Food proteins

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Food proteins

  1. 1. Food Proteins
  2. 2. -Amino Acid Sequence -Protein Conformation -Levels of Protein Structure -Primary structure -Secondary structure -Tertiary structure -Quaternary structure -Classification of Proteins -Denaturation of Protein
  3. 3. Peptide Linkage Formation
  4. 4. Peptides and Proteins  Peptides and proteins are polymers of twenty amino acids connected to each other by peptide bonds.  Oligopeptide is formed of (2 –10) amino acids: 2 amino acids 3 amino acids 4 amino acids dipeptide, tripeptide, tetrapeptide ….etc.  Polypeptide is formed of more than 10 amino acids.
  5. 5. In proteins, almost all carboxyl and amino groups are combined in peptide linkage and not available for chemical reaction (except for hydrogen bond formation).
  6. 6. Food Proteins -Like peptides, proteins are formed from amino acids through amide linkages. -Covalently bound hetero constituents can also be incorporated into proteins. For example, phosphoproteins such as milk casein or phosvitin of egg yolk contain phosphoric acid esters of serine and threonine residues. -The structure of a protein is dependent on the amino acid sequence (the primary structure) which determines the molecular conformation (secondary and tertiary structures). -Proteins sometimes occur as molecular aggregates which are arranged in an orderly geometric fashion (quaternary structure). -The sequences and conformations of a large number of proteins have been elucidated and recorded in several data bases.
  7. 7. -Glycoproteins, such as casein, various components of egg white and egg yolk, collagen from connective tissue and serum proteins of some species of fish, contain one or more monosaccharide or oligosaccharide units bound O-glycosidically to serine, threonine or hydroxylysine or Nglycosidically to asparagine.
  8. 8. Amino Acid Sequence
  9. 9. 1-Amino Acid Composition, Subunits -Sequence analysis can only be conducted on a pure protein. -First, the amino acid composition is determined after acidic hydrolysis. -The procedure (separation on a single cation-exchange resin column and color development with ninhydrin reagent) has been standardized and automated (amino acid analyzers). -As an alternative to these established methods, the derivatization of amino acids with the subsequent separation and detection of derivatives is possible (pre-column derivatization). Various derivatization reagents can be selected, such as: • 9-Fluorenylmethylchloroformate (FMOC) • Phenylisothiocyanate (PITC) • Dimethylaminoazobenzenesulfonylchloride (DABS-Cl) • Dimethylaminonaphthalenesulfonylchloride (DANS-Cl) • 7-Fluoro-4-nitrobenzo-2-oxa-1,3-diazole (NBDF) • 7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBDCl) • o-Phthaldialdehyde (OPA)
  10. 10. Amino acid chromatogram. Separation of a mixture of amino acids (10 nmol/amino acid) by an amino acid analyzer.
  11. 11. Amino Acid Composition, Subunits -It is also necessary to know the molecular weight (MW) of the protein. -MW could be determined by gel column chromatography, ultracentrifugation or electrophoresis. -It is necessary to determine whether the protein is a single molecule or consists of a number of different polypeptide chains (subunits) associated through disulfide bonds or non-covalent forces. -Dissociation into subunits can be accomplished by a change in pH, by chemical modification of the protein, such as succinylation, or with denaturing agents (urea, guanidine hydrochloride, sodium dodecyl sulfate SDS). -Disulfide bonds, which are also found in proteins which consist of only one peptide chain, can be cleaved by oxidation of cystine to cysteic acid or by reduction to cysteine with subsequent alkylation of thiol group to prevent reoxidation. -Separation of subunits is achieved by chromatographic or electrophoretic methods.
  12. 12. 2-Terminal Groups -N-terminal amino acids can be determined by treating a protein with l-fluoro-2,4-dinitrobenzene (Sanger’s reagent) or 5dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride). -Another possibility is the reaction with cyanate, followed by elimination of the N-terminal amino acid in the form of hydantoin, and separation and recovery of the amino acid by cleavage of the hydantoin . -The N-terminal amino acid (and the amino acid sequence close to the N-terminal) is accessible by hydrolysis with aminopeptidase, in which case it should be remembered that the hydrolysis rate is dependent on amino acid side chains and that proline residues are not cleaved. -A special procedure is required when the N-terminal residue is acylated (N-formyl- or N-acetyl amino acids).
