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Proteins

PEPTIDE

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Peptide Bond
Joins amino acids 40% double bond character
Caused by resonance

Resonance forms of peptide bonds. The peptide bond (C) is a hybrid of A and B, giving it a partial double bond character

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Planar nature of the peptide bond. The partial double bond characteristic prevents free rotation around the C-N bond; keeping it in the same plane with the attached O and H atoms. These planar bonds can pivot around the shared Ca atom
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Trans conformation; most common and sterically favored

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Cis conformation; found rarely with Pro, sterically unfavorable 9

Peptide bond
Joins amino acids 40% double bond character
Caused by resonance Results in shorter bond length

Peptide bond
Joins amino acids 40% double bond character
Caused by resonance Results in shorter bond length Double bond disallows rotation

Amino Acids

been chemically modified after they have been incorporated into a polypeptide and AAs that occur in living organisms but are not found in proteins e.g. proline & lysine hydroxyproline & hydroxylysine glutamate -carboxyglutamate

Non-standard AAs consists of AA residues that have

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Polypeptides

1. 2. 3. 4. 5. 6. 7. 8.

9.

Enzymatic catalysis enzymes involved in different metabolic pathways Transport & Storage hemoglobin Coordinated motion Actin & Myosin (muscle contraction) Mechanical support Collagen & Keratin Immune protection Antibodies (gamma globulins) Generation & Transmission of nerve impulses neurotransmitters Control of Growth & differentiation Repressor proteins (control elements in specific DNA of a cell) Hormones Insulin, Thyrotropin, Somatotropin (GH), Luteinizing hormone, & Follicle stimulating hormone (FSH) Biologic membranes
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I.

Based on composition, physical, & chemical properties

A. Simple proteins: 1. Albumin 5. Albuminoids & Scleroproteins 2. Globulins 6. Histones 3. Glutelins 7. Protamines 4. Prolamines

B. Conjugated proteins:
1. 2. 3. Nucleoproteins 4. Chromoproteins Glycoproteins & Mucoproteins 5. Lipoproteins Phosphoproteins 6. Metalloproteins

C. Derived proteins:
1. Primary derived 2. Secondary derived
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II.

Based on the shape & certain physical characteristics of the protein

A. Fibrous proteins Tough Insoluble in water Arranged around a single axis to form a fiber Involved in structural functions e.g. Collagen & keratin (bone & ligaments) B. Globular proteins Involved in mobile & dynamic functions e.g. Enzymes, hemoglobin, plasma proteins

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III.

Based on biologic functions

A. Enzymes dehydrogenases, kinases B. Storage proteins ferritin, myoglobin C. Regulatory proteins DNA-binding proteins,

peptide hormones D. Structural proteins function in brick & mortar" roles e.g. Collagen, proteoglycans E. Protective proteins blood clotting factors, immunoglobulins, interferon F. Transport proteins hemoglobin, plasma lipoproteins, transferrin G. Contractile or motile proteins actin, myosin, tubulin
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Each has a central carbon called -carbon 4 different groups:

1) A basic amino group (-NH2)

2) An acidic carboxyl group (-COOH) 3) A hydrogen atom (-H)

4) A distinctive side chain (-R)

Common AAs are known as -AAs because they have a primary amino group & a carboxylic acid group; the sole exception is PROLINE, which has a secondary amino group (-NH-), referred to as -imino acid
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H H2N
C

COOH

General Amino Acid Structure At pH 7.0

H +H3N
C

COO-

At physiological pHs (7.0-7.4), both the carboxyl and amino groups are charged

Only L-amino acids are found in proteins

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all protein AAs share the absolute configuration of L-glyceraldehyde and thus are L--AAs D-AAs are usually found in nonmammalian peptides & certain antibiotics human brain also contain D-serine & D-aspartate CW of gm-pos bacteria D-alanine & D-glutamate

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Amino Acids
Chiral

D-glyderaldehyde

L-glyderaldehyde

Amino Acids
Chiral 20 naturally occuring; distinguishing side chain

The properties of each AA are dependent on its side chain (-R)

they are the functional groups, hence the major determinants of the conformation & function of proteins (as well as electrical charge) knowledge for methods of analysis, purification, & identification of proteins

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Amino Acids
Chiral 20 naturally occurring; distinguishing side chain Classification:
Non-polar (hydrophobic) Charged polar Uncharged polar

