Protein chemistry

chemistry of proteins , amino acids and its relevance to medicine , useful for medical and dental students

1. Protein chemistry Dr.S.Sethupathy,M.D., Ph.D ., Rajah Muthiah Medical College, Annamalai University.

2. Proteins Greek word – proteios which means of supreme importance. ¾ of the the total dry body weight. Composed of mainly carbon , hydrogen, oxygen and nitrogen Sulphur and phosphorus as minor constituents. Nitrogen content of proteins is 16% by weight The proteins are polymers of amino acids.

3. Amino acids They have a carboxylic group (-COOH) and primary amino group (-NH 2 ) attached to alpha carbon - carbon next to carboxyl carbon . Based on R group the amino acids vary from each other.

4. Peptide bond Amino acids in proteins are linked by peptide bonds. α- carboxyl group of one amino acid reacts with α- amino group of another to form a peptide bond by removal of water. ie : - CO- NH – bridge.

5. Peptides and proteins Two amino acids form dipeptide and three amino acids form tripeptide . 10-50 amino acids form polypeptide More than 50 amino acids form proteins. The sequence of amino acids determine the structure of proteins .

6. Neutral amino acids They have one amino and one carboxylic group . They may be aliphatic or aromatic amino acids. Simple Aliphatic amino acids Glycine and alanine . Glycine is the smallest and optically inactive Branched chain amino acids are valine , leucine , isoleucine

7. Nonpolar aliphatic , simple A.A

8. Branched chain A.A (VIL)

9. Neutral amino acids Hydroxy amino acids are serine and threonine Sulphur containing amino acids are cysteine, methionine Aromatic amino acids are phenylalanine, tyrosine and tryptophan.

10. Hydroxy amino acids (Polar)

11. Sulphur A.A Non polar - Polar

12. Aromatic non polar - polar A.A

13. Acidic amino acids and their amides Aspartic acid glutamic acid – they have one amino and two carboxyl groups The amide derivatives of these acidic amino acids are asparagine and glutamine respectively.

14. Acidic amino acids

15. Amide derivatives of Asp, Glu

16. Basic amino acids are lysine (two-NH2 group), arginine (has a guanidino group) in the aliphatic chain and histidine has an imidazole group. Imino acids : proline is an imino acid. It has a secondary imino group (-NH-)

17. Basic amino acids

18. Imino acid- non polar

19. Nutritionally Essential amino acids They are methionine, tryptophan, threonine , valine , isoleucine, leucine , phenylalanine and lysine . They cannot be synthesized in the body and they have to be supplied in the diet. Their deficiency causes nutritional disorders.

20. Histidine and arginine are semi essential as their synthesis in the body is inadequate especially during growth and recovery from illness and needs dietary supply then. Other amino acids are nutritionally non- essential as they can be synthesized in the body.

21. Glucogenic and ketogenic A.A The amino acids which can be converted to glucose are called glucogenic amino acids. Eg . Glycine, alanine, glutamate etc. The amino acids which cannot be converted to glucose but can form acetyl units are called ketogenic amino acids The only ketogenic amino acid is leucine whereas Phenylalanine, tyrosine, tryptophan, and lysine are both ketogonic and glucogenic .

22. Derived amino acids present in proteins Hydroxy proline and hydroxy lysine do not have genetic code . They are important constituents of collagen . Non protein amino acids Derived amino acids are found free in the cells. They are produced during metabolism of amino acids. Ex : Ornithine, citrulline and homocysteine .

23. Non alpha amino acids Gamma amino butyric acid is derived from glutamic acid. Beta alanine is a constituent of pantothenic acid. Amino acids having non polar side chains Alanine , valine , isoleucine , leucine ,methionine , proline , phenyl alanine and tryptophan . They have hydrophobic groups .

24. GABA β -alanine

25. Amino acids having non-ionic polar side chains Glycine, serine, threonine, cysteine, tyrosine, glutamine and asparagine are hydrophilic in nature. Amino acids having ionic polar side chains Acidic amino acids are aspartic acid and glutamic acid. Basic amino acids are lysine, arginine and histidine . They are hydrophilic in nature.

26. Amino acids Exist as NH 3 + - cationic form at acidic pH Exist as COO - - anionic form at alkaline pH Isoelectric pH ( pI ) It is the pH at which amino acids or proteins having equal number of positive and negative charges – zwitterionic form Do not move in electric field due to zero net charge.