  13. 13. -Determination of C-terminal amino acids is possible via the hydrazinolysis procedure recommended by Akabori: -The C-terminal amino acid could be then separated from the amino acid hydrazides by a cation exchange resin.
  14. 14. Terminal Groups -The C-terminal enzymatically by amino acids can be removed - Carboxypeptidase A which cleaves amino acids with aromatic and large aliphatic side chains, - Carboxypeptidase B which cleaves lysine, arginine and amino acids with neutral side chains or - Carboxypeptidase C which cleaves with less specificity but cleaves proline.
  15. 15. 3- Partial Hydrolysis -Long peptide chains are usually fragmented. The fragments are then analyzed for amino acid sequences. -Selective enzymatic cleavage of peptide bonds is accomplished primarily with Trypsin, which cleaves exclusively Lys-X- and Arg-X-bonds, and Chymotrypsin, which cleaves peptide bonds with less specificity (Tyr-X, Phe-X, Trp-X and Leu-X). -The enzymatic attack can be influenced by modification of protein. For example, -Acylation of the amino group of lysine limits tryptic hydrolysis to Arg-X, -Substitution of the SH-group of cysteine residue with an aminoethyl group introduces a new cleavage position for trypsin into the molecule “pseudolysine residue”
  16. 16. Partial Hydrolysis -Also suited for the specific enzymatic hydrolysis of peptide chains is the endoproteinase Glu-C from Staphylococcus aureus. It cleaves Glu-X bonds as well as Glu-X plus Asp-X bonds. -The most important chemical method for selective cleavage uses cyanogen bromide (BrCN) to attack Met-X-linkages.
  17. 17. Partial Hydrolysis -Hydrolysis of proteins with strong acids reveals a difference in the rates of hydrolysis of peptide bonds depending on the next amino acid side chain. -Bonds involving amino groups of serine and threonine are particularly sensitive to hydrolysis. -This effect is due to N→O-acyl migration via oxazoline and subsequent hydrolysis of the ester bond. -Hydrolysis of proteins with dilute acids cleaves aspartyl-X-bonds.
  18. 18. Partial Hydrolysis -Separation of peptide fragments is achieved by gel and ionexchange column chromatography using a volatile buffer as eluent (pyridine) which can be removed by freeze-drying of the fractions. -The separation of peptides and proteins by reversed-phase HPLC has gained great importance, using volatile buffers mixed with organic, water-soluble solvents as the mobile phase. -The fragmentation of the protein is performed by different enzymic and/or chemical techniques, at least by two enzymes of different specifity. -The arrangement of the obtained peptides in the same order as they found in the protein is accomplished with the aid of overlapping sequences.
  19. 19. 4- Sequence Analysis -The classical method is the Edman degradation reaction. -It involves stepwise phenylisothiocyanate. degradation of peptides with -The resultant phenylthiohydantoin is identified directly. -The stepwise reactions are performed in solution or on peptide bound to a carrier, i. e. to a solid phase. -Both approaches have been automated (“sequencer”). Carriers used include resins containing amino groups (e.g., amino polystyrene) or glass beads treated with amino alkylsiloxane:
  20. 20. Sequence Analysis -The peptides are then attached to the carrier by carboxyl groups (activation with carbodiimide as in peptide synthesis) or by amino groups. -For example, a peptide segment from the hydrolysis of protein by trypsin has lysine as its C-terminal amino acid. It is attached to the carrier with phenylene-diisothiocyanate through amino groups. -Mild acidic treatment of the carrier under conditions of the Edman degradation splits the first peptide bond. -The Edman procedure is then performed on the shortened peptide through second, third and subsequent repetitive reactions:
  21. 21. Protein Conformation
  22. 22. Protein molecule can be formed of one or more polypeptide chains which may vary in the number and sequence of amino acid residues.
  23. 23. Extended Peptide Chains -Information about conformation is available through X-ray crystallographic analysis of protein crystals and by measuring the distance between selected protons of the peptide chain by means of H-NMR spectroscopy in solution. -X-ray structural analysis of a fully extended peptide chain reveal the lengths and angles of bonds -The peptide bond has partial (40%) double bond character with electrons shared between the C-O and C-N bonds. -The resonance energy is about 83.6 kJ/mole Structure of an elongated peptide chain.
  24. 24. Levels of Protein Structure  Primary structure  Secondary structure  Tertiary structure  Quaternary structure
  25. 25. Primary structure  It is the amino acid sequence of the polypeptide chain linked by peptide bonds.  It is characteristic for every protein.  All proteins have an  N-terminal end (with a free amino group) and  C-terminal end (with a free carboxyl group).  Polypeptide chain sequence is written according to the sequence of amino acid residues from the N to C terminus amino acids.
  26. 26. Secondary structure  Is the local spatial arrangement of the polypeptide’s backbone (peptide bond) atoms without regard to the conformations of its side chains.  Peptide bonds contain polar amide hydrogen atoms (with a partial positive charge) and polar carbonyl oxygen atoms (with a partial negative charge).  This allows hydrogen bonds to form between peptide bonds in different parts of the chain.  The polypeptide chain can take different shapes or patterns in different parts of the chain, and these patterns are called the secondary protein structure.  There are 2 types of secondary structure: Alpha helix (α-helix) Beta-pleated sheet (β-pleated sheet).
  27. 27. Secondary structure Alpha helix • A spiral, compact, rod like structure • Mostly right handed α-helix, with R groups protruding outside • Stabilized by numerous hydrogen bonds which are formed between carbonyl oxygen (C=O, hydrogen acceptor) and peptide nitrogen (NH, hydrogen donor). • Forms about 100% of fibrous protein -keratin -80% of the globular protein; hemoglobin.
  28. 28. Alpha helix Alpha helix is disrupted by: • Proline: its imino group is not geometrically compatible with α- helix. • Large numbers of bulky amino acids e.g. tryptophan because of steric interference. • Large numbers of branched amino acids e.g. valine and isoleucine because of steric interference. • Large numbers of acidic and basic amino acids because they form ionic bonds or electrically repel each other.
  29. 29. β- PLEATED SHEET • Almost fully extended and its surface appear pleated. • Found in fibrous and globular protein. • Formed of 1 or polypeptide chains. more • Stabilized by hydrogen bonds between peptide bonds.
  30. 30. Types of β-PLEATED SHEET 1. Parallel β-pleated sheet: formed of 2 or more polypeptide chains running in the same direction (Nterminals are on the same side) 2. Anti-parallel β-pleated sheet: formed of one or more polypeptide chains running in opposite directions (N and C terminals are alternating).
  31. 31. Comparison of -helix and -sheet -helix -sheet Structure 1 polypeptide chain 1 or more polypeptide chains polypeptide Coiled Almost fully extended Hydrogen bonds - Formed between 2 - Formed between amino acids peptide bonds of 4 amino which has no relation in primary acids apart in the primary structure. structure. - Parallel to the axis of - Perpendicular to the axis of polypeptide chain. polypeptide chain. R groups - Protrude outside the helix - Project above and below the plane of the sheet
  32. 32. SECONDARY STRUCTURE OF PROTEIN α- helix
  33. 33. Tertiary structure • Is the three dimensional structure of a single polypeptide chain giving protein its characteristic shape. I- Globular proteins (enzymes) • Approximately spherical shape- water Soluble. II- Fibrous proteins (structural proteins) • • • • Rod-like shape Poor water solubility. Cross links and bonds in 3ry structure: S-S bond, Ionic, Hydrophobic interactions and H-bonding. Globular protein Fibrous protein
  34. 34. Tertiary structure
  35. 35. Forces that stabilize tertiary structure These are bonds that form between side chains of amino acids of the same polypeptide chain: 1. Disulfide bonds. 2. Hydrophobic interactions. 3. Hydrogen bonds. 4. Ionic interactions. 5. Van der Waal’s forces.
  36. 36. Forces that stabilize tertiary structure Disulfide bonds: covalent bond between 2 SH groups of 2 cysteine residues forming an S~S bond of cystine residue. Hydrophobic interaction: non covalent bonds between amino acids with non-polar side chains that are located in the interior of polytpeptide chain away from water. Hydrogen bonds: non covalent bond between a hydrogen atom attached to nitrogen or oxygen and another oxygen or nitrogen atom. Ionic interaction: non covalent bonds between negatively charged groups in acidic amino acids (as carboxilic group in the side chain of aspartate or glutamate) and positively charged groups in basic amino acids (as amino group in the side chain of lysine) Van der Waal’s forces: non covalent bonds occurring when two adjacent atoms come into closer distance.
