whole proteins are electro sprayionization (ESI) and matrix assisted laser desorption/ionization (MALDI).•Mass spectrometer has three main parts for characterizing the protein –1. An ionization source2. A mass analyzer3. An ion detector
  22. 22. PROTEIN SEQUENCINGProtein sequencing by MassSpectrometry22PHYSICALMETHODIONIZATION SOURCEMASS ANALYZERDETECTORFig no.6. PARTS OF MASS SPECTROMETER
  23. 23. PROTEIN SEQUENCINGFig no.7. Mass Spectrometry 23PHYSICALMETHOD
  24. 24. PROTEIN SEQUENCINGProtein sequencing by MassSpectrometry24PHYSICALMETHODDigested the peptide bonds .By protease enzymeIonization by MALDI(matrix assisted laser desorption/ionization)or electro spray.Then ionized molecules are introduced into mass- analyzer.Using the mass spectrometer the masses of each of these ionizedpeptides is calculated.Using the masses it is now possible to know the amino acidsequence of these protein.
  25. 25. PROTEIN SEQUENCINGADVANTAGES –•Stable isotopic labeling of proteins may be used todiscriminate between contaminants and original partnersusing MS (mass spectrometry) techniques.•MS (mass spectrometry) has also made advantages in theanalysis of membrane proteins.DIS-ADVANTAGES-•Miscalibration is one of the main errors of MS spectrometry.•MALDI doesn’t favor the identification of hydrophobicpeptides.25MASSSPECTROMETRY
  26. 26. PROTEIN SEQUENCING1. mRNA/protein analysis and coding SNP (single nucleotideprotein) scoring tools.2. Knowledge of the sequence of amino acids in a protein canoffer insights into its three dimensional structure and itsfunction in cellular location and evolution.3. Certain amino acid sequences serve as signals that determinethe cellular location chemical modification on an half life ofa protein.4. Protein sequences can elucidate the history of life on Earth.26APPLICATION
  27. 27. PROTEIN SEQUENCING• The protein is a polypeptide chain which is made up of aminoacid monomers polymerization.• Protein sequencing is the sequencing of monomer of peptidechain which is amino acid.• For sequencing of protein firstly we determined the amino acidsequences which are done by hydrolysis, separation andquantitative analysis.• The protein sequencing is done by two methods which arechemical and physical method.27SUMMARY
  28. 28. PROTEIN SEQUENCING28CONCLUSION• Protein sequencing also gives information regarding whichconformation the protein adopts.• Discovering the structures and functions of proteins in livingorganisms is an important tool for understanding cellular processes.• Not all proteins contain all the amino acid, nor do the amino acidsoccur with equal frequency.• The amino acids have N-terminal end and C-terminal end.
  29. 29. PROTEIN SEQUENCINGCox M. Michael andNelson L. David 2008 Principles of Biochemistry, 5th EditionLehninger L.Albert 2009 Biochemistry, 2nd EditionStryer Lambert 2009 Principle of Biochemistry, 2nd Edition29REFERENCES
  30. 30. THANK YOU30

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