27. Isoelectric pH

28. The isoelectric point ( pI ) for neutral amino acids can be calculated : pI = pk1 + pk2 / 2 Glycine - pk1 – 2.34 pk2 - 9.6 pI = 2.34+ 9.6 /2 = 5.97 The isoelectric point ( pI ) for more Acidic amino acids can be calculated: pI = pk1 + pkR / 2 Glutamic acid – pI = acidic group and R group /2 pk1 – 2.19 pk2 – 9.67 pkR - 4.25 pI = 2.19 + 4.25 / 2 = 3.22

29. Dibasic amino acids - pI The isoelectric point ( pI ) for more amino, monocarboxylic amino acids can be calculated: pI = pk2 + pkR / 2 Lysine – pk1-2.2, pk2- 8.9 , pkR - 10.5 pI = 8.9+ 10.5 = 9.7 pK1 =Dissociation constant of (-COOH) group pK2 =Association constant of (-NH2) group pKR =Dissociation (or) Association constant of “R” side chain

30. Isoelectic pH ( pI ) pK 1 is due to carboxyl group and pK2 is due to amino group. pI can be calculated from the pK values. Eg : glycine pI = pk1 + pk2 /2 ie : 2.4 +9.8 / 2 = 6.1. Buffering capacity is minimum at pI . The pK value of histidine is 6.4 and is an effective buffer at physiological pH of 7.4.

31. Optical activity Except glycine all amino acids have asymmetric carbon and are optically active . With reference to alpha carbon, D,L isomers are formed. Natural amino acids are L- isomers. D-amino acids are constituents of certain antibiotics - Actinomycin -D, valinomycin and bacterial cell wall peptidiglycans .

32. Decarboxylation The amino acids will undergo alpha decarboxylation to form amines. Histidine Histamine + CO 2 Tyrosine tyramine + CO 2 Tryptophan tryptamine + CO 2 glutamic acid GABA + CO 2 (gamma amino butyric acid )

33. Gamma Amino Butyric Acid (GABA ) from glutamate

34. Amide formation The carboxyl group of aspartic acid and glutamic acid (other than alpha carboxyl group) reacts with ammonia to form their amide derivatives. Aspartic acid + NH 3 asparagine Glutamic acid + NH 3 glutamine

35. Glutamine formation

36. Transamination A pair of an amino acid and α - keto acid react to form a new pair of α - keto acid and amino acid by transfer of amino group. It helps in pooling of amino groups of the different amino acids on α keto glutarate to form glutamate and helps in transport of ammonia to liver. It also helps in the synthesis of non essential amino acids.

37. Transamination – PLP coenzyme

38. Oxidative deamination Glutamic amino acid undergoes oxidative deamination and releases ammonia and α - keto acid.

39. Oxidative deamination

40. Carbamino compound Carbon dioxide adds to the alpha amino group of amino acids to form carbamino compounds. By this mechanism, hemoglobin transports CO 2 from tissues to lungs for elimination. Hb – NH 2 + CO 2 Hb –NH- COOH ( Carbamino Hb )

41. Carbamino compound

42. Transmethylation S- adenosyl methionine is the active donor of methyl group and after donating methyl group, it becomes S- adenosyl homocysteine .

43. Transmethylation

44. Ester formation Serine ,threonine residues can form ester linkage with phosphoric acid. It plays a role in the regulation of many enzyme activities by phosphorylation and dephosphorylation of serine or threonine residues in them.

45. Ester formation

46. Glycosidic linkage Hydroxyl groups of serine and threnoine can form O- glycosidic bonds with carbohydrate residues in glycoproteins. Amide group of asparagine and glutamine can form N- glycosidic bonds with carbohydrate residues in glycoproteins.

47. Glycosidic linkages

48. Cysteine and cystine

49. Reaction of SH group of cysteine It can form disulphide linkage in peptides which may be either intra chain or inter chain . Two molecules of cysteine react to form Cystine or dicysteine .

50. Ninhydrin test

51. Biuret test

52. Xanthoproteic test

53. Lead sulphide test

54. Millon’s test

55. Sakaguchi test

56. Hopkin’s cole reaction

57. Cyanide- nitroprusside test

58. Methionine - negative Cysteine and cystine give this test positive but methionine gives negative because sulphur in methionine is not released due to methyl group attached to it. Casein and gelatin contains methionine and insignificant amount of cysteine , gives the cyanide – nitroprusside test negative.

