Amino Acids exam I

• Glycine
• Gly
• G
• Nonpolar, aliphatic R group
• simplest, smallest, ONLY ACHIRAL

• Alanine
• Ala
• A
• Nonpolar, aliphatic R group

• Proline
• Pro
• P
• Nonpolar, aliphatic R group
• Imino group, KINKS IN PROTEINS

• Valine
• Val
• V
• Nonpolar, aliphatic R group

• Leucine
• Leu
• L
• Nonpolar, aliphatic R group

• Isoleucine
• ile
• I
• Nonpolar, aliphatic R group
• 2 chiral carbons

• Methionine
• Met
• M
• Nonpolar, aliphatic R group
• Methylated S (non terminal)

• Phenylalanine
• Phe
• F
• Aromatic group
• most hydrophobic/least useful at 280 nm to assay proteins

• Tyrosine
• Tyr
• Y
• Aromatic group
• most ionizable, used for protein assay at 280nm

• Tryptophan
• Trp
• W
• Aromatic group
• best for protein assay at 280nm

• Serine
• Ser
• S
• Polar uncharged group
• weak acid

• Threonine
• Thr
• T
• Polar uncharged group
• 2nd chiral carbon, weak acid

• Cysteine
• Cys
• C
• Polar uncharged group
• SH group, DISULFIDE BOND, HOLDS LOOPS IN PROTEINS TOGETHER, can ionize

• Asparagine
• Asn
• N
• Polar uncharged group
• in asparagus

• Glutamine
• Gln
• Q
• Polar uncharged group

• Lysine
• Lys
• K
• Positively charged group (basic)

• Histidine
• His
• H
• Positively charged group
• imidazole group, ONLY FUNCTIONAL GROUP WITH pKa NEAR 7.0, blood buffer

• Arginine
• Arg
• R
• Positively charged group
• GUANINDINO GROUP, UREA SYNTHESIS

• Aspartate
• Asp
• D
• Negatively charged group (acidic)

• Glutamate
• Glu
• E
• Negatively charged group (acidic)

organic compounds only produced by living organisms (life begets life)

• lab synthesis of urea
• NH3 + N=C-OH --> N=C-O- NH4+  --> H2N-C(=0)-NH2
• ammonia + cyanic acid ---> ammonium cyanate ---> urea

• compound of carbon (bad b/c CO2 is inorganic)
• SO now they say must have C-C or C-H bond, but urea doesn't.

• reactivity: ionic more, bonds break easier
• mp/bp: hydrocarbon lower (liquid at room temp) b/c only has London forces.  
• electrical conductivity: ions are mobile, hydrocarbons don't conduct
• solubility in aqueous solvents: ions yes, hydrocarbons no1/6

• OH, alcohol
• intra/inter molecular dehydration (C=C, ether)
• Oxidation(-CHO)

• aldehyde -CHO, ketone R-C(=O)-R'
• Reduction (aldehyde), oxidation (COOH)

• carboxylic acid -COO(H)
• Reduction (aldehyde), esterification (+OH = COO), thioester +SH, anhydrides (+COOH or POOH), +amine = amide

• NH3, Amine
• +COOH amide
• protonize = protonated amine

amide, C=O-NH3

• thiol, -SH
• +COOH = thioester
• Oxidation = disulfide bond

phosphate PO4

COO

COS

-O-

R-C(=O)OP(=O)OH

amide bond, aa only

• same molecular formula, different arrangement
• Most dissimilar of all isomers, b/c functional groups can be different

• same groups of atoms but differ in arrangment in space
• sub-categories (diastereomers, enantiomers)

stereoisomers that are non-mirror image.  Include geometric, conformational

cis/trans.  Diastereomers, stereoisomers.  Hindered around double bond.

rotation around C=C bond, eclipsed/staggered.Diastereomers, stereoisomers

mirror image stereoisomers.  Chiral center.

• enantiomers where spatial arrangement is fixed.  L vs D, rotate light differently (optically active except when racemic).  Stereospecific.  Humans usually L
• Put COO at the top.  NH3 on L = L, NH3 on R= D

should be alkane but it is alkyne b/c of pi bonds

nucleophiles.  Unshared pairs cause nucleophilic attacks.  Makes new pathway with lower activation energy.

