Study BMB 401H Chapter 7 Flash Cards

 
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BMB 401H Chapter 7

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Hydrazinolysis
-can be used to determine C terminal amino acid
-treated w/ hydrazine (H2N-NH2)
-all peptide bonds cleaved
-yield hydrazine resideues except for C terminus aa
-only free aa is C terminus
retention times
for Edman's reagent
HPLC chromatogram
Edman degradation
-Phenylisothiocyanate
-Removes one aa at a time
-only takes off N-terminal aa
-IDs 20-30 amino acids from the N terminus
-compare retention times on HPLC
-Edman can be repeated automatically to determine aa sequence
dansyl chorlide
-reacts w/ N terminus
-acid hydrolysis breaks all peptