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Twice Nobel Prize Winner

Twice Nobel Prize Winner. FREDERICK SANGER HARD WORK IS PAID IN FORM OF AWARDS Prasanna Khandavilli. Curiosity is the key for Scientific Discovery. Frederick Sanger. The Nobel Prize in Chemistry 1958. "for his work on the structure of proteins, especially that of insulin ”.

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Twice Nobel Prize Winner

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  1. Twice Nobel Prize Winner FREDERICK SANGER HARD WORK IS PAID IN FORM OF AWARDS Prasanna Khandavilli

  2. Curiosity is the key for Scientific Discovery

  3. Frederick Sanger The Nobel Prize in Chemistry 1958 "for his work on the structure of proteins, especially that of insulin”

  4. The Nobel Prize in Chemistry 1980 “for their contributions concerning the determination of base sequences in nucleic acids” Frederick Sanger Walter Gilbert

  5. Frederick Sanger • Born: August 13, 1918 • Place of Birth: Rendcombe, Gloucestershire, England • Residence: U.S.A./Great Britain • Affiliation: MRC Laboratory of Molecular Biology, Cambridge

  6. Basic Principles of Protein Chemistry Proteins - Amino Acid residues Physical and Biological Properties- Arrangement of the Amino Acid residues

  7. Bergmann and Niemann Periodic arrangement of Amino Acids Pure protein – A random mixture of similar individuals

  8. Chibnall Studies on Insulin: • Simpler composition • Tryptophan and Methionine absent • Accurate analysis

  9. Van Slyke Procedure • High content of free α-amino groups • Short Polypeptide chains Jensen & Evans: Phenylalanine at the end of one of the chains

  10. Molecular weight of Insulin • Physical methods 36,000 to 48,000 • Gutfreund 12,000 • Harfenist & Craig 6,000

  11. Dinitrophenyl (DNP) method 1:2:4 flourodinitrobenzene (FDNB) *Alkaline conditions

  12. DNP method contd. Hydrolysis of DNP protein with Acid

  13. DNP method contd. Extraction with Ether Fractionation (Partition Chromatography) Comparison of Chromatographic rates (Silica-gel Chromatography or Paper Chromatography) Identification and Estimation Calorimetrically

  14. DNP labeling of Insulin Three yellow DNP-derivatives • ε-DNP-lysine (not extracted with Ether) • DNP-phenylalanine • DNP-glycine

  15. Edman phenyl isothiocyanate method Standard method for studying N-terminal residues

  16. Disulphide bridges • Cystine residues • Reduction to –SH derivatives Polymerization gave insoluble products How to break these Disulfide bridges?

  17. Oxidation with Performic Acid

  18. Precipitation of Oxidized Insulin • Fraction A : N-terminal residue Glycine Acidic Simpler composition (Lys, Arg, His, Phe, Thr, Pro were absent) • Fraction B: N-terminal residue Phenylalanine Basic Amino acids

  19. Acid hydrolysis of DNP-Phenylalanine

  20. Conclusions • Position of residues • Only two types of chains • Molecular weight 12,000

  21. Fractionation Paper Chromatography for Fractionation of small peptides Consden, Gordon, Martin & Synge worked on pentapeptide Gramicidin-S

  22. Fraction B studies • Ionophoresis, Ion-exchange Chromatography, Adsorption on Charcoal • 5-20 peptides • Paper Chromatography • Analysis of the constituent Amino Acids

  23. Results

  24. Conclusions Five sequences present in Phenylalanine Chain

  25. Problems How the 5 sequences are joined ? Hurdles in solving this mystery: • Technical difficulty in fractionating peptides with non-polar residues (Tyr & Leu) • Acid lability of the bonds involving Serine and Threonine

  26. Solution is……… Enzymatic Hydrolysis: Use of Proteolytic enzymes More specific than acid hydrolysis

  27. Proteolytic Enzymes Pepsin – Peptide Bp3 fragment Phe (CySO,H, Asp, Glu, Ser, Gly, Val, Leu, His) Trypsin, Chymotrypsin studies

  28. Fraction A studies Problems in applying fraction B studies to fraction A: • Few residues that occur only once • Less susceptible to enzymatic hydrolysis • Water soluble peptides- difficult to fractionate on paper chromatography

  29. Paper Ionophoresis • pH 2.75 -COOH groups uncharged -SO3H groups negative charge -NH2 groups positive charge • pH 3.5 -COOH groups charged

  30. Results of Paper Ionophoresis

  31. Sequence of Fraction A

  32. Acid Hydrolysis Ammonia produced from Amide groups on Aspartic and Glutamic acid residues • Position of Amide groups: Ionophoretic rates Amide contents of peptides

  33. Arrangement of Disulphide bridges Assumptions and hypothesis: Harfenist & Craig Mol Wt 6000 Two chains with three disulphide bridges: Two bridges connecting the two chains One intrachain bridge in fraction A

  34. Disulphide interchange reaction

  35. Disulphide interchange reaction Contd. • Two types of disulphide interchange reactions • In neutral & alkaline solution catalyzed by –SH compounds

  36. Enzymic Hydrolysis • Chymotrypsin action -CySO3H.AspNH -Leu.Val. CySO3H.Gly.Glu.Arg.Gly.Phe.Phe

  37. Cystine peptide structure

  38. The Structure of Insulin

  39. Sequenced Insulin supports Protein chemistry theories • Hofmeister & Fischer – Classical peptide hypothesis No evidence of periodicity Random order Unique & most significant order

  40. Insulin from different species

  41. Determination of Nucleotide Sequences • Smallest DNA molecule - Bacteriophage φX174 – 5,000 nucleotides • tRNA - 75 nucleotides

  42. Fractionation of 32P-labelled oligonucleotides G.G.Brownlee and B.G.Barrell method: • Partial degradation by enzymes • Separation of smaller products • Determination of sequence • Applied to RNA sequences

  43. Disadvantages • Slow and tedious • Requires successive digestions and fractionations • Not easy to apply to larger DNA molecules

  44. Copying Procedures • C.Weissmann: Bacteriophage Qβ -Qβ Replicase – Complementary copy -Pulse-labeling with radio actively labeled nucleotides • DNA Polymerase substitutes Replicase -Primer, Triphosphates containing 32P in α position - Sanger

  45. Copying Procedure

  46. Primer Source • Synthetic Oligonucleotides • Restriction enzymes

  47. Copying procedure Results • Short specific regions of labeled DNA were obtained • Unable to obtain individual residues for sequencing How to obtain individual nucleotide residues?

  48. Solution is ……… Incorporation of ribonucleotides in DNA Sequence by DNA Polymerase Splitting of ribonucleotide residues later by action of alkali Technique put forth by Berg, Fancher & Chamberlin

  49. The ‘Plus and Minus’ method α[32P]-dNTP labeling and sequence specific termination J.E.Donelson - Ionophoresis of products on acrylamide gels

  50. The Dideoxy method Quicker and more accurate • φX174 • Bacteriophage G4 • Mammalian mitochondrial DNA

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