• group transfer: xfer a group from one molecule to another, catalyzed by transferase, kinase transfers phosphoryl
• redox: catalyzed by oxidoreductase
• intramolecular rearrangement: isomerization with isomerase, btween structural isomers
• cleavage: splits or joins without water.  Breaks C-C/C-O/C-N bond, may form =, catalyzed by lyase
• condensation: joins 2 while eliminating water (dehydration synthesis)/hydrolysis, hydrolase.

group transfer

redox

intramolecular rearrangment

cleavage

joins 2 while eliminating water (dehydration synthesis)/hydrolysis, hydrolase.

protein, lipid, carbohydrate, nucleic acid

• forces of attraction between molecules
• ionic bond (salt bridge), ion-dipole, dipole-dipole, hydrogen bond (stronger when not bent like H2O), dipole-induced dipole/dipole polarizability, london forces, hydrophobic forces,

attraction between opposite charges or repulsion between like charges (ion pair/salt bridge).

ion and partial opposite charge, attraction

attraction between partial opposite charges (increases bp of dimethyl ether above propane)

particularly strong dipole-dipole.  H attached to very strong electronegative (F, O, N), so H is very +

permanent dipole deforms cloud of electrons, attracts them.  Nonpolar gases are weakly soluble

two temporary dipoles.  WEAKEST.  Increases as Mr increases (bigger = stronger)

2 nonpolar will join together in aqueous environment.  entropy increases as aqueous shell is released. Not due to attraction

• high heat of fusion (organisms don't freeze)
• high heat of vaporization (sweating cools without dehydration)
• high specific heat (minimize temp fluctuations)
• less dense as solid than liquid (ice floats)

• hydrophobic and hydrophilic parts
• hydrophobic fold together, form micelles or bilayers
• common in proteins, membrane lipids, fatty acids, vit A and D

= to molarity b/c ionizes completely

• n x M
• moles times molarity
• strong acid like HCl, N = M b/c only 1 H.  H2SO4, N=2M

• V_AN_A = V_BN_B
• 10.0 mL (N_A) = 7.2 mL (0.10N)
• N_A = 0.072 N = 7.2 x 10^-2 N = [H+]
• pH = - log (7.2 x 10^-2) = 1.1
• HCl = monoprotic (one H)
• NaOH = monohydroxy base
• so n = 1, N = M

• effective at physiological pH (6-8)
• pH doesn't change with dilution or changes in temp
• chemically unreactive
• don't diffuse across bio membranes
• don't absorb light at 240-700 nm

• buffers (borate, phosphate, tris)
• "Good Buffers" (bicine, mops, hepes, pipes)
• Developed by Dr. Normal Good

• pKa.  Stronger acid has higher Ka and lower pKa
• So Phosphoric stronger than hydrogen phosphate

• more than one H to give away, more than one step to hydrolyze, more than one Ka.
• phosphoric acid is triprotic
• carbonic acid is diprotic

•    may ignore bottom x
• pKa = -log K_a

• X = K_a x HA (x = [H+])
• CH₃COOH <--> H⁺ + CH₃COO^-

• Ka = 1.74 x 10^-5 = x²/0.100 (ignore -x)
• 
• = 1.32 x 10^-3

-log = 2.88

• Add half molar amount of base, so [A-] = [HA].  Half equivalence point, half titration point, inflection point.  Where pH = pKa.  
• less than half molar  = pH<pKa
• more than half molar = pH > pKa.  So pH goes with [A-]
• polyprotic have inflection/pKa for each ionization

pH increases above 7.4, tetany (7.8)

blood pH decreases below 7.4, coma (7.0)

watch for solutions that are 7.0-7.39 = basic but acidotic

• weak acid and conjugate base pair
• best at +/- 1 from pKa
• higher concentration makes more effective

• can sub mole ratio for molar ratio.  VOlumes of HA and A- are same
• 
• 
• pH = pKa + log frac{A^-}{HA}
• equimolar can use volume fraction

•   = mole fraction
• 4.76 (pKa) + log 2.76 = 5.20 pH

• total moles of acetate = 0.30 mol/L x 5.0L = 1.5 mol
• 
• 4.46 = 4.76 + log...
• -0.30 = log...
• 0.501/1 = A^-/HA
• mole fraction: X_A^- = 0.501/1.501
• X_HA = 1.00/1.501
• X x moles = moles per part.  
• all 1.5 moles came from HA.  1.5mol x 2.0mol/L = 0.75L = 750 mL HOAc.  
• Titrate with 0.501 moles KOH to convert 0.501 mol HA to 0.501 mol A-
• 0.501mol KOH x 2.5mol/L = 0.20 L = 200 mL KOH

• total moles of phosphate buffer = 2.00L x 0.200mol/L = 0.400 moles
• Use pKa closest to pH of the problem (6.86)
• 7.20 = 6.86 + log 
• = mol fraction, get g of each.

• 7.8 = 8.1 + log [A-]/[HA]
• each mol fraction (portion over whole) x 0.20 M

[A or Tris^o] + 0.030M H⁺ --> HA⁺

• 0.0668 M - 0.030 M H+ = 0.0368 M Tris^o
• 0.133 M + 0.030 M H+ = 0.163 M Tris+

• pH = pKa + log...
• 8.1 + log (0.037M/0.163M)
• 8.1 + log 0.227
• 8.1-0.64 = 7.46 = 7.5

• pH = - log [H+]
• - log 0.030M
• = 1.5

• P_i
• pKa 6.86, ~80mM intracellular

Hb, H₂CO₃

H₂CO₃ (15x higher concentration than Pi)

• CO₂ (g) in lungs <-(K₃)-> CO₂(d) in blood + H₂O <-(K₂=K_h)-> H₂CO₃ <-K₁=Ka->HCO₃^- + H⁺
• Kh is equilibrium constant for hydration of CO2 (d)
• pKa for the ionization of H2CO3
• open system, respiration rate controlled by brainstem controls pH

more CO2 exhaled, Rx moves to left

basic, shift right, breathe out less CO2

• decreased CO2, blowing off too much, shift left, consume H+, when H+ depleted, pH increases, basic, respiratory alkylosis
• Breathe into a paper bag, rebreathe CO2 to equalize

increased CO2, not blowing off, shift right, respiratory acidosis.  More dangerous thatn alkylosis.

asthma, pneumonia, obstruction, collapsed trachea, CF, COPD, CHF

• pH = pKa + log...
• pH = 6.1 + log 50 = 7.79897~7.8.  
• Alkylosis

dipolar ion

• put COOH up.  NH3 is left in L amino acids.  Not necessary levorotary
• This system based on glyceraldehyde. 
• ALL AMINO ACIDS IN PROTEINS ARE L (evolution.  Could all be D, mix doesn't work)

gly, ala, val, leu, ile, pro, met

phe, tyr, trp

ser, thr, cys, asn, gln

asp, glu

lys, arg, his

• 
•      I        II        III      IV        V

• pH at which the net charge is 0 
•   for diprotic
• for triprotic, isoelectric species and average pKa just before and after.

• set molecules up so N terminus on L, carboxyl on R 
• hydrolysis.  Endergonic.  Stable, high activation energy
• usually linear.

2-10 amino acid residues long

2-10 amino acid residues long.

11-50 residues long

> 50 residues long

calculate first NH3 and last COOH, then any ionizable R groups.  Ignore nonpolar groups.  Start protonated, titrate up until pI is reached, use pKa before and after.

• glucagon (controls blood glucose levels)
• vasopressin (increases blood pressure by stimulating reabsorption of H2O by kidneys and vasoconstriction)
• oxytocin (uterine contraction during labor and milk duct contraction during lactation)

• both are cyclic due to disulfide bond making ring
• both have amide derivatives at end instead of amine
• vaso has a phe and an arg
• oxy has a ile and a leu
• Arginine is biggest difference--polar.

proteins

non-amino acid portion of protein that is necessary to function

amino acid part of conjugated protein, does not include prosthetic groups.

contains groups other than amino acids (apoprotein +prosthetic groups)

includes only amino acids

conjugated protein, with carbohydrate prosthetic group (IgG)

conjugated protein with heme prosthetic group (Hb, Mb, cytochromes)

conjugated proteins with Fe²⁺, Zn²⁺, Cu⁺, etc as prosthetic.  Hb, Mb, cytochromes

conjugated proteins with lipid prosthetic group, like HDL, LDL, which transport cholesterol.

amino acids listed in alphabetical order, with commas

amino acids listed in order

shape, folding of protein

each individual folded polypeptide.  Extended/unfolded are not subunits (like in collagen)

multi-subunit, several subunits in one protein

multimeric protein where some subunits are identical.  The subunits are called protomers

subunits in an oligomeric protein

homologous subunits

heterologous subunits

• molar mass.  
• Average amino acid residue in protein = 110 g/mol.  
• protein molar mass/110

• unit of molecular mass (=1amu).  
• Can also become kilodaltons

relative molar mass.  Same, just unitless

• can do large volumes, cheap materials
• centrifugation: Includes cell lysis and subcellular fractionation, then selective preciptitation by acetone, ammonium sulfate or isoelectric precipitation

• acetone: dentatures membranes protecting hydrophobic proteins
• ammonium sulfate: "salting out" by competing with ammonium
• isoelectric precipitation: change pH to create pI, no charge is less water soluble and will precipitate out.

no net charge so nonpolar, less water soluble and will precipitate out, a type of crude separation

• later in purification, use expensive materials and work better on more homogenous substances.  
• chromatography, electrophoresis

• mixture in mobile phase passes over separating material in stationary phase
• separation occurs via distribution in between phases
• types characterized by nature of separating material
• 1. TLC (thin layer chromatography) for lipids
• 2. paper - amino acids and peptides
• 3. column for amino acids, peptides and proteins

• concentrating purification for amino acids, peptides and proteins.  
• ion-exchange chromatography, separated by net charge.  Can be anion exchanger (DEAE-Sephadex, binds - charges) or cation exchanger (Dowex-50, binds + charges)
• like charges bind, neutral or opposite charge wash off
• increase salt or change pH to elute.

"un-stick"

increasing, makes cation into acid so protein comes off

anion

decreasing, make anion into base to accept proton, must lower solution to make it acid.

it can't, they wash off together.

• separation by size and shape, aka size exclusion chromatography, molecular sieve chromatography
• VERY SMALL VOLUME (<5%)
• stationary phase is porous beads (Sephadex), large molecules go around and elute first, small molecules enter all beads and come out last.  Compact molecules move more slowly than elongated, "effective radius"

• in gel filtration chromatography, elongated molecules move like they are large spheres with the molecule as the diameter.  
• They therefore come out earlier/move faster than they should
• This is a source of error.

• insulin
• oxytocin
• heptapeptide
• tripeptide
• amino acid

• separates by specific binding to another molecule (or ligand), attached covalently to stationary phase.  
• substrate/enzyme, hormone/receptor, Ag/Ab.
• unbound wash off, then elute with free ligand, dialysis

a molecule that specifically and reversibly binds to a protein

dialysis

• separation based on polarity.  Stationary phase can be polar (bind polar molecules, elute by increasing polarity) or nonpolar (binds nonpolar, elute by decreasing polarity)
• tightly packed column, high pressure, great resolution, fast.

• by rate of reaction, follow appearance of product or disappearance of reactant by spectrophotometer
• 1 unit enzyme activity = amt of enzyme that converts 1 micromol S to P/min at 25C
• after each step acn deterimine recovery, calculate cumulative yield of activity, specific activity, cumulative purification factor per step.

• stepwise % yield = (units recovered at end of step)/units recovered at end of previous step x 100
• stepwise purification factor = specific activity at the end of step/specific activity at end of previous step.  
• If a substance is pure the specific activity won't increase.

• molecular weight markers (proteins of known M_r)
• Blue Dextran (large polysaccharide excluded by beads, determines V_o = void volume)
• elution volume of your protein via standard curve

• proteins migrate in electric field according to net charge.  Stationary is polyacrylamide gel, mobile is proteins dissolved in buffer.  
• mobility (charge/friction), strength of electric field (higher volts is faster, % acrylamide (higher = smaller pores, slower), net charge (larger moves faster), shape (smaller=faster), shape (elongated is slower)
• SDS-PAGE helps determine Mr, bids to make all same charge density and shape, migrate to anode with rate dependant on size.
• Run with bromophenol blue, anionic small dye.  
• Detect via Coomassie Blue
• Good for subunit composition, 1 or few bands = pure

• Blue dextran is large polysaccharide which skips beads in gel filtration chromatography
• bromophenol blue is an anionic small dye run with MW standards on SDS-PAGE
• Coomassie Blue binds proteins via peptide/Ab binding for western blot on SDS-PAGE

• determines pI
• isoelectric focusing gel has ampholytes, mix of organic acids and bases.  When placed in electric field, pH gradient forms

molecules with both acidic and basic groups.

• @ pH 7 most organic acid = COO-, migrates towards +
• @ pH 7 organic bases are mostly protonated, NH3+, migrates to -

no net charge won't migrate

equilibrium technique, combines isoelectric focusing (1st) and SDS-PAGE (2nd) to resolve complex mixtures.  FIrst separation along one part of tube gel, 2nd by running those bands into slab gel, get 2D protein spots (molecular weight and pI

isoelectric focusing must be first because SDS-PAGE makes everything the same charge.

tests 2 things, unlikely that two compounds have same pI and M_r

amino acid sequence and disulfide bond location

local folding, reapeated pattern (alpha or beta)

overall folding of molecule

subunit association

• 6M HCl with no O2, complete hydrolysis, leaving free amino acids (spectophotometry)
• Then cation exchange chromatography (aa bind column at low pH when + charged, elute by increasing pH.  least + elute first (asp), then hydrophobic (gly-phe), then basic/most +.  
• HPLC
• N-terminal analysis: reagents react with free alpha amino group, form derivative stable to acid hydrolysis.  ID'd by HPLC
• Edman degredation: automated sequencing method.  Can "walk down" polypeptide chain N--->C up to 50-60 aas.

• break disulfide by oxidation or reduction (prevent reformation by covalent block like iodoacetate)
• amino acid composition (spectrophotometry)
• N-terminal analysis
• specific internal cleavages by 2 reagents, overlapping fragments (not Edman beyond 60)
• sequence fragments, assemble like a puzzle.  
• Locate disulfide via 2-D paper electrophoresis

• used to sequence polypeptides
• mass spectrometer separates mix of peptides
• one product is cleaved, makes a mixture of singly-cleaved products
• cleavate products are separated in second mass spectometer
• sequence can be read off spectrum
• can be performed off a few 100ng of protein extracted from SDS
• leu and ile are hard to distinguish (small molecular mass)

the spatial arrangement of groups in a molecule that are free to assume different positions due to free rotation about simple bonds

• the conformation of a protein that is biologically active.
• Only marginally stable, held together by weak interactions (except S-S), considered DYNAMIC (so that they work)

unfolded proteins that are biologically inactive.  Some can refold spontaneously in better conditions.

• proteins have 1 or few native conformations, those with lowest G/most stable
• many synthetic proteins spontaneously form into native conformation
• some denatured proteins spontaneously renature

• folded polypeptide chains (spherical-ish or globular in shape)
• enzymes, Ig's, transport molecules, hormones

• polypeptide chains arranged in long sheets or strands
• structural proteins such as collagen, keratin, silk fibroin

hydrophobic forces

• hydrophobic forces
• ionic bonds/salt bridges
• H-bonds
• London forces
• disulfide bonds

• occur between polar groups trapped in the middle with nonpolar.  These bonds are in lieu of bonds that would be made with water on the outside.  
• DO NOT ADD TO DELTA G, EVEN EXCHANGE.

only covalent interaction, strongest bond.  Holds loops of proteins in place and chains of insulin together.