Classification of AAs Based on Polarity I. AAs with nonpolar or hydrophobic R groups

- they are also referred to as neutral AAs contg. hydrocarbon R groups (do not bear positive or negative charges) - interact poorly with H20, nonpolar AAs play an important role maintaining the 3-dimensional structure of proteins - 2 types of hydrocarbon side chains: aromatic & aliphatic (Phe & Trp aromatic ring structures) - Alanine - Phenylalanine - Leucine - Glycine - Tryptophan - Valine - Proline - Methionine
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II. Amino acids with uncharged polar R groups

- they have functional groups capable of hydrogen bonding (easily interact with H20) - Ser, Thr, & Tyr contain polar hydroxyl groups, which enables them to participate in hydrogen bonding (an important factor in protein structure) - other functions of -OH group: a) formation of the phosphate ester of Tyr (regulatory mechanism) b) serve as points of attaching CHOs (Ser, Thr) c) amide derivative to Asp & Glu from Asparagine & Glutamine - Serine - Glutamine - Threonine - Asparagine - Tyrosine
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III. AAs with positively charged R groups - Lysine - Arginine - Histidine IV. AAs with negatively charged R groups - Aspartic acid - Glutamic acid

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Twenty Amino acids

TYR: Amphipathic

GLY: Unclassifiable
Hydrophobic (non polar)

Polar

Aliphatic
(ALA, VAL, LEU, ILE, MET, PRO)

Aromatic
(PHE, TRP)

Polar Neutral

Charged

Amide
(ASN, GLN)

-OH
(THR, SER)

-SH

Acidic

Basic

(CYS) (ASP, GLU)

(HIS, LYS,ARG)

Asparagine Glutamate Tyrosine Glycine

Asparagus wheat (gluten)

tyros (cheese)
glykos (sweet)

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Functions of AAs: 1. Building blocks of proteins 2. AAs are precursors of nitrogen-contg. molecules
a) Glycine heme, purines, creatinine, glutathione, hippuric acid b) Glutamic acid GABA (-aminobutyric acid) c) Phenylalanine & Tyrosine melanin, dopamine d) Tryptophan niacin, serotonin, indole & skatole, melatonin e) Histidine Histamine
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3. Source of energy 4. Special AAs, as components of certain types of proteins (post-translational modifications)

a) Hydroxyproline & Hydroxylysine collagen b) N-methyllysine myosin c) Gammacarboxyglutamic acid prothrombin d) Desmosine derivative of lysine, found in elastin e) N-acetyllysine found in histones that are associated with chromosomes
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5. Phosphorylation & dephosphorylation of Serine, Threonine, and Tyrosine play major role in the signal
transduction pathways

6. L--AAs in low MW peptides play additional roles as hormones 7. Both D- and L--AAs are present in polypeptide antibiotics elaborated by microorganisms 8. -AAs or their derivatives act as chemical messengers (neurotransmitters) e.g. Glycine, GABA,
serotonin, & melatonin 9. Hormones Thyroxine, Epinephrine, & Norepinephrine are derived from Phe & Tyr

10. Several Standard & non-standard AAs act as metabolic intermediates e.g. Arg, Citrulline, Ornithine components of the urea cycle 49
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Physical properties of AAs

Solubility 2. Melting points 3. Taste 4. Appearance 5. Optical property
1.

- for all standard AAs (except Glycine), the carbon is asymmetric, bonded to four different substituent groups. - -carbon is a chiral center; all molecules with a chiral center optically active [they can rotate the plane-polarized light either to the right (dextrorotatory) or to the left (levorotatory)]

6.

UV absorption spectrum of aromatic AAs

- Phe, Tyr, Trp (Aromatic AAs) all possess absorption maxima in the near-UV. - The absorption bands arise from the interaction of radiation with electrons in the aromatic rings

7.

