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| splitting by adding water |
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| polymers formed by monomers formed by? |
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| that portions of physics that is concerned with energy and how eneryis interconverted from one form to another |
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| that part of the universe that is under study |
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| the operation of a force through a distance |
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| a push or pull, can be molecular |
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| energy that is manifestation of the random motion of (atoms) or molecules |
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| the total amount of matter and energy in the universe is constant |
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| the degree of disorder of the universe (entropy) is constantly increasing, this has implications in terms of predicting the spontaneity of a process such as a rxn |
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| tend to proceed from a state of order to a state of disorder |
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| ground state electronic configuration of oxygen |
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| O-H covalent bond distance (water) |
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| van de Waals radius of O (water) |
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| Van der Waals radius of H (water) |
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| excellent solvent for polar moecular |
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| causes nonbonded interactions - H-bonds |
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| tetrahydral sp3 orbitals of the O atoms , dipole |
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| directional intermolecular association, ~0.5 A |
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| tetrahedral, 6-membered ring, density = 0.92 g/mL |
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| denisty = 1.00 g/mL, 85% of the H-bonds of ice, dynamic lifetimes of 2 X 10-11 s, irregular reorientation of each molecule ~1/10-12 s, rapid fluctuation of H-bonds |
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| covalent, polar, nonpolar |
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| partially charged, oxygen |
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| carboxylate>>>amine, electrostatic, nonbonded interaction, noncolaent (ionic interation), aka: salt linkage |
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| van der waals interactions |
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| H-bond, diople-dipole, London dispersion forces |
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| covalent vs. non covalent |
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| stronger bond... weaker bond |
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| 2 componants of interations, 2 nonpolar, weakest bond, significant only in close contact, extremely important for structure, momentary |
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| polar & polar interactions |
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Definition
| interactions between permanent dipoles |
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| polar & nonpolar interaction |
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| dipole-induced dipole interactions |
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| biochem is done in water near __ pH |
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| 99.5% of biochem. is done in __ phase |
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| thermo activity of water is assigned to be __, because we use water as our solvents |
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| required biosynthesis & energy |
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1) fermentation as a source of energy
2) development of photosynthesis
3) evolution of aerobic metabolism |
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| differentiated & become multicellular |
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exothermic
(enthalpicaly favored) |
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endothermic
(enthalpically disfavored) |
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| life obeys the Laws of Thermodynamics |
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1) living organisms are open systems
2) living things maintain a steady state not equilibrium
3) enzymes catalyze biological reactions |
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| aggregation of nonpolar molecules in water |
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Definition
1) hydrophobic olecules are not dispersed (not solvated)
2) entropy is increased
3) decrease in the molecule
4) increase in water |
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Term
| orientation of water molecules around a nonpolar solute |
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Definition
1)maximizing H bonds capacity
2) H bond network around the molecule
3) increaseof order in water molecule |
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1) very slightly ionize
2) H+ acutally exists as H2O+ (hydronium ion)
3) porton jumping |
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1) Adenine
2) Guanine
-larger |
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1) cytosine
2) uricil
3) thymine
-smaller |
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Definition
1) planar
2) aromatic
2) heterocyclic molecules |
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-nucleotides
-contain adenosine |
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1) phosphodiester bonds
2) 5' -> 3' direction
-polynucleotides: mono-, di-, oligo- |
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Definition
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| bases in tautomeric forms |
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Definition
| -can easily be converted isomers that differ only in H positions |
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1) charged phosphate groups
2) monovalent cations
2) divalent cations = specific binding to pohosphate groups |
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| fractionation of nuclceic acids |
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Definition
1) chromotography
2) electrophoresis
2) untrlacentrifugation |
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Definition
1) α carbon
2) carboxylic acid group
3)amino group
4) R group |
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Definition
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Definition
1) condensation of the 2 amino acids
2) amino acid residue
3) amino terminus (N-terminus)
4)carboxyl terminus (C-terminus)
4) an amide linkage |
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| values depend on nearby groups |
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| optically active molecules |
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Definition
nonsuperimposable mirror image
chiral centers |
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Definition
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Definition
| isomer that differs at a single chiral center |
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Definition
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Definition
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Definition
| do not indicate its ability to rotate the plane of polarized light |
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| primary protein structure |
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Definition
| the amino acid dequence of its polypeptide chain |
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Definition
1) at least 40 residues
2) vast majority are between 100 - 1000 residues
3) monomeric and multimeric
4) amino acid composition: average occurance in proteins |
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Definition
quantitative detection
-specific, sensitive, & convenient |
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Definition
1)substrate & product
2) physiological & artificial substrates
2) coupled |
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Definition
1)antibody
2) radioimmunoassay (RIA)
3) enyzme-linked immunosorbent assay (ELISA) |
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Definition
protein absorption at 280 nm
-chromophore absorb light in the visible region |
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bradford assay
-coomassie brilliant blue
-at 595 nm |
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Definition
1) purification of target protein by fractionation procedures
2) selective elmination of the other components
3) using physicochemical properties of proteins |
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Definition
1) proteins are polyelectrolytes (polyionic polymers)
2) proteins are least soluble when its net charge is 0 |
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Definition
mobile phase: liquid
station phase: porous solid matrix |
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| ion-exchange chromotography |
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Definition
anion exchanger: diethylaminoethyl (DEAE)
cation exchanger: carboxyl-methyl (CM) |
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Definition
SDS: sodium dodecylsulfate
uniform binding of SDS to portein and denaturation
net charge os equal in every portein
separation depends on size |
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| capillary electrophoresis |
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Definition
1) electrophoresis in very thin capollary tubes (20-100 um diameter)
2) dissipate heat and permit high electric fields
3) fast separation |
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Definition
1)isoelectric focusing (IEF) & SDS-PAGE
2) application in proteomics |
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Definition
1)analytical
2)preparative
3)density gradient centrifugation (zonal centrifugation): sucrose, Percoll
3) equilibrium density gradient centrifugation: CsCl |
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Definition
1) usuually required for studies of its function
2) try to purify a solute that <0.1% of dry weight of tissue to al least ~98% purity
3)moleucular cloning techs can be very helpful in boosting starting lelvel of protein
4) unless the protein is secreted by cells, must disrupt cells to get out the protein of interest. cells can be disrupted by grinding, sonic oscillation, freezinf, and thawing
5) cell debris may be removed by filtration or centrifugation
6)if protein is membrane-associated, may feed detergent extraction by commercially avaible detergent solution you may not know this at first. membrane-bound proteins are usually harder to purify than water-soluble proteins
7) in order to purify a protein, you must know where it i. In other words, you must have a higly sensitive, specific assay for protein |
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Definition
| important since it determines changes of side chains of amino acids that in turn influence surrounding groups and cause changes in intra molecular forces, usually try to control pH with buffers |
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| protein solubility: ionic strength: |
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Definition
| can influence strucutre (subunit interaction) |
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| protein solubility: temp.: |
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Definition
| most proteins unfold (denature) at high temps.. a few denature at low temps, but freezing is more likely to cause a porblem than colling, usually we purify porteins in a cold room (~4 C) |
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Term
| protein solubility: hydrolytic enzymes: |
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Definition
| breaking open cells also releases digestive enzymes such as proteases and nucleases, much minimize this effec (codl, inhibitors,...) |
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Term
| protein solubility: surface denaturation: |
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Definition
| some proteins can be absorbed on plastic, some on flass, causing nenaturation and/or mechanical loss. minimize foeaming and keep protein as concentrated as possible |
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| radioimmunoassays (RIA's) |
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Definition
| indirectly determine the amount of the antigen protein by determining how much fo a standad radioactively labeled standard protein is displaced from the antibody |
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Definition
| proteins have no net charge at pI and tend to be less soluble, can precipitate (may not be reversible) |
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| protein stability: ionic strength |
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Definition
| most water solublew proteins get more soluble as salt is added at low concentrations. The salt prevents electrostatically-driven aggregation (salting in). Even the sme protein will tend to get less oluble at higher concentrations of salts (esp. sulfates) (salting out). In the latter case, solvation of the salt leaves less bulk H2O to solvate the portein and it precipitates (often reversible). |
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Definition
| has high H2O solubility at O C (3.9 M) |
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| protein stability: solvent polarity |
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Definition
| H2O is a high dielectric constant colvent. Less polar H2O miscible solvents (eg. ethanol or acetone) have lower dielectric constants and can cause proteins to precipitate from solution if added judiciously (sometimes reversibly). not as effective as ionic strenth. |
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Term
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Definition
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| Iso-electric focusing (IEF) |
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Definition
1) mix of proteins is electrophoresis thought a solution or gel that has a stable pH gradient
2) each protein moves to point in gradient pI and then stops |
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Term
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Definition
1)use mercaptans such as 2-mercaptoethanol
2) irreversible process |
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Term
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Definition
1) prevents disulfides from reforming by air oxidation
2) iodoacetate = can substitue a radioactive molecule
3) downside = use both dis redu. and akyl. |
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Term
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Definition
| requires hydrolysis of protein, acid degrades SER, THR, TRY and TRP, converts ASN and GLN to ASP and GLU. bases destroys CYS, SER, THR and ARG and causes deamination, hydrolysis by HPLC |
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Definition
!) # of peptides
2) Sanger's reagent (2,4-Dinitrofluoro-benzene {DNFB})
3) yellow color |
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Term
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Definition
1)0 peptides longer than 40-100 residues must be fragmented into smaller peptides
2) Endopeptidase or Endoproteases (not exopeptidases) can be used to generate fragments |
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Definition
the most specific protease available
-any amino acid residue, but not Pro |
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Term
| subtractive edman degredation |
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Definition
1) uses Edman's reagent to sequence peptides
-PITC = phenyl isothiocyanate
-produces PTH = phenylthio hydanlio in amino acid derivative |
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Term
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Definition
1)useful in several ways in characterizaing macromoleculaes
2) most accurate mass determinations done by electrospray ionization (ESI)MS
3) high vacuum, low pressure
4) vacuum makes molecules expand, also freeze rapidly
5)N amkes molecule freeze and move down vacuum tube
6) allows you to detect the migration by a measurement of time, before ions connect ot detector block
7) ESI = soft ionization can separate large ions, but not not have salt
8) MALDI = TOF-MS soft ionization, without fragmenting ion, uses a laser, 25,169 do +/- 0.01% |
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Term
| immunoaffinity chromo. and metal chelate affinity chromo. |
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Definition
| -possible using recombinant DNA methods to ass His-Tag |
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Definition
| comparision to purified standard "marker" proteins |
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Definition
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Term
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Definition
usually circular, self-replicating duplex DNA molecules, such as plasmids
-genes of interest from foreign DNA are introduced into the vector for propogation of the gene
-both the cloning vector and the foreign DNA are digested with the sme restriction endonuclease
-the sticky ends generated by this treatment are all complementary and can be covalently joined by a DNA ligase
-chrimeric DNA is formed by ligating a portion of he foreign DNA to the cloing vector
-such a contruct can be used to replicate a foreign gene inside a host cell |
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Term
| cloning with bacteriophage "lambda" |
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Definition
-a nonessential portion of a phage genome is removed
-a segment of foreign DNA can be inserted into the cleaved lambda DNA
-chimeric DNA can be packaged into an infectious phage particle only if the insert DNA has the appropriate size. if so the chimeric DNA can be propagated in host cells |
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Term
| site-sirected mutagenesis |
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Definition
-a primer is synthesized contained mismatches corresponding to the desired mutations and hybrided to the wild-type DNA
-the mismatched primer is extened by DNA polymerase, generating the mutated gene, the altered gene can be inserted into a suitable cloning vector to be amplified, expressed, or used to generate a mutant organism |
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Definition
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Definition
lack 3'-hydroxyl groups
-terminate the growth of DNA chains |
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Term
| PCR (Polymerase Chain Reaction) |
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Definition
amplifies
-cycle 1: initial reaction mixture
-target DNA
-primers must be complementary to the region b eing amplified
dNTPs are precursors for new DNA synthesis
-thermostable DNA pol. makes the new DNA strands
-Cycle 1: denaturation
-the temp. is raised to greater than 90 degrees
-high temp. denatures the target DNA making single strands
-cycle 1: annealing
-the temp. is lowered to annealing temp. of the primers as determined by their lengths and base compositions
-the primers anneal to complementary strands at specific sequences via base pairing
cycle 1: extension
-the temp. is set to optimize the action of the DNA poly. (72 C)
-thermostable DNA pol. isolated from bacteria which live at high temp. are not denatured as most DNa pol. would be
-cycle 2
-there are now twice as many single-stranded templates as in the first cycle
-end up with 4 X as cycle 1 at the end of cycle 2
1/4 of the strands now end at primer positions at boths ends
1/4 of the strands are original templates
1/2 of the strands extend beyond the primer sequence at one end |
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Term
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Definition
a primer that contains a few mismatches can still anneal to its target DNA so as to permit the initiation of DNA synthesis
-the amplified produce contains segments that are exactly complementary to the mismatched primer (which is no longer mismatched)
-this is a convenient method to engineer sequence changes into a target DNA |
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Term
| the strength of association of ionic groups of oppostie charge depends on: |
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Definition
1) the chemical nature of the ions
2) the distance between them
3) the polarity fot he medium |
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Term
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Definition
| noncovalent associations between neutral molecules arise from electrostatic interactions among permanent or induced dipoles |
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Term
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Definition
| occurs more rapidly than direct molecular migration, accounting for the observed high ionic mobilities of hydronium ions in aqueous solutions |
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