  37. 37. Forces that stabilize tertiary structure
  38. 38. Quaternary structure  Many proteins are composed of two or more polypeptide chains which are loosely associated through noncovalent interactions (hydrogen bonds, ionic bonds and hydrophobic interactions).  An individual polypeptide is termed subunit or monomer.  According to the number of subunits, proteins are either: dimeric (2 subunits), trimeric (3 subunits), tetrameric (4 subunits; e.g. HB) oligomeric (many subunits).
  39. 39. Examples of globular proteins
  40. 40. Classification of Proteins Simple proteins 1. Albumin 2. Globulins 3. Histones Conjugated proteins 1. 2. 3. 4. 5. 6. Phosphoproteins Glycoproteins Chromoproteins Lipoproteins Nucleoproteins Metalloproteins Derived proteins Results from denaturation or cleavage of native proteins by the action of acids, alkali or enzymes.
  41. 41. Conjugated Proteins Proteins can be modified to include other chemical groups “prosthetic groups” besides amino acids: Class Prosthetic group (s) Example •Lipoproteins •Lipids •VLDL •Glycoproteins •Carbohydrates •Immunoglobulin G •Phosphoproteins •Phosphate groups •Casinogen of milk •hemoproteins •Heme (iron porphyrin) •Hemoglobin
  42. 42. Denaturation of Protein
  43. 43. Denaturation of Protein -The term denaturation denotes a reversible or irreversible change of native conformation (tertiary structure) without cleavage of covalent bonds (except for disulfide bridges).  The primary structure of the protein is not changed because the peptide bonds are not affected  Denaturing agents include: 1. 2. 3. 4. 5. 6. Heat Changes in pH (concentrated acids or alkali) Ultraviolet rays X ray High salt concentration Heavy metals.
  44. 44. Denaturation -Denaturation is possible with any treatment that cleaves hydrogen bridges, ionic or hydrophobic bonds. This can be accomplished by: changing the temperature, adjusting the pH, increasing the interface area, or adding organic solvents, salts, urea, or detergents such as sodium dodecyl sulfate. -Denaturation is generally reversible when the peptide chain is stabilized in its unfolded state by the denaturing agent and native conformation can be re-established after removal of the agent. -Irreversible denaturation occurs when the unfolded peptide chain is stabilized by interaction with other chains (as occurs for instance with egg proteins during boiling). During unfolding reactive groups, such as thiol groups, that were blocked, may be exposed. Their participation in the formation of disulfide bonds may also cause an irreversible denaturation.
  45. 45. Denaturation
  46. 46. Effects of Denaturation -Denaturation destroys the native conformation of protein. -Denaturation destroys the biologic activity of protein, there is loss of hormonal, enzymatic and antibody activity. Applications of protein denaturing 1- Boiling eggs: Change in albumin shape and solubility. 2- Cooking meat: Easily chewable, digestible. 3- Swabbing skin with alcohol (disinfectant): Denatures/kills bacteria and viruses. 4- HCl in our stomach: denatures proteins and making it easily digestible by enzymes - So, eating cooked eggs, meat and liver is more useful to humans than eating them raw
  47. 47. Denaturation of Protein: Examples in Food -An aggregation of the peptide chains caused by the folding of globular proteins is connected with reduced solubility or swellability. -Thus the part of wheat gluten that is soluble in acetic acid diminishes as heat stress increases. -As a result of the reduced rising capacity of gluten caused by the pre-treatment, the volume of bread made of recombined flours is smaller. Solubility of gluten (wheat) in diluted acetic acid after various forms of thermal stress
  48. 48. Denaturation of Protein: Examples in Food -In the case of fibrous proteins, denaturation, through destruction of the highly ordered structure, generally leads to increased solubility or rising capacity. One example is the thermally caused collagen-to-gelatin conversion, which occurs when meat is cooked. -The thermal denaturation of the whey proteins βlactoglobulin and α-lactalbumin has been well-studied. -Denaturation of biologically active proteins is usually associated with loss of activity. The fact that denatured proteins are more readily digested by proteolytic enzymes is also of interest.

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