Proteins Working molecules of the cell Most abundant and functionally diverse molecules High molecular weight Generally

complex molecules of great variety that play a vital role in the organization function of a living organism Polymers of amino acids, connected by peptide bonds Minimum of 2 amino acid residues (ex: GLUTATHIONE) *NO SINGLE AMINO ACID EXISTS ALONE.
*PROLINE the only amino acid that is complete by itself and does not need the skeleton.

There are 20 naturally occurring amino acids in proteins which are encoded in our genes. An amino acid is made up of: COOH, NH2 (except proline), H atom and R group [ 4 substituents] joined to an alpha Carbon.

Primary Structure sequence or order of the amino acids. R group gives amino acids its distinctive characteristics (size, shape charge). The R-group give its extra negative charge. The hydrophobic part of proteins is located in its interior while the charged part are exposed to solvents. This what makes proteins soluble.

GRAPH of pH against no. of moles of OH / no. Of moles of amino acids

Isoelectric pH pH at which proteins precipitate and are isolated; basis of separation of proteins Isoelectric Precipitation isolation of protein throught the principle of precipitation, encouraging the isoelectric point to obtain isoelectric pH *Casein solid protein. Major Protein in Milk; used for Cheese production *Whey In Yakult. Separated from milk by Isoelectric Precipitation.

Amino Acids- optically active, except Glycine. Essential Amino Acids have nutritional quality; not produced by the body but can be supplemented. *pH of a body or a living system = 7.4 GRAPH involving isoeelctric point FORMULA involving the skeleton (explains why NH3+ becomes NH2 which is uncharged) [ H of the NH3 is deprotonated by the base. This involves the titration curve]

Formula that shows isoelectric pH at the center and what charged structures/ions prevail when it is basic or acidic. [Predominant Alanine at pH 12 = negative] [Predominant Alanine at pH 2 = positive]

CLASSIFICATION OF PROTEINS

A. According to Biochemical Function 1. 2. 3. 4. 5. 6. 7. 8. 9. Catalytic (Biocatalyst) Enzymes -most proteins are enzymes except RNA which is a nucleic acid Transport Hemoglobin, Transferin, Albumin (all these are found in blood) Storage Ovalbumin?, Casein, Gliadin, Zein? (in Gluten which is isolated from starch) Contraction actin, myosin (found in muscles) Gene Regulation Histones, non-histone (nuclear?) Regulatory Hormones, Repressor proteins Immune Protection antibodies, fibrin Structural collagen, elastin, keratin Neurotransmission receptor proteins

B. Composition 1. Simple Protein contains amino acids and derivatives a. b. c. d. e. Albumin egg albumin, serum, albumins, legumelin, leucosin Globulins egg globulin, fibrinogen, myosin Albuminoids or Scleroproteins collagen, elastin, keratin Glutellins glutenin Prolamine Giladin, zein, hordein

2. Complex or Conjugated Protein a. b. c. d. e. f. g. Chromoprotein contains pigments ( haemoglobin, cytochromes [in respiration], flavoproteins ) Nucleoprotein contains nucleic acids (histones, prolamine) Glycoprotein contains a carbo group Y=gamma ? (mucin in saliva and globulin) Phosphoprotein contains phosphate group (casein, ____) Lipoprotein contains fatty acids (apoB 100 ? ) Lecithe Proteins or Lecithane) with lecithin (milk) Metalloproteins with a metal atom ( Ferritin [a transport protein, iron]; cytochromes, flavoprotein)

3. Derived Proteins by digestion or Hydrolysis from other proteins Acid Hydrolysis addition of an acid Enzyme Hydrolysis addition of an enzyme Coagulated Proteins formed by heat ( a denaturing agent which denatures protein and makes it small) Secondary Derivatives intermediate products of protein digestion Proteans result from short action of acids or enzymes Metaproteins formed by the actions of acids or bases Cyanogen Bromide (CNBr) for cleaving ?the peptide terminal

C. Shape 1. Fibrous Protein polypeptide chain in parallel form along a single axis; forming long, thin sheets of fibers Examples: elastin, collagen, keratin, silk fibroin Collagen structural ____ 2. Globular Proteins polypeptide chains tightly folded into spherical or globular shape Examples: Enzymes, Albumin, Globulins, Hormones Hemoglobin oxygen treatment Myoglobin storage of oxygen Ribonuclease Enzyme Lysozyme Enzyme (bacterial with hydrolysis ?) Cytochrome C electron transport Immunoglobulin defense/ antibodies Actin Movement

DTERMINATION OF THE PRESENCE OF AMINO ACIDS

Ninhydrin Test Ninhydrin reagent + Amino Acid Purple Pigment General Test for Proteins Proteins have amino group like Proline With Imino group = Yellow __ blue ? Sanger s test Reagent = Carboxyl Terminal? Dansyl Chloride Determines presence of amino acid sequences Two terminal residues = with one peptide bond; a dipeptide

ASPARTAME methyl ester derivative marketed as a sugar substitute under the trade name Nutrasweet

Synthetic, not good for the body (but still, it has commercial importance) Dipeptide but still, physiologically active

GLUTATHIONE tripeptide; commonly occurring in nature

With a considerable physiological importance. It is a scavenger for oxidizing agents

*Two Pentapeptides in the brain ENKEPHELINS are naturally occurring analgesics. They differ only in their C-terminal amino acids.

1. Y-G-G-F-L

Leucine enkephalin

2. Y-G-G-K-M Methiaonine Enkephalin

OXYTOCIN- induces labor in pregnant women and controls contraction of uterine muscles.

plays an important rule in stimulating the milk flow in nursing mothers.

VASOPRESSIN controls blood pressure, regulates contraction of smooth muscle

Released by the action of hypothalamus which is located on the posterior pituitary gland Transported by the blood specific receptors LEVELS OF PROTEIN STRUCTURE

Primary Structure - amino acid sequence, order, arrangement - most stable (covalent bond) and peptide bond - most important, dictates all other structures - connected by peptide bond ( straight, rigid, planar with des. Of restriction - rotation about the alpha-carbon o psi alpha carbon = carbonyl carbon o phi alpha carbon = N ? - composed of single blocks of monomers with amino acids

Secondary Structure - Repetitive; regular folding - Spatial arrangement of atoms in polypeptide chain - Interaction = H-bond between amide proton and carbonyl oxygen N H ........ O = C a. Alpha helix  intramolecular H bond  Pitch: 5.4 armstrong  3.6 amino acid residues per Turn Illustration Types of Alpha helix y Right handed Alpha-helix illustration y Left-handed Alpha Heli x illustration *The R-group is located outside the helix Destabilizers of Alpha Helix ( Helix Breakers ) y y Ex: proline (cyclopentane) and hydroxyproline bulky, sterile effect Adjacent, similarly charged amino acid residues

Adjacent amino acids residues with bulky R groups (aromatic and aliphatic side chains)

b. Beta pleted sheets y Parallel-pleated sheets not parallel ang H-bonds y Anti-parallel pleated sheets straight ang H-bonds Illustration c. Other Secondary Structures y Random coils y Reverse turning? Or beta bends Tertiary Structure - 3D arrangement - Important aspect: arrangement of the side chains of amino acid residues - R groups form ionic bonds ( R groups are charged groups ) - Example of ionic bond: disulfide bonds - globular?, subunits (alpha and beta subunits) FORCES INVOLVED IN PROTEIN FOLDING Folding caused by many interactions (ionic, covalent, disulfide linkages) A. Hydrophobic Bond LIVAGMWFP and aromatic rings and aliphatic side chains B. Van der Waals

TYPES OF PROTEIN CONFORMATION A. Globular Proteins alpha and pleated B Sheets ( spherical, globules) B. Fibrous Proteins elastin, collagen (most abundant in vertebrae), fibrins, keratin ( in nailm hair, animal skin and feathers) y Filamentous, elongated

Quaternary Structure

Spatial arrangement of polypeptide subunits (tetramer?) With subunits different from tertiary structure Interactions: same with tertiary structure

*Additional Interactions protein can undergo folding and become smaller

Organization of Proteins

Structure and functions of proteins

1. COLLAGEN -has a triple helix structure -most abundant protein in animals -water insoluble fibers (hydrophobic) -great tensile strength -major role: stress-bearing component of connective tissues Primary Structure - (-X-Y-G) - X-Pro/Hyp; (Y is any amino acid) (Hyp = hydroxyproline) -each chain is about 800 amino acid residues Secondary Structure Left-handed alpha helix (a-helix) 3.3. amino acids/turn Pitch: 10 armstrong

Tertiary Structure Three chains parallel and wind each other in a right-handed manner to form a triple-helical structure H-Bond involving Hyp (hydroxyproline) and Hyl (hydroxyleucine?) residues Intramolecular and intermolecular ____ covalent _____