Acid-base properties amino & carboxylic acid groups of AAs readily ionize
- pK values of -carboxylic acid = 2.2

- pK values of -amino = 9.4

-at physiologic pH (7.4), the amino groups are protonated & the carboxylic acid groups are in their conjugate base (carboxylate) form

Those -AAs having a single amino group & a single carboxyl group crystallize from neutral aqueous solutions as fully ionized species known as zwitterions (hybrid ions), each having both a positive & a negative charge. These ions are electrically neutral & remain stationary in an electrical field

Isoelectric Point (pHI, pI, or IpH)

- that pH at which an AA bears no net charge & hence does not move in an electrical field - that pH exactly at the midpoint between the pK values on either side of the zwitterion species

General Rule on IpH

the IpH of neutral AAs are around 6.0; that of acidic AA very much below 6.0; that of basic AA very much above 6.0 the physical properties of AAs are influenced by the ionic states of the carboxyl & -amino groups, and any ionizable groups in the side chains (7 of the common AAs have ionizable side chains) at a given pH, AAs frequently have different net charges; the ionic states of AA side chains strongly influence the 3-D structures & biochemical functions of proteins

Titration of AAs
Because AAs contain ionizable groups, the predominant ionic form of these molecules in solution depends on the pH. Titration illustrates the effect of pH on AA structure; it is also a useful tool in determining the reactivity of AA side chains e.g. alanine titrate with a strong base (NaOH)

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the isoelectric point for ALA may be calculated as follows: IpH = pK1 + pK2 = 2.4 + 9.8 = 6.1 2 2 as titration continues, the ammonium group loses its proton, leaving an uncharged amino group. ALA then has a net negative charge because of the carboxylate group

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Planar nature of the peptide bond. The partial double bond characteristic prevents free rotation around the C-N bond; keeping it in the same plane with the attached O and H atoms. These planar bonds can pivot around the shared Ca atom

Peptide Bond Steric Restrictions

The planar nature of the peptide bond restricts the possible conformations that a protein can assume. This can be predicted by the angle (above or below the peptide bond plane) of the two bonds between the a-carbon of the constituent amino acids. These phi (f) and psi (y) angles can be used to predict and define some higher order protein structures.

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Peptides
1. Glutathione (-glutamyl-L-cysteinylglycine)
- it contains an unusual -amide bond, wherein the -carboxyl group of the glutamic acid residue, not the -carboxyl group contribute to the peptide bond - involved in biological processes: a. protein & DNA synthesis b. drug & environmental toxin metabolism c. AA transport d. reducing agent (-SH group of cysteine residue) * accumulation of H202, oxidation of Fe Fe *with glutathione peroxidase, it prevents the accumulation of methemoglobin

O NH3 O O O O-C-CH-CH2-CH2-C-NH-CH-C-NH-CH2-C-O CH2 SH GLUTATHIONE

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2. Oxytocin The AA sequences of these 3. Vasopressin 2 peptide hormones differ only by 2 residues. They both contain 9 AAs & are produced by the cleavage of polypeptide precursors w/in diff. specialized cells in the hypothalamus Oxytocin stimulates uterine muscle contraction & ejection of milk (); testosterone synthesis () Vasopressin ADH; secreted in response to low pressure or a high Na conc. (water retention)
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CYSTYRILEGLNASNCYSPROLEUGLYNH2 I________S__S__________I OXYTOCIN

CYS-TYR-PHE-GLN-ASN-CYSPROARGGLYNH2 I________S__S__________ I VASOPRESSIN

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4. MET-Enkephalin They belong to a group 5. LEU-Enkephalin of peptides called Opioid peptides, found predominantly in nervous tissue cells.
- molecules that relieve pain & produce pleasant sensations; they bind to receptors in brain cells & induce analgesia - they represent one of the bodys own mechanism for pain control - enkephalin receptors also bind morphine, heroin, and other addicting opiate drugs

TYR GLY GLY PHE MET (Met-enkephalin) TYR GLY GLY PHE LEU (Leu-enkephalin)
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6. Atrial Natriuretic Factor a peptide w/ 28 AA residues produced by specialized cardiac cells & the NS; it stimulates the production of a dilute urine (effect opposite that of ADH)
SER-LEU-ARG-ARG-SER-SER-CYS-PHE-GLY-GLY-ARG-MET-ASP-ARGILE-GLY-ALA-GLN-SER-GLY-LEU-GLY-CYS-ASN-SER-PHE-ARG-TYR