59. Purpose of transamination

60. A.A derivatives of clinical importance Gamma amino butyric acid (GABA )- inhibitory neuro transmitter ,formed from glutamic acid by glutamate decarboxylase using pyridoxine as coenzyme . Pyridoxine deficiency, GABA not formed , convulsions occur. Histamine - mediator of allergic reactions. Thyroxine maintains Resting Metabolic Rate (RMR). Cycloserine , a derivative of serine is used as antituberculous drug. Azaserine is an anticancer drug.

61. Proteins Proteins are polymers of amino acids linked by peptide bond. Alpha amino group of one amino acid reacts with alpha carboxyl group of another amino acid forming a peptide linkage with removal of water. Peptides have amino terminal (Free alpha amino group) and carboxyl terminal (Free alpha carboxyl group) . Biologically important peptides are glutathione, oxytocin , antidiuretic hormone, insulin etc.

62. Peptide bond

63. Structural organization of proteins Primary structure : It denotes the number and sequence of covalently linked amino acid residues in a polypeptide. Bonds are peptide bonds and disulphide linkages. Peptide 1- Gly -Val-Lys Peptide 2 - Val – Lys – Gly The sequence determines structure. Free alpha amino group is the N terminal amino acid The free alpha carboxyl terminal amino acid is the last amino acid. The higher levels of structural organization are dependent upon the primary structure.

64. Structural organization of proteins

65. Bonds responsible for protein structure Covalent bonds are peptide and disulphide linkages . Non covalent bonds are Hydrogen bonds, Hydrophobic bond, electrostatic or ionic bonds and weak Van der Waals’ forces.

66. Bonds

67. Structural organization of proteins

68. Secondary structure It is the folding and twisting of polypeptide chain forming a two dimensional structure due to interaction between adjacent amino acid residues in the linear sequence of the peptide. Eg . Alpha – helix is a right handed helix Beta pleated sheet is mainly due to inter or intra chain interactions which may be antiparallel or parallel sheets.

69. Tertiary structure Peptides further fold into a three dimensional structure due to interaction between amino acid residues which are far away in the linear sequence of the peptide. Non-covalent forces-Hydrogen bonds, electrostatic bonds, hydrophobic bonds and weak van der Waals forces - secondary and tertiary structure Domain is the compact functional unit of a protein. Eg : catalytic domain of an enzyme , regulatory domain of a protein.

70. Quaternary structure More than one polypeptides interact and form a single functional assembly. Eg : Hemoglobin is a tetramer. (α2β2 - four polypeptides) . Each polypeptide chain is called as subunit or monomer . Creatine kinase is a dimer .If a dimer is composed of similar copies of polypeptides, it is called as homodimer . Eg : CK-MM. If both copies are different, they are called as heterodimers Eg : CK-MB.

71. Classification of proteins Based on its functions Structural proteins Eg : collagen is the most abundant protein in mammals. Pepsin as an enzyme , Transferrin as a Transport protein , Ferritin as a Storage protein and insulin as hormone and immunoglobulis as the mediator of immunity are examples.

72. Based on shape Globular proteins: They are oval in shape and are easily soluble. Eg : albumin, globulin. Fibrous proteins: They are elongated and resist digestion . Their solubility is minimum. Eg ; collagen, keratin, elastin. Axial ratio (Length/Breadth) is more than 10 for fibrous proteins and for globular proteins it is less than 10.

73. Based on nutritional value Nutritionally rich proteins: Complete proteins or first class proteins and with all essential amino acids in required proportions. Eg : Egg white , Casein of milk. Incomplete proteins: Lack one essential amino acid. Eg : Cereals lack lysine and pulses lack methionine. Poor proteins: Lack many essential amino acids . Eg : Zein from corn lacks tryptophan and lysine.

74. Based on compostion Simple proteins : They are made up of amino acids only. Eg . Albumin and Globulins are soluble in water and heat coagulable proteins. Histones,, Protamines .

75. Conjugated proteins They are proteins combined with the non-protein substance called prosthetic group. Eg . Glycoproteins- blood group antigens, lipoproteins- chylomicrons, nucleoproteins – histones with DNA, phosphoproteins – casein of milk and vitellin of egg yolk, metalloproteins – hemoglobin , cytochrome.

76. Derived proteins Derived proteins: They are derived from simple and conjugated proteins . Denatured or coagulated proteins are called primary derivatives . Partial hydrolysis of proteins leads to secondary derivatives - proteoses , peptones and peptides.

77. Biologically important peptides Thyrotropin releasing hormone (TRH) is a tripeptide and releases thyroid stimulating hormone . Glutathione is a tripeptide and helps in the maintenance of RBC membrane integrity and keeping certain enzymes in an active state. Oxytocin and antidiuretic hormone (ADH or Vassopressin ) , Angiotensin I are peptide hormones. Gramicidin S is an antibiotic produced by Bacillus brevis It is circular ,composed of 10 amino acids and has D- phenyl alanine.

78. Estimation of proteins The protein sample is digested by boiling with concentrated sulfuric acid at 360 o C in the presence of copper sulfate and sodium sulfate as catalysts. Ammonia liberated is measured. Proteins contain 16% nitrogen, it can be calculated. ie Weight of nitrogen x 100 /16 or nitrogen x 6.25. Accurate procedure but it is tedious and takes longer time.

79. Biuret method Cupric ions chelate with peptide bonds in alkaline medium to produce violet color and measured colorimetrically . Compared with known concentration of protein standard treated similarly with Biuret reagent . It is simple and widely used in clinical laboratory. But its sensitivity is less and not suitable for estimation of mg quantities.

80. Lowry’s method Reduction of Folin - Ciocalteau reagent ( Phospho molybdic acid and phosphotungstic acid ) by the tyrosine and tryptophan residues of protein. A blue color is developed and can be compared with a known standard. This is very sensitive and microgram quantity can be measured. If the tryptophan and tyrosine content is different between test protein and standard protein, the accuracy is lost.

81. Spectrophotometric method Proteins absorb UV light at 280 nm . This is due to tyrosine and tryptophan content. It is compared with known standard protein solution. The method is accurate, simple and sensitive up to micrograms . Since reagent is not added, the sample can be used for other tests. But the instrument is expensive.

82. Nephelometry The detection of scattered light by turbid particles in solution. Antigen-antibody complexes can be detected. A beam of light is passed through the solution and the scattered light detected at an angle of 30-90 o (preferably 60 o ). This is rapid and suitable for automation but the instrument is expensive. Microgram quantity can be measured.

83. Turbidimetry The proteins in biological fluids such as urine, CSF can be estimated by adding precipitating agents such as sulfosalicylic acid and the turbidity is measured. It is simple, cheaper but less accurate. Detector is kept at an angle of 180 o . RIA and ELISA : Protein of Nanogram and picogram quantities can be estimated by Radio immuno assay (RIA) and Enzyme linked immuno sorbent assay (ELISA).

84. Steps for determination of primary structure N-Terminal amino acid determination Dansyl chloride combines with N-terminal amino acid The tagged polypeptides are hydrolyzed by boiling at 110 o C in 6H HCl for 18- 36 hours. The number of dansyl amino acids indicate the number of polypeptides. N- terminal amino acid can also be determined using Sanger’s reagent –1, Fluoro 2,4-dinitro bezene (1-F, 2,4-DNB).

85. Ends of the peptide

86. Dansyl chloride

87. Sanger’s method

88. C-terminal amino acid C- terminal amino acid can be determined using carboxypeptidase A and B. Carboxy peptidase A will not act if the C-terminal amino acid is Arginine, proline or lysine B will act only when the penultimate amino acid residue is proline .

89. Breaking up larger peptides into smaller peptides Larger peptides are broken into smaller peptides using 1. Trypsin hydrolyzes peptide bonds formed by alpha carboxyl group of lysine or arginine. 2. Chymotrypsin hydrolyzes peptide bonds formed by alpha carboxyl group of Phenyl alanine, tyrosine , tryptophan or leucine . 3. Cyanogen bromide hydrolyzes peptide bonds formed by alpha carboxyl of methionine .

90. Sequencing of peptides The peptides with a length of 10-30 amino acids can be sequenced using Edman’s degradation technique. Edman’s reagent is phenyl iso thio cyanate . N- terminal amino acid reacts with this and phenyl thiohydantoin derivative is formed . By this reaction, sequentially amino acids of a peptide can be identified. The amino acids composition of the peptide subjected to various hydrolyzing agents are arranged By overlapping technique ,primary structure can be elucidated

91. Enzymatic hydrolysis Trypsin, chymotrypsin B A H J U E K I D L I F T G B A H J U E K I D L I F T G B A H J U E K I D L I F T G By overlapping, the whole sequence is determined.