• Defined mathematically by Ramachandran, PHI AND PSI
• limited due to steric hindrance, otherwise O and H collide

• specialized right handed spiral (clockwise when viewed from above), favored by L amino acids.
• 3.6 acids per turn/0.54 nm axial distance is repeat length.  
• Each NH is H-bonded to CO
• R groups stick out from helix
• BACKBONE NOT EXTENDED - springy.  Hair or wool

asp-arg, phe-leu 3-4 amino acids apart

• bulky R near each other (ser, thr, cys, asn)
• gly (slips due to free rotation)
• pro (ring interferes with NH)
• charge repulsion

• polypeptide chains, almost fully extended in zig-zag
• beta pleated sheets when chains line up side by side.  Can be PARALLEL OR ANTIPARALLEL
• H-bonds between NH-CO, perpendicular in antiparallel, not in parallel
• R groups alternate up and down, ROOM FOR BULK
• Flexible but inelastic, HIGH TENSILE STRENGTH
• silk fibroin, spider web

• beta
• alpha
• beta

• a reverse in direction in polypeptide chain that permits folding.
• beta-turn (hairpin turn), requires 4 amino acids to go 180 degrees.  H-bond between 1 and 4.  Pro forces turn, gly at 2.

• break alpha helix
• appear in beta turn

• simple, no teriatry or quaternary, pure alpha helix or beta sheet, not folded or turned.  
• insoluble in water (lots of nonpolar aas, don't fold)
• structural proteins, rope or rod-like, fibers, resist denaturation, STRONG
• alpha keratin, collagen

• 2 alpha helixes wrap into LEFT handed coil, two chains interact via hydrophobic interactions
• chains held by S-S bond (like a perm)
• hair stretches when wet because water breaks H-bond
• hard keratin (nails, horns, claws) have more S-S bonds than soft (hair, wool)

left handed (2 rights make a left, two lefts make a right)

• major structural protein in mammals (1/3 body protein, 35% gly)
• mostly gly, pro, ala, hyp (hydroxyproline)
• typical sequences of gly-X-pro or gly-X-hyp
• left handed helix (hyp and pro are good in left, break right), 2x as extended as alpha so little stretch, high tensile strength.  
• gly at inside of triple right helix so as small as possible = tensile strength
• exterior nonpolar, water insoluble
• peptide bonds face inside so resist proteolysis

• ascorbic acid protects protein hydroxylase and prevents F2+ from becoming F3+
• hydroxyl group stabilizes triple helix in collagen
• vitC deficiency causes poor assembly of collagen, bleeding gums, lesions, etc

• tightly wound with gly inside
• resists proteolysis because peptide bond is inside and water insoluble
• covalent cross-links so that even if chain is nicked, structure doesn't fall apart.

• covalent crosslinks via hyl and lys stabilize structure 
• happens post-translation, after secretion into extracellular space
• form between chains of triple helix and between helices after fibrils assemble

substitute bulkier amino acid for gly, causes connective tissue defects

• supersecondary structure
• recurring theme or pattern of secondary structure
• alpha-helix-turn-alpha-helix (180 degrees)
• beta-strand-turn-beta-strand (180 degrees)

• alpha-helix-turn-alpha-helix (alpha alpha)
• 180 degrees

• beta-strand-turn-beta-strand (beta beta)
• 180 degrees
• antiparallel

• beta-strand-alpha-helix-beta-strand
• 360 degrees
• parallel beta sheet formed

• region of polypeptide that maintains conformation and function independantly of rest of polypeptide.  
• made of motifs
• include antibody molecules, substrate binding sites of some enzymes

• loss of biological activity.  
• decreases water solubility to cause precipitation
• caused by affecting noncovalent bonds,  
• detergent, urea, organic solvents that stabilize nonpolar groups at the exterior, cause unfolding
• change in pH, increased temp, high salt concentration
• some renature, especially small with few S-S bonds

decreases, harder to make right combo.  Smaller helps.