7. Substance P They stimulate the perception 8. Bradykinin of pain (protective mech.), an effect opposed by the opioid peptides.
ARG-PRO-LYS-PRO-GLN-PHE-PHE-GLY-LEU-MET-NH2 (Substance P) ARG-PRO-PRO-GLY-PHE-SER-PRO-PHE-ARG
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(Bradykinin)
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9. Glucagon a pancreatic hormone w/ 29 AA residues; opposes the action of insulin 10. Corticotropin contains 39 AA residues; a hormone of the anterior pituitary gland that stimulates the adrenal cortex
11. L-aspartylphenylalanyl methyl ester a commercially synthesized dipeptide w/c is an artificial sweetener (ASPARTAME or NUTRASWEET)
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Structure helps us understand function Many disorders are due to aberrant protein structure

Sickle cell anemia

Can aid in design of therapeutics

Disruption of structure causes disruption of function

denaturation

The normal structure found in an organism Functional structure

Human Insulin

AAs can be polymerized to form chains; the process can be represented as a condensation reaction (elimination of water molecules) The resulting CO-NH linkage, an amide linkage, is known as the peptide bond The individual AAs in a peptide chain (monomeric units) = AA residues Linear polymer = head-to-tail fashion

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z 7

Evidences that a peptide linkage exist in proteins:

1. Positive reaction to the Biuret test 2. Relatively few titratable amino & carboxyl functions 3. Hydrolyzed by enzymes w/c are specific for the peptide bond 4. X-ray diffraction studies proved the existence of peptide linkages between AAs in hemoglobin & myoglobin 5. Synthesis of insulin

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Primary structure = the complete set of covalent bonds within a protein

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PRIMARY: the linear sequence of amino acids linked together by peptide bonds SECONDARY: regions within polypeptide chains with regular, recurring, localized structure stabilized by H-bonding between constituent amino acid residues
- two types: a) coils or helices (intrachain H-bonding) b) sheets or pleats (interchain H-bonding)

TERTIARY: the overall threedimensional conformation of a protein QUATERNARY: the three-dimensional conformation of a protein composed of multiple polypeptide subunits THE PRIMARY AMINO ACID SEQUENCE IS THE ULTIMATE DETERMINANT OF FINAL PROTEIN STRUCTURE

To know the molecular basis of activity of the protein. To know sequence/structure relationships. Molecular Pharmacology to know which AA is at fault e.g. Sickle Cell Anemia

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Disulfide bonds
Form between two intraor interchain cysteine residues, product called cystine - Stabilizes/creates protein conformation - Prevalent in extracellular/ secreted proteins

Ex: INSULIN

2o Structure:

a-helix

each oxygen of a carbonyl group of a peptide bond forms a H-bond with the hydrogen atom attached to a nitrogen in a peptide bond 4 amino acids further along the chain; very stable structurally; prolines will disrupt helix formation

1. Stabilized by inter-residue H-bonds formed between the H atom attached to a peptide N & the carbonyl O of the residue fourth in line behind in the primary structure. 2. Each peptide bond participates in the H-bonding. This confers maximum stability. 3. An -helix forms spontaneously as it is the lowest energy, most stable conformation for a polypeptide chain. It is relatively stable, provided the R groups are uncharged & incapable of H bonding. 4. There are about 3.6 AA residues per turn & the pitch (distance between corresponding points per turn) is 0.54nm or 5.4A. The spacing per residue is about 1.5A or 0.15 nm. 5. AA R groups extend outward from the helix *property of cooperativity in folding *side chain affects the stability of the a-helix (ALA, GLY, 84 4/22/2013 PRO)

1. Presence of adjacent similarly charged AAs 2. Presence of adjacent bulky R groups of AAs 3. Presence of Proline contains a rigid ring that prevents the N-C bond from rotating; it has no N-H group available to form intrachain H-bonds

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In this secondary structure, each amino acid residue is rotated 180o relative to its adjacent residue. Occur most commonly in anti-parallel directions, but can also be found in parallel. H-bonds between adjacent chains aid in stabilizing the conformation.