Quaternary Structure Procollagen Tropocollagen Collagen Fibrils Diseases related to collagen defects Scurvy deficiency of vitamin C Enlers- Danlos Syndrome Stretching of the skin

2. ELASTIN -

Connective tissue that has elastic properties Nonpolar amino acid residues 1/3 G, 1/3 V + A ... rich in P Random coil conformation ( these can stretch or relax) Intramolecular and intermolecular desmosine crosslinks

3. HEMOGLOBIN and MYOGLOBIN

Globular, hemoproteins Major role: hemoglobin oxygen transport Major role: myoglobin storage of oxygen *beef muscle redder than pork meat because it has more protein than fats

a. Hemoglobin -tetramer: A2B2 (2 alpha anad 2 beta) subunits four tetramer all in all -each subunit contains one heme - responsible for the red coloration of blood - prosthetic group - prophylin ring -centrally bound with FE +2 Primary Structure: Alpha = 141 amino acids Beta = 146 amino acids Secondary Structure- helical Tertiary Structure globular Quaternary Structure four-folded ( chain in tetrahedral arrangement) b. Myoglobin - no quaternary structure - monomer, only one polypeptide chain - contains heme - cys, asp, glu, gly, pre, ala Primary Stucture 153 amino acids Secondary Structure alpha helix (helical) Tertiary Structure Globular

AFFINITY OF OXYGEN TO HEMOGLOBIN AND MYOGLOBIN Hemoglobin sigmoidal curve Myoglobin hyperbolic curve Graph of partial pressure of O2 against Saturation with O2

Sickle Cell Anemia -

genetically altered hemoglobin Alteration occurs in the 6th amino acid of the beta subunit ( Glu becomes Val . ) Affected hemoglobin is called hemoglobin S Results in poor transport of oxygen and poor nutrition

4. INSULIN AND GLUCAGON - For carbohydrate metabolism or homeostasis - Regulatory proteins - Endocrine hormones - Both produced by the islets of langerhans in pancreas - Insulin: by Beta Cells ( for hyperglycemia, type II diabetes) - Glucagon: by Alpha Cells ( for hypoglycaemia ) a. Insulin

- Forms 2 polypeptide chain connected by 2 cys molecules and a disulfide linkage at the 7th and __ a.a. - 51 amino acid residues - Intermolecular at intramolecular disulfide linkage - Hypoglycaemic hormone? b. Glucagon - Monomer, only one polypeptide molecule - 29 amino acid residues - Simpler than insulin but still possess important functions - Increases blood level of glucose 5. IMMUNOGLOBULINS (IGa, IGe) - Defense proteins, antibodies - Produced by B-lymphocytes - Y-shaped, tetramer - Intermolecular and intramolecular disulfide linkages - Light chain and heavy chain - Constant region and variable region

6. MACROPHAGES - Autoimmune deficiency BUBBLE BOY. Inherited immunodeficiency - HIV

DENATURATION OF PROTEINS Loss of high level of structural organization of protein (sec, tert and quaternary structure) Use of heat changing pH ( positive ions are more dominant in acidic while negative are dominant in basic ??) use of organic solvents like alcohol and urea salts of heavy metals peroformic acid and 2-mercaptoethanol

SEPARATION AND CHARACTERIZATION TECHNIQUES FOR PROTEINS AND AMINO ACIDS I. Protein Isolation a. Selection of starting material - Source: animal or plant tissue (example: microorganisms) b. Criteria for choosing a sample - Ease of obtaining sufficient quantity of tissue - Amt of biomolecules in tissue - Any properties peculiar to the biomolecules of choice ( solubility, etc) c. Methods of Solubilization (lysis, or breaking them apart)

By hemogenization or solubilisation? Use of detergent (____name______) Trituration with mortar and pestle (to lyse the cells) o Types of solutions used: -isotonic, hypotonic (solute is greater inside) and hypertonic

Separation Techniques based on characteristics of the biomolecules. Solubility, molecular weight, size, density, affinity, charge *Glycine = smallest ___

Based on Solubility Change in pH Isoelectric pH / Precipitation -pH of mixture is adjusted to pHi of protein to be isolated as a ppt to minimize its solubility Principle used: dif. In solubility at isoelectric pH (pHi) Graph of pH against solubility Change in Ionic Strength -