• large proteins can't renature, they are kinetically blocked, too many wrong ways, can't find right way.  
• in vivo they are folded during translation (stepwise, each step makes next step) or have chaperones

• polypeptide bonding proteins, usually nonpolar bond
• block incorrect interactions (prevent nonpolar from being buried before partner is translated)
• guide correct folding of new proteins

• 4 weak noncovalent interactions, occasional S-S bond
• aided by chaperones

• more complex structure than fibrous proteins
• all have tertiary, some quaternary
• more easily denatured than fibrous (fewer covalent crosslinks, less twisted)

• globular protein, binds and stores O2 in muscle fro use in cellular respiration
• relatively small, one polypeptide chain, no cys
• alpha helix and loops (no beta or s-s)
• 1 domain "globin fold" around heme, polar facing out, nonpolar face in, hydrophobic pocket
• no quaternary
• prosthetic group: heme (porphyrin ring with Fe2+)

• oxidation of Fe2+ = Fe3+, met Mb (brown).  
• Fe3+ = ferriMb or metMb can't bind O2.  
• When protein is denatured this happens

• Fe2+ has 6 bonding orbitals
• 4 bind porphyrin N (planar)
• 1 binds proximal his N (perpendicular)
• 1 binds O2 reversibly (perpendicular to heme plane).  Distal his shields O2

• toxic.  Competes for O2 site in Mb, Hb, cytochrome a3
• CO binds heme with 20,000x affinity of O2, so distal his is bent  to reduce affinity.  Binds with Mb at 25x and Hb with 200x

right to form MbO2

• Mb + O2  <--->  MbO2
• deoxymyoglobin     Oxymyoglobin

left to form deoxymyoglobin

storage depot for O2

• Constant.  
• pO2 at which 50% of ligand-binding sites on Mb are occupied.  
• dissociation constant (Kd is inverse of Ka, lower means tighter binding)
• p50 of Mb is very low, so high affinity for O2

hyperbolic

binds and TRANSPORTS O2 throughout body.

4 non-identical subunits, alpha and beta (like Mb), tetrahedral, a-b by exposed hydrophobic, hemes buried and apart.  Changes shape on binding O2.  Each subunit can bind O2 (4 each)

• "other shape".  Hb changes shape upon binding O2.  
• deoxy form: O2sits out of porphyrin plane, T-state, low affinity for O2, salt bridges
• oxy form: Fe sits in porphyrin plane, R-state, high O2 affinity, broken salt bridges

Fe2+ sits out of porphyrin plane, T-state "taut", salt bridges, low O2 affinity

Fe2+ sits out of porphyrin plane, "taut", deoxy, salt bridges, low O2 affinity

R-state, "relaxed", Fe2+ sits in porphyrin plane, broken salt bridges, high affinity for O2

• sickle cell anemia.  1 amino acid substitution on beta chain (glu to val).  neg charged to nonpolar, now hydrophobic interactions, form aggregates/tetramers, shape changes, decreases solubility, short lifespan, block capillaries.  
• Tell by gel electrophoresis or PCR (moves less far toward anode than HbA)

valine sub for glutamate, less negatively charged.

• 
• pKa higher pH than tissues on deoxy side, pH of lungs higher than pKa on oxy side.  
• When O2 binds, conformational change, breaks salt bridges, lowers pKa and releases protons

• O2 binding to 1 subunit of Hb, conformational change from T to R, conformational change in other three subunits, increase O2 affinity
• As a result the saturation curve is sigmoidal, not hyperbolic like Mb

• sigmoidal (due to cooperativity/affinity)
• Allows release of O gradually, even distribution throughout body, no "dump"

Mb goes down to 80 for land mammals, Hb goes down to 10 because it's purpose is delivery, not storage.

affinity for O2

• subcategory of ligand
• a molecule that binds to a protein at a site separate from the protein's functional binding site and modulates activity of protein.  
• Protons are effector in Hb