Anti-Parallel
(more stable; colinear H-bond)

Parallel

Repeat distance is 7.0 R group on the Amino acids alternate up-down-up above and below the plane of the sheet 2 - 15 amino acids residues long 2 - 15 strands per sheet Ave of 6 strands with a width of 25 parallel less stable than anti-parallel Anti-parallel needs a hairpin turn Tandem parallel needs crossover connection which has a right handed sense

1. Hydrogen bonds are interchain 2. R groups lie above or below the zigzagging planes of the pleated sheet & are nearly perpendicular to them 3. AAs with less bulky R groups are present

1. Bulky R groups 2. R groups w/ like charges

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 they are combinations of secondary structure (-helix & -pleated sheet) that appear in a number of different proteins they may have a particular function or may simply occur as part of a larger functional unit (domain) Motifs may be encoded by distinct segments of DNA that are combined in different ways; have roughly 10 40 residues each
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b-bend

Certain amino sequences have patterns to their folding. A. bab motif, B. b hairpin C. aa motif

Super-secondary structures commonly found in some DNA-binding proteins

b-Barrel

Bundle

Saddle

1. Helix-loop-helix or -units

- one of the simplest supersecondary structures - contains 2 successive -helices separated by a loop or nonhelical segment - occurs in a number of Ca-binding proteins, where glutamate & aspartate residues provide the Ca-binding site - it also provide the oligonucleotide-binding portion of DNA-binding proteins (repressors & transcription factors) - it is also referred to as the EF hand
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loop regions connecting alpha-helical segments can have important functions e.g. EF-hand and DNA-binding EF hand loop ~ 12 residues polar and hydrophobic a.a. conserved positions Glycine is invariant at the sixth position The calcium ion is octahedrally coordinated by carboxyl side chains, main chain groups and bound solvent

2. Coiled-coil motif

3. bab unit

- composed of two, three, or four amphipathic -helices wrapped around one another - hydrophobic side chains project like knobs from one helix & interdigitate into the gaps or holes between the hydrophobic side chains of the other helix consists of two parallel beta strands linked to an intervening alpha-helix by two loops - The helix connects the carboxyl end of one beta strand to the amino end of the next & usually runs parallel to the 2 strands
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4. Hairpin

5. Zinc finger

- consists of two adjacent antiparallel beta strands connected by a hairpin loop - composed of three secondary structures, an -helix & two -strands w/ an antiparallel orientation, w/c form a fingerlike bundle held together by a zinc ion - this motif is commonly found in proteins that bind RNA or DNA - another DNA-binding domain - the knobs on the leucine zipper represent leucine side chains
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6. Leucine zipper

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7. Greek key

8. -meander pattern

- it is formed when an antiparallel beta-sheet doubles back on itself in a pattern that resembles a common Greek pottery design - it links 4 or more antiparallel beta strands

- two antiparallel -sheets are connected by polar AAs & glycine to effect an abrupt change in direction of the polypeptide chain - it is also called reverse or -turns

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Glyceraldehyde-3phosphate dehydrogenase

Binding NADH in the Rossmann fold.

Tertiary Structures of Proteins

- refers to the unique 3-D structure of the protein - results from the folding of a polypeptide w/c may already possess some regions of alpha-helix and/or beta-sheet, into a closely packed, nearly spherical shape - it indicates how secondary structural features (helices, sheets, bends, turns, & loops) assemble to form domains, and how these domains relate spatially to one another

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Ex: Tertiary Structure

Ex: Quaternary Structure

Myoglobin

b-subunit Hemoglobin

The amino acid sequences of myoglobin and hemoglobin are similar (or, highly conserved) but not identical Their polypeptide chains fold in a similar manner Myoglobin is found in muscles as a monomeric protein; hemoglobins are found in mature erythrocytes as multisubunit tetrameric proteins. Both are localized to the cytosol

(Surface helix) Myoglobin Hba (horse) Hbb (horse) Hba (human) Hbb (human) Hbg (human) Hbd (human) (Internal helix) Myoglobin Hba (horse) Hbb (horse) Hba (human) Hbb (human) Hbg (human) Hbd (human)

At the tertiary level, surface residues prevent one myoglobin from binding complementarily with another myoglobin; thus it only exists as a monomer. Each monomer contains a heme prosthetic group: a protoporphryin IX derivative with a bound Fe2+ atom. Can only bind one oxygen (O2) per monomer The normal physiological [O2] at the muscle is high enough to saturate O2 binding of myoglobin.