SALTING IN - solubility of protein at low ionic strength (concentration) generally increases with the salt concentration - NaCl and KCl - salt: for preservation and storage

SALTING OUT -Uses ammonium sulfate -Ionic concentration -In myoglobin, increased concentration of ammonium sufate to lower / isolate myoglobin -decrease in solubility of proteins -result of competition between added salt ions and other dissolved solutes for molecular salvation *proteins aggregate -solution used = free(?) ammonium and sulfate ions Based on molecular Size CENTRIFUGATION - Process of subjecting a suspension of sample in a greatly increased gravitational field (centrifugal force) by rapidly rotating a receptacle containing the sample which will lead to the sedimentation of particles. Application: Differential Centrifugation different speed are used. Principles is dif. In size (lower size, larger biomolecules first). Used in food mixtures Ultracentrifugation tiniest molecules can be separated (organelles) (Cu mitochondria from Zn mitochondria) Refrigerated Centrifuge DIALYSIS

-movement of molecules by diffusion from high conc to lower conc. - uses semi-permeable membranes -dif in molecular size ULTRAFILTRATION -when macromolecular solution is forced under pressure througha semi-permeable bag or disc. LACTRILE (B17) AMYGDALIN injected to cancer patients GEL FILTRATION CHROMATOGRAPHY - size occlusion chrom - column is packed with porous beads - small molecules enter the beads and are retarded while large molecules cannot enter and they migrate faster - larger molecules will go with the eluent to produce the eluate - collected first = solvent (interstitial fluid) , then larger molecules - based on dif. In molecular size - larger molecules are eliminated first Based on Affinity Chromatography depends on affinity to stationary and mobile phase a. Normal phase Chromatography TLC, paper chrom b. Reversed Phase Chromatpgraphy HP liquid chromm Affinity Chrom based on ability of proteins to interact with specific molecules Based on Charge ELECTROPHORESIS -separation of charged particles in an electric field through a support medium. Types of Support Medium A. Polyacetamide Gel- for sep of proteins B. Agarose Gel sep of nucleic acids ISOELECTRIC FOCUSING involves gel electrophoresis of protein mixtures through stable pH gradient medium. Protein migrate to region where pH is equal to pHi. GEL ELECTROPHORESIS - Based on charge, size and size + charge SDS (sodium dodicyl sulfate) - Detergent for isolating proteins - Mask the intrinsic charge of proteins due to large negative charge it imparts on it - Based on molecular weight. - Straightens the protein to cut/cleave it. ION-EXCHANGE CHROM - Same with aaffinity chrom (both use ligands) - Based on net charge - Column is packed with resin that has ligands (either neg or positive) - Cation exchange chrom neg charge so it attracts pos ions - Anion exchange chrom cellulose, paper. Pos charged so it attracts neg ions.  Reference point = attracted ions

ELUTE OF HIGH SALT (eliminate them) - eluted first polar substance (Asp, Glu, Thr and those with OH that forms H Bond with H2O); acidic with OH and COOH - eluted last nonpolar molecules Gly, Ala, val, Met Nonpolar; with aliphatic side chains RETENTION TIME __ the amino acids are eluted

DETERMINATION OF THE PRIMARY STRUCTURE OF PROTEINS a. Qualitative and Quantitative Analysis of Amino Acids 1. Hydrolyse Peptide with 6 N HCl at 110 degree C for 24 hours 2. Separate mixture by amino acid analyzer *Amino acids are isolated or analyzed by changing pH. *Dissociate disulfide linkage by using a reducing agent. b. Determination of Amino Acid Sequence 1. For identification of N-terminal (first amino acid) A.A. residue a. Sanger s Reagent (DNFB Dinitrofluorobenzene) b. Dabsyl Chloride and Dansyl Chloride c. Edman Degradation - phenythio____ to produce phenylthiohydantoin? *a, b and c are all chemical treatments. 2. For identification of C-terminal (last AA) A.A. Residue a. Carboxy Peptidase (enzyme) b. Hdrazinolysis 3. Cleavage of Protein into Peptides. a. Chemical Method i. Cyanogen Bromide (CNBr) cleaves at Carboxyl side of Met? b. Enzymatic method i. Trypsin cleaves on carbpxyl side of Arg and Lys (both are basic AA) ii. Chymotrypsin cleaves at carboxyl side of aromatic AA *Both do not work when Pro is present, which is a destabilize of a-helix.