• acts as effector
• enhances uptake of O2 at lungs and delivery to tissues via Bohr effect.  
• Higher H+ = lower pH.  
• at lungs shifts equation to right so CO2 can be blown out.  Low CO2 increases affinity for O2
• high CO2 at tissues, binds and stabilizeses deoxy Hb via salt bridge, shifts eq to left, O2 released.  Higher CO2 at tissues decreases affinity of Hb for O2

low CO2 at lungs increases affinity of Hb for O2

high CO2 at tissues decreases affinity of Hb for O2

at low pH, Hb has less affinity for O2 so curve shifts to right, less O2 bound at same pO2

mechanism of CO2 transport, binds ~13% in blood, formed when Hb binds CO2 at N-terminal (releases H+), usually at tissues

• carbamate (little)
• HCO3- (LOT)
• dissolved CO2 (least)

• VERY negative, made in RBC, crosslinks 2 beta chains of deoxyHb via salt bridge (to lys/his). 
• Shifts O2 binding equilib to left, decreases affinity of Hb for O2 (shifts right).  Stabilizes T form
• CAUSES SIGMOID CURVE, enables unloading of O2

• increase 2,3-BPG (or increased RBC).  
• LOWERS affinity, lowers saturation, but makes proportion/delivery close to sea level.

• evolutionary response to low O2 in utero, HbF has HIGHER AFFINITY for O2 than HbA, takes O2 from mom.  Myoglobin even higher.  
• O2 in utero: HbA --> HbF--> MbF

family of Y-shaped proteins produced and secreted by B-lymphocytes with binding sites for antigens, effector that determines response.

• tetramer H2L2 joined by disulfide bonds.  
• Domains made of antiparallel beta pleated sheets called IMMUNOGLOBULIN FOLDS
• Variable domains (N-term of V_H and V_L, with Ag sites), constant domains of H chains (C_H, same per class, contain effector), constant domains of L chains (C_L, determines two types (kappa and lamda), both in all classes of Ig)

can be cleaved at hing region into 2 fragments: Fab (antigen binding domains) and Fc (fragment crystallizable)

• antigenic determinant
• site on Ag recognized by Ab (idiotype)
• very specific

• site on Ab that recognizes Ag (epitope)
• antigenic determinant. 
• specific

• epitope to idiotope
• specific but based on shape so cross-reactivity.  TIGHT, high affinity for Ag.

large polymers/foreign cells (or haptens linked to polymer), contain epitope to be recognized and bound

• antibodies can cross-link 2 antigens
• can also be multivalent and make larger aggregates that precipitate out of solution

antibody to antigen

small molecules made antigenic by linking to a large polymer.

• produced first (primary immune response), activates complement
• low affinity individ but high avidity due to structure
• 5 units in circle, linked by J chains/disulfide bonds

total binding strength--more weak bonds in IgM

• MOST, produced after IgM, main serum ab.  
• Crosses PLACENTA and goes into MILK, passive immunity
• activates macrophages/neutrophils/complement

cascade of proteases activated by Ab-Ag complexes (inflammation, lyses pathogen)

• major secretory Ig, monomer (blood) or dimer (secretions, J chain)
• transcytosis (enters epithelial cells, secreted via apical membranes)
• defense against infection

binds mast cells in tissues and basophils to release histamine, eosinophils for parasites.

cell surface form (naive B cell with IgM) and secretory form (binds basophils and induces antimicrobial and B-cell-activating factors.  NOT HISTAMINE release

• enzyme-linked immunosorbant assay
• assay for Ag or Ab
• bind to polystyrene, wash, block unoccupied, wash, add Ag or Ab, wash, substrate forms color.  
• HIV test

• pure HIV Ag bound in production.  add blood as Ab.  Positive suggests infection, INDIRECT, not definitive.  Testing for Ab not Ag.
• False positives due to cross-reactivity, so there is a more specific

tests for Ab in blood, not Ag.

capture assay.  Ab on plate, Ag binds, another Ag on top, sandwich.

• specific HIV test, 3 different matches make false positive/negative unlikely.  (PCR too)
• SDS gel electorphoresis
• nitrocellulose membrane, innoculated with Ab.  Specific for antigenicity and MW.