Heme-Fe2+

Protein-Heme Complex with bound oxygen

At the tertiary level, the surface residues of the a and b subunits form complementary sites that promote tetramer formation (a2b2), the normal physiological form of hemoglobin. Contains 4 heme groups, so up to 4 O2 can be bound Its physiological role is as a carrier/transporter of oxygen from the lungs to the rest of the body, therefore its oxygen binding affinity is much lower than that of myoglobin. If the Fe2+ becomes oxidized to Fe3+ by chemicals or oxidants, oxygen can no longer bind, called Methemoglobin

1. Electrostatic/ionic 2. Hydrogen bonds

3. Hydrophobic interactions 4. Disulfide bonds

Protein disulfide isomerase - catalyzes formation of disulfide bonds Peptidyl Prolyl Cis-Trans Isomerases - this class of enzymes, also called rotamases, facilitates the conversion of Pro residues to cis-conformations (originally made in a trans conformation)

Chaperones - a large group of proteins, also termed heat shock proteins and chaperonins, their precise regulation and mechanisms of action remain largely undefined. During the folding process, they function to prevent unfavorable protein interactions with other potentially complementary surfaces (like other proteins, carbohydrates, lipids, nucleic acids, etc.) Many of these proteins are ATPases (use hydrolysis of ATP as an energy source).

Electrophoresis Chromatography: Gel filtration, ion exchange, affinity Mass Spectrometry, X-ray Crystallography, NMR

pH matters as well as the pI of the protein. Can be run at several pH values depending on proteins. DNA can also be separated on agarose gels. Genomic sized DNA can also be separated but requires more sophisticated equipment.

Presence of SDS, a detergent, denatures and linearizes a protein (Na and sulfate bind to charged amino acids, the hydrocarbon chain interacts with hydrophobic residues). An applied electric field leads to separation of proteins based on size through a defined gel pore matrix. For electrophoresis in the absence of SDS, separation is based on size, charge and shape of the protein (proteins are not denatured and can potentially retain function or activity)

Separation of proteins based on their size is linear in relation to the distance migrated in the gel. Using protein standards of known mass and staining of the separated proteins with dye, the mass of the proteins in the sample can be determined. This is useful for purification and diagnostic purposes.

Add sodium dodecyl sulfate, a 12 carbon detergent to give a negative charge to the protein. SDS also denatures the protein and collapses into a globular ball.

The proteins are separated by molecular mass

Analytical methods used to separate molecules. Involves a mobile and a stationary phase.

Mobile phase is what the material to be separated is dissolved in.

Stationary phase is a porous solid matrix which the mobile phase surrounds. Separation occurs because of the differing chemistries each molecule has with both the mobile and stationary phase. Chemistries are different depending on the specific method.

Gas - liquid: Mobile phase is gaseous, stationary phase is liquid usually bound to a solid matrix. Liquid - Liquid: Mobile phase is gaseous, stationary phase is liquid usually bound to a solid matrix. If separation is based on ionic interaction the method is called Ion Exchange chromatography. If separation is based on solubility differences between the phases the method is called adsorption chromatography. If the separation is base on size of molecule the method is called gel filtration or size exclusion. If the separation is base on ligand affinity the method is called Affinity chromatography.

Separation is based on protein size. Dextran or polyacrylamide beads of uniform diameter are manufactured with different pore sizes. Depending on the sizes of the proteins to be separated, they will enter the pore if small enough, or be excluded if they are too large.

Hydrophobic Chromatography
Proteins are separated based on their net content of hydrophobic amino acids. A hydrocarbon chain of 4-16 carbons is the usual type of resin.

Separation of proteins based on the net charge of their constituent amino acids. Different salt concentrations can be used to elute the bound proteins into tubes in a fraction collector. As shown below, resins for binding (+) or (-) charged proteins can be used

Based on the target proteins ability to bind a specific ligand, only proteins that bind to this ligand will be retained on the column bead. This is especially useful for immunoaffinity purification of proteins using specific antibodies for them. Example:

The sequence of a protein (or peptide) is determined using sophisticated Mass Spectrometry procedures. The three dimensional structures of proteins are determined using X-ray crystallographic and NMR (nuclear magnetic resonance) spectroscopic methods. Protein sequence data banks useful for structural and sequence comparisons Please note that the new discipline termed Proteomics is evolving to incorporate crossover analysis of sequence data banks, Mass Spec methodology, and living cells

Metabolic defects: Phenylketonuria (Phe), Tyrosinemias (Phe, Tyr), Maple Syrup Urine Disease (Leu, Val, Ile), Alkaptonuria (Tyr) Absorption/transport defects: Cystinuria (Cys), Hartnup disease , Fanconis Syndrome These diseases are generally diagnosed from indicators in the urine or plasma.

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