92. Overlapping technique Hydrolysis of peptides using different agents to produce fragments

93. Edman degradation

94. NMR Nuclear magnetic resonance (NMR) Spectroscopy is useful in determining functional group and change in conformation after binding with ligand. X- ray diffraction study is useful in studying the structure of pure , crystallized protein. Chemical synthesis of peptides by solid phase technique: Here the carboxy terminal amino acid is attached to insoluble polystyrene beads so that washing between reactions is rapid. The other amino acids are added sequentially. Insulin was first synthesized by this method.

95. Precipitation of proteins Salting out: When a neutral salt such as ammonium sulphate or sodium sulphate is added to protein solution, the shell of hydration is removed and protein gets precipitated. The globulins are precipitated by half saturation with ammonium sulphate where as albumin is precipitated by full saturation because it is more hydrated than globulin.

96. Proteins are least soluble at iso electric pH . Organic solvents such as alcohol cause precipitation of proteins by removing water from their shell. Precipitation by heavy metal ions In alkaline solution, heavy metal ions are cations and combine with anionic proteins to form metal proteinates and get precipitated . Salts of Copper, zinc, cadmium, mercury, lead are toxic because they precipitate proteins. Based on this principle , raw egg can be used as antidote for mercury poisoning.

97. Precipitation by alkaloidal agents Tungstic acid, phosphotungstic acid, tannic acid, picric acid, trichloro acetic acid and sulphosalicylic acid are powerful protein precipitating agents. Tannic acid is useful in tanning process in leather industry. Techniques of protein separation Chromatography It is a physical method of separation in which the components to be separated are continuously distributed between two phases stationary phase and mobile phase

98. Paper chromatography Paper (cellulose) acts as supporting medium. Water adsorbed to paper acts as stationary phase and the non polar solvent such as butanol act as mobile phase. When the mobile phase moves, non polar compounds are carried by them where as polar compounds are retained by polar stationary phase. Rf value (ratio of fronts) = distance traveled by solute / distance traveled by solvent Other types are thin layer Chromatography, ion exchange Chromatography, Gas Chromatography, affinity Chromatography, high performance liquid Chromatography ,gel filtration Chromatography, etc.

99. Electrophorsis It is a technique for the separation of the charged particles by means of electric current. In an electric field, a charged particle migrates towards cathode or anode based on its net charges. Paper, agar, polyacrylmide gel electrophoresis are the different types.

100. Biological significance of A.A They are involved in protein synthesis. Energy yield is 4 kcal / gm. Glycine is required for purine, haem , creatine , glutathione synthesis. Cysteine is needed for glutathione synthesis. Methionine is involved is one carbon transfer reactions.

101. Tyrosine is the precursor of thyroid hormones, catecholamines ,dopamine, melanin pigment. Tryptophan is the precursor for niacin, serotonin and melatonin. Glutamic acid is the precursor of gamma amino butyic acid (GABA). Aspartate , glutamine are needed for purines and pyrimidines synthesis.

102. Denaturation Change in native conformation of proteins due to altered non covalent forces is called denaturation. There will be loss of activity of the proteins. Primary structure is not altered. Denaturation involves the secondary, tertiary, quaternary structure of the molecules.

103. Causes of denaturation Chemical agents such as Acid, alkali, mercury, lead, alcohol Physical agents such as heat, UV rays, ionizing radiation Mechanical means like vigorous shaking, grinding etc . It can be reversible some times.

104. uses Denaturation causes unfolding of proteins. It helps in the digestion of dietary proteins by proteolytic enzymes. Denatured proteins form aggregates and precipitation occurs. Helps in the isolation of proteins of interest. Ex : Precipitation of proteins by ammonium sulfate in the initial stages of isolation of proteins.

105. Floccculation Denatured protein at isoelectric pH gets more precipitated forming floccules which still can be reversible. Coagulation If the proteins are permanently and irreversibly disorganized, it results heavy precipitates called as coagulum and coagulation is irreversible. Eg : Heat coagulation.

106. Ultracentrifugation Proteins can be separated based on size, shape and molecular weight based on their sedimentation velocities. Using ultracentrifuge-lipoproteins can be separated by high speed centrifugation . The ultracentrifuge - high as 2,00,000 g. Preparative ultracentrifuge is used to separate cellular organelles. Analytical centrifuge is used to obtain information about the shape, conformation and size distribution of the molecules.

107. Thank you