Amino Acids exam I

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    • 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
    • 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
    • Aspartate
    • Asp
    • D
    • Negatively charged group (acidic)
    • Glutamate
    • Glu
    • E
    • Negatively charged group (acidic)
  1. vitalists
    organic compounds only produced by living organisms (life begets life)
  2. Freidrich Wohler
    • lab synthesis of urea
    • NH3 + N=C-OH --> N=C-O- NH4+  --> H2N-C(=0)-NH2
    • ammonia + cyanic acid ---> ammonium cyanate ---> urea
  3. modern def of organic cmpd
    • 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.
  4. hydrocarbons vs ionic
    reactivity, mp/bp, electrical conduction, solubility in aqueous
    • 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
  5. hydroxyl
    • OH, alcohol
    • intra/inter molecular dehydration (C=C, ether)
    • Oxidation(-CHO)
  6. carbonyl
    • aldehyde -CHO, ketone R-C(=O)-R'
    • Reduction (aldehyde), oxidation (COOH)
  7. carboxyl
    • carboxylic acid -COO(H)
    • Reduction (aldehyde), esterification (+OH = COO), thioester +SH, anhydrides (+COOH or POOH), +amine = amide
  8. amino
    • NH3, Amine
    • +COOH amide
    • protonize = protonated amine
  9. amido
    amide, C=O-NH3
  10. sulfhydryl
    • thiol, -SH
    • +COOH = thioester
    • Oxidation = disulfide bond
  11. phosphoryl
    phosphate PO4
  12. Ester
  13. Thioester
  14. ether
  15. mixed anhydride
  16. what constitutes a peptide bond?
    amide bond, aa only
  17. structural isomers
    • same molecular formula, different arrangement
    • Most dissimilar of all isomers, b/c functional groups can be different
  18. stereoisomers
    • same groups of atoms but differ in arrangment in space
    • sub-categories (diastereomers, enantiomers)
  19. diastereomers
    stereoisomers that are non-mirror image.  Include geometric, conformational
  20. geometric isomers
    cis/trans.  Diastereomers, stereoisomers.  Hindered around double bond.
  21. conformational isomers
    rotation around C=C bond, eclipsed/staggered.Diastereomers, stereoisomers
  22. enantiomers
    mirror image stereoisomers.  Chiral center.
  23. configurational isomers
    • 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
  24. alkane/alkene/alkyne, who is most reactive?
    should be alkane but it is alkyne b/c of pi bonds
  25. why are alcohols, amines, thiols more reactive than hydrocarbons, if their polar bonds are stronger?
    nucleophiles.  Unshared pairs cause nucleophilic attacks.  Makes new pathway with lower activation energy.
  26. types of chemical transformations
    • 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.
  27. xfer a group from one molecule to another, catalyzed by transferase, kinase transfers phosphoryl
    group transfer
  28. catalyzed by oxidoreductase
  29. isomerization with isomerase, btween structural isomers
    intramolecular rearrangment
  30. splits or joins without water.  Breaks C-C/C-O/C-N bond, may form =, catalyzed by lyase
  31. condensation
    joins 2 while eliminating water (dehydration synthesis)/hydrolysis, hydrolase.
  32. 4 classes of macromolecules
    protein, lipid, carbohydrate, nucleic acid
  33. intermolecular forces
    • 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,
  34. ionic bond
    attraction between opposite charges or repulsion between like charges (ion pair/salt bridge).
  35. ion=dipole
    ion and partial opposite charge, attraction
  36. dipole-dipole
    attraction between partial opposite charges (increases bp of dimethyl ether above propane)
  37. hydrogen bond
    particularly strong dipole-dipole.  H attached to very strong electronegative (F, O, N), so H is very +
  38. dipole-induced dipole dipole polarizability
    permanent dipole deforms cloud of electrons, attracts them.  Nonpolar gases are weakly soluble
  39. london forces
    two temporary dipoles.  WEAKEST.  Increases as Mr increases (bigger = stronger)
  40. hydrophobic forces
    2 nonpolar will join together in aqueous environment.  entropy increases as aqueous shell is released. Not due to attraction
  41. important properties of water
    • 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)
  42. amphipathic
    • hydrophobic and hydrophilic parts
    • hydrophobic fold together, form micelles or bilayers
    • common in proteins, membrane lipids, fatty acids, vit A and D
  43. How is [H+] of strong acid calculated?
    = to molarity b/c ionizes completely
  44. N
    • n x M
    • moles times molarity
    • strong acid like HCl, N = M b/c only 1 H.  H2SO4, N=2M
  45. 7.2 mL of 0.10 M NaOH required to titrate a 10.0 mL sample of gastric juice from patient.  What is the pH of gastric juice?
    • VANA = VBNB
    • 10.0 mL (NA) = 7.2 mL (0.10N)
    • NA = 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
  46. biochemically desirable properties of good buffers
    • 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
  47. use of weak inorganic acids
    • buffers (borate, phosphate, tris)
    • "Good Buffers" (bicine, mops, hepes, pipes)
    • Developed by Dr. Normal Good
  48. weakness/strength of acid determined by
    • pKa.  Stronger acid has higher Ka and lower pKa
    • So Phosphoric stronger than hydrogen phosphate
  49. polyprotic
    • 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
  50. calof pKa of weak acids
    •    may ignore bottom x
    • pKa = -log Ka
  51. calculate pH of 0.100 M solution of CH3COOH
    • X = Ka 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
  52. Henderson-Hasselbalch Equation
  53. How is pKa determined?
    • 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
  54. alkalosis
    pH increases above 7.4, tetany (7.8)
  55. acidosis
    blood pH decreases below 7.4, coma (7.0)

    watch for solutions that are 7.0-7.39 = basic but acidotic
  56. buffer
    • weak acid and conjugate base pair
    • best at +/- 1 from pKa
    • higher concentration makes more effective
  57. calculate the ratio of 0.100 M sodium acetate (NaOAc) to 0.100 M acetic acid (HOAc) needed to prepare 0.100 M sodium acetate buffer, pH 5.00
    • 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
  58. What is the pH of buffer made from 138 mL of 0.100 M NaOAc + 50.0 mL of 0.100 M HOAc?
    •   = mole fraction
    • 4.76 (pKa) + log 2.76 = 5.20 pH
  59. calculate the ratio of 0.100 M sodium acetate (NaOAc) to 0.100 M acetic acid (HOAc) needed to prepare 0.100 M sodium acetate buffer, pH 5.00
    • 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: XA- = 0.501/1.501
    • XHA = 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
  60. Prepare 2.00 L of 0.200 M phosphate buffer pH 7.20 from solid K2HPO4 (dibasic) (F.W.174.18 g/mol) and solid KH2PO4 (monobasic) (FW = 136.09 g/mol)
    • 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.
  61. 0.20 M tris buffer, pH 7.8, pKa 8.1.  Calculate  mole fraction of Tris+ [HA+] and Triso [A] at start
    • 7.8 = 8.1 + log [A-]/[HA]
    • each mol fraction (portion over whole) x 0.20 M
  62. 0.20 M tris buffer, pH 7.8, pKa 8.1, enzyme produces 0.030M H+.  what is the reaction of Tris+ [HA+] and Triso [A] that maintains pH?
    [A or Triso] + 0.030M H+ --> HA+
  63. 0.20 M tris buffer, pH 7.8, pKa 8.1.  Enzyme produces 0.030M H+.  Calculate  mole fraction of Tris+ [HA+, 0.133M] and Triso [A, O.0668M] at end
    • 0.0668 M - 0.030 M H+ = 0.0368 M Triso
    • 0.133 M + 0.030 M H+ = 0.163 M Tris+
  64. 0.20 M tris buffer, pH 7.8, pKa 8.1.  Enzyme produces 0.030M H+.  Calculate pH at end of Tris+ [HA+, 0.163M] and Triso [A, 0.0368M].
    • pH = pKa + log...
    • 8.1 + log (0.037M/0.163M)
    • 8.1 + log 0.227
    • 8.1-0.64 = 7.46 = 7.5
  65. 0.20 M tris buffer, pH 7.8, pKa 8.1.  Enzyme produces 0.030M H+.  Calculate pH at end if no tris was present.   Tris+ [HA+, 0.163M] and Triso [A, 0.0368M].
    • pH = - log [H+]
    • - log 0.030M
    • = 1.5
  66. Major intracellular buffer
    • Pi
    • pKa 6.86, ~80mM intracellular
  67. blood buffers
    Hb, H2CO3
  68. most important blood buffer
    H2CO(15x higher concentration than Pi)
  69. equilibrium equations for respiration
    • CO2 (g) in lungs <-(K3)-> CO2(d) in blood + H2O <-(K2=Kh)-> 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
  70. What is effect of increase in H+ from lactic acid produced in skeletal muscle during anaerobic exercise?
    more CO2 exhaled, Rx moves to left
  71. What is the effect of an increase in NH3 produced in protein catabolism?
    basic, shift right, breathe out less CO2
  72. What is the effect of increased respiration rate on blood pH?  What do you do?
    • 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
  73. What is the effect of decreased respiratory rate on blood pH.
    increased CO2, not blowing off, shift right, respiratory acidosis.  More dangerous thatn alkylosis.
  74. Respiratory diseases that could cause acidosis
    asthma, pneumonia, obstruction, collapsed trachea, CF, COPD, CHF
  75. if the [HCO3-][CO2(d)] ratio is 50, what is the blood pH and what is the person suffering from?
    • pH = pKa + log...
    • pH = 6.1 + log 50 = 7.79897~7.8.  
    • Alkylosis
  76. zwitterion
    dipolar ion
  77. How to tell L from D in amino acid
    • 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)
  78. nonpolar aliphatics
    gly, ala, val, leu, ile, pro, met
  79. aromatic group
    phe, tyr, trp
  80. polar uncharged
    ser, thr, cys, asn, gln
  81. negative, acidic aa's
    asp, glu
  82. positive, basic aa's
    lys, arg, his
    •      I        II        III      IV        V
  83. Isoelectric point (pI)
    • pH at which the net charge is 0 
    •   for diprotic
    • for triprotic, isoelectric species and average pKa just before and after.
  84. synthesis of peptides
    • set molecules up so N terminus on L, carboxyl on R 
    • hydrolysis.  Endergonic.  Stable, high activation energy
    • usually linear.
  85. Peptide
    2-10 amino acid residues long
  86. oligopeptide
    2-10 amino acid residues long.
  87. polypeptide
    11-50 residues long
  88. protein
    > 50 residues long
  89. pI of peptide
    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.
  90. Peptide hormones (3)
    • 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)
  91. comparison of the structures of vasopressin and oxytocin
    • 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.
  92. most versatile macromolecules
  93. prosethetic groups
    non-amino acid portion of protein that is necessary to function
  94. apoprotein
    amino acid part of conjugated protein, does not include prosthetic groups.
  95. conjugated protein
    contains groups other than amino acids (apoprotein +prosthetic groups)
  96. simple protein
    includes only amino acids
  97. glycoprotein
    conjugated protein, with carbohydrate prosthetic group (IgG)
  98. hemoprotein
    conjugated protein with heme prosthetic group (Hb, Mb, cytochromes)
  99. metalloproteins
    conjugated proteins with Fe2+, Zn2+, Cu+, etc as prosthetic.  Hb, Mb, cytochromes
  100. lipoproteins
    conjugated proteins with lipid prosthetic group, like HDL, LDL, which transport cholesterol.
  101. composition of amino acids
    amino acids listed in alphabetical order, with commas
  102. sequence of amino acids
    amino acids listed in order
  103. conformation of amino acids
    shape, folding of protein
  104. subunit
    each individual folded polypeptide.  Extended/unfolded are not subunits (like in collagen)
  105. multimeric protein
    multi-subunit, several subunits in one protein
  106. oligomeric protein
    multimeric protein where some subunits are identical.  The subunits are called protomers
  107. protomers
    subunits in an oligomeric protein
  108. identical subunits are called
    homologous subunits
  109. nonidentical subunits are called
    heterologous subunits
  110. How to estimate the number of amino acids in a protein
    • molar mass.  
    • Average amino acid residue in protein = 110 g/mol.  
    • protein molar mass/110
  111. Daltons
    • unit of molecular mass (=1amu).  
    • Can also become kilodaltons
  112. Mr
    relative molar mass.  Same, just unitless
  113. crude separation
    • can do large volumes, cheap materials
    • centrifugation: Includes cell lysis and subcellular fractionation, then selective preciptitation by acetone, ammonium sulfate or isoelectric precipitation
  114. types of selective precipitation in crude separation
    • 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.
  115. why will a protein be less water soluble at it's pI?
    no net charge so nonpolar, less water soluble and will precipitate out, a type of crude separation
  116. examples of sophisticated separation techniques and when to use them
    • later in purification, use expensive materials and work better on more homogenous substances.  
    • chromatography, electrophoresis
  117. chromatography
    • 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
  118. Column chromatography
    • 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.
  119. elute
  120. will increasing or decreasing pH elute a protein bound to a cation exchanger.
    increasing, makes cation into acid so protein comes off
  121. will a protein with a net negative charge bind to a cation or anion exchanger?
  122. will a negative protein be eluted by increasing or decreasing pH?
    decreasing, make anion into base to accept proton, must lower solution to make it acid.
  123. How does a cation exchanger separate net negative from neutral
    it can't, they wash off together.
  124. gel filtration chromatography
    • separation by size and shape, aka size exclusion chromatography, molecular sieve chromatography
    • 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"
  125. "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.
  126. arrange the following in the order they will emerge from a gel filtration column
    amino acid
    insulin (51aa's)
    oxytocin (9aa's)
    • insulin
    • oxytocin
    • heptapeptide
    • tripeptide
    • amino acid
  127. affinity chromatography
    • 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
  128. ligand
    a molecule that specifically and reversibly binds to a protein
  129. how would you separate the protein from bound ligand once complex is eluted from affinity chromatography column?
  130. High performance liquid chromatography
    • 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.
  131. enzyme assay as a means of product detection
    • 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.
  132. How to assess effectiveness of purification 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.
  133. Ways to standardize gel filtration chromatography column
    • molecular weight markers (proteins of known Mr)
    • Blue Dextran (large polysaccharide excluded by beads, determines Vo = void volume)
    • elution volume of your protein via standard curve
  134. Polyacrylamide Gel Electrophoresis (PAGE)
    • 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
  135. Coomassie Blue
    Blue Dextran
    Bromophenol Blue
    • 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
  136. Isoelectric focusing
    • determines pI
    • isoelectric focusing gel has ampholytes, mix of organic acids and bases.  When placed in electric field, pH gradient forms
  137. ampholytes
    molecules with both acidic and basic groups.
  138. Why will ampholyte with pH 3 migrate to anode and pH 9 migrate to cathode?
    • @ pH 7 most organic acid = COO-, migrates towards +
    • @ pH 7 organic bases are mostly protonated, NH3+, migrates to -
  139. why do proteins migrate to position where pH = pI
    no net charge won't migrate
  140. two-dimensional electrophoresis
    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
  141. which method should be applied first in two-dimensional elecrtophoresis?
    isoelectric focusing must be first because SDS-PAGE makes everything the same charge.
  142. why is two-dimensional electrophoresis more effective than any one method
    tests 2 things, unlikely that two compounds have same pI and Mr
  143. primary structure of proteins
    amino acid sequence and disulfide bond location
  144. secondary structure of proteins
    local folding, reapeated pattern (alpha or beta)
  145. tertiary structure of proteins
    overall folding of molecule
  146. quaternary structure of proteins
    subunit association
  147. best way to determine aa sequence of short polypeptides
    • 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.
  148. best way to determine aa sequence of a protein
    • 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
  149. tandem mass spectometry (MS/MS)
    • 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)
  150. conformation of proteins
    the spatial arrangement of groups in a molecule that are free to assume different positions due to free rotation about simple bonds
  151. native conformation of proteins
    • 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)
  152. denatured proteins
    unfolded proteins that are biologically inactive.  Some can refold spontaneously in better conditions.
  153. evidence that amino acid sequence --> conformation --> function (3)
    • 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
  154. globular proteins
    • folded polypeptide chains (spherical-ish or globular in shape)
    • enzymes, Ig's, transport molecules, hormones
  155. fibrous proteins
    • polypeptide chains arranged in long sheets or strands
    • structural proteins such as collagen, keratin, silk fibroin
  156. most significant contributor to delta G folding
    hydrophobic forces
  157. interactions that maintain protein conformation
    • hydrophobic forces
    • ionic bonds/salt bridges
    • H-bonds
    • London forces
    • disulfide bonds
  158. ionic bonds/salt bridges and H bonds role in delta G of protein folding
    • 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.  
  159. Disulfide bonds in proteins
    only covalent interaction, strongest bond.  Holds loops of proteins in place and chains of insulin together.
  160. bond angles in protein folding
    • Defined mathematically by Ramachandran, PHI AND PSI
    • limited due to steric hindrance, otherwise O and H collide
  161. alpha helix
    • 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
  162. Things that stabilize an alpha helix
    asp-arg, phe-leu 3-4 amino acids apart
  163. alpha helix breakers
    • bulky R near each other (ser, thr, cys, asn)
    • gly (slips due to free rotation)
    • pro (ring interferes with NH)
    • charge repulsion
  164. beta conformation
    • 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
  165. alpha or beta?  
    more tensile strength
    more flexibility
    bulky R groups
    • beta
    • alpha
    • beta
  166. bends and loops in proteins and most common type
    • 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.
  167. ser, asn, pro, gly are most likely to
    • break alpha helix
    • appear in beta turn
  168. fibrous proteins
    • 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
  169. alpha keratin
    • 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)
  170. Two alpha helixes will wrap into what kind of coil?
    left handed (2 rights make a left, two lefts make a right)
  171. collagen
    • 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
  172. vitamin C and proline hydroxylase in collagen
    • 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
  173. How does collagen resist denaturation
    • 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.
  174. collagen crosslinks
    • 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
  175. genetic mutations of collagen
    substitute bulkier amino acid for gly, causes connective tissue defects
  176. motifs
    • 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
  177. domain
    • 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
  178. denaturation
    • 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
  179. as S-S bonds increase, chance of renaturation ______
    decreases, harder to make right combo.  Smaller helps.
  180. large proteins and renaturation
    • 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
  181. molecular 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
  182. quaternary structure is held together by
    • 4 weak noncovalent interactions, occasional S-S bond
    • aided by chaperones
  183. globular proteins
    • more complex structure than fibrous proteins
    • all have tertiary, some quaternary
    • more easily denatured than fibrous (fewer covalent crosslinks, less twisted)
  184. myoglobin structure
    • 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+)
  185. why must heme in myoglobin stay in the hydrophobic pocket?
    • oxidation of Fe2+ = Fe3+, met Mb (brown).  
    • Fe3+ = ferriMb or metMb can't bind O2.  
    • When protein is denatured this happens
  186. How Mb binds O2
    • 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
  187. Mb and CO
    • 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
  188. At high O2 concentration, myoglobin equilibrium shifts to _____ to form _________ and a fresh supply of O2 is delivered to muscle from lungs via arteries.
    right to form MbO2
  189. myoglobin respiration equation
    • Mb + O2  <--->  MbO2
    • deoxymyoglobin     Oxymyoglobin
  190. At low O2 concentration, myoglobin equilibrium shifts to ________ to form _________ and muscle uses up O2 via cellular respiration
    left to form deoxymyoglobin
  191. what is myoglobin's #1 function?
    storage depot for O2
  192. P50
    • 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
  193. shape of Mb's saturation curve
  194. function of hemoglobin
    binds and TRANSPORTS O2 throughout body.
  195. structure of hemoglobin
    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)
  196. allosteric
    • "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
  197. deoxy form of Hb
    Fe2+ sits out of porphyrin plane, T-state "taut", salt bridges, low O2 affinity
  198. T-state of hemoglobin
    Fe2+ sits out of porphyrin plane, "taut", deoxy, salt bridges, low O2 affinity
  199. oxy form of Hb
    R-state, "relaxed", Fe2+ sits in porphyrin plane, broken salt bridges, high affinity for O2
  200. HbS
    • 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)
  201. why does HbS not move as far toward the anode as HbA?
    valine sub for glutamate, less negatively charged.
  202. Hb O2 binding equation
    • 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
  203. cooperativity (Hb) and result
    • 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
  204. Hb saturation curve is
    • sigmoidal (due to cooperativity/affinity)
    • Allows release of O gradually, even distribution throughout body, no "dump"
  205. Low point of saturation curve for Mb and Hb
    Mb goes down to 80 for land mammals, Hb goes down to 10 because it's purpose is delivery, not storage.
  206. On saturation graph, curve more to the right has ______________ than curve on the left
    affinity for O2
  207. effector
    • 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
  208. Effect of H+ in Hb pathway
    • 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
  209. Low CO2 at lungs and Hb
    low CO2 at lungs increases affinity of Hb for O2
  210. High CO2 at tissues and Hb
    high CO2 at tissues decreases affinity of Hb for O2
  211. at low pH, Hb __________O2 so ____________
    at low pH, Hb has less affinity for O2 so curve shifts to right, less O2 bound at same pO2
  212. Carbamate
    mechanism of CO2 transport, binds ~13% in blood, formed when Hb binds CO2 at N-terminal (releases H+), usually at tissues
  213. three ways that CO2 is transported through the blood
    • carbamate (little)
    • HCO3- (LOT)
    • dissolved CO2 (least)
  214. 2,3 BPG (bisphosphoglycerate)
    • 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
  215. physiological responses to low environmental O2
    • increase 2,3-BPG (or increased RBC).  
    • LOWERS affinity, lowers saturation, but makes proportion/delivery close to sea level.
  216. Fetal Hb
    • 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
  217. Immunoglobulins
    family of Y-shaped proteins produced and secreted by B-lymphocytes with binding sites for antigens, effector that determines response.
  218. Immunoglobulin structure
    • tetramer H2L2 joined by disulfide bonds.  
    • Domains made of antiparallel beta pleated sheets called IMMUNOGLOBULIN FOLDS
    • Variable domains (N-term of VH and VL, with Ag sites), constant domains of H chains (CH, same per class, contain effector), constant domains of L chains (CL, determines two types (kappa and lamda), both in all classes of Ig)
  219. cleavage of Igs
    can be cleaved at hing region into 2 fragments: Fab (antigen binding domains) and Fc (fragment crystallizable)
  220. epitope
    • antigenic determinant
    • site on Ag recognized by Ab (idiotype)
    • very specific
  221. idiotype
    • site on Ab that recognizes Ag (epitope)
    • antigenic determinant. 
    • specific
  222. antigen binding
    • epitope to idiotope
    • specific but based on shape so cross-reactivity.  TIGHT, high affinity for Ag.
  223. antigens
    large polymers/foreign cells (or haptens linked to polymer), contain epitope to be recognized and bound
  224. bivalent
    • antibodies can cross-link 2 antigens
    • can also be multivalent and make larger aggregates that precipitate out of solution
  225. strongest known affinity
    antibody to antigen
  226. haptens
    small molecules made antigenic by linking to a large polymer.
  227. IgM
    • 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
  228. avidity
    total binding strength--more weak bonds in IgM
  229. IgG
    • MOST, produced after IgM, main serum ab.  
    • Crosses PLACENTA and goes into MILK, passive immunity
    • activates macrophages/neutrophils/complement
  230. complement
    cascade of proteases activated by Ab-Ag complexes (inflammation, lyses pathogen)
  231. IgA
    • major secretory Ig, monomer (blood) or dimer (secretions, J chain)
    • transcytosis (enters epithelial cells, secreted via apical membranes)
    • defense against infection
  232. IgE
    binds mast cells in tissues and basophils to release histamine, eosinophils for parasites.
  233. IgD
    cell surface form (naive B cell with IgM) and secretory form (binds basophils and induces antimicrobial and B-cell-activating factors.  NOT HISTAMINE release
  234. ELISA
    • 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
  235. ELISA for HIV
    • 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
  236. Indirect ELISA
    tests for Ab in blood, not Ag.
  237. Sandwich ELISA
    capture assay.  Ab on plate, Ag binds, another Ag on top, sandwich.
  238. Western Blot/immunoblot
    • 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.

Card Set Information

Amino Acids exam I
2014-10-14 02:13:28

Exam one: Standard amino acid structures, need names, 3-letter and 1-letter abbreviations, + Ch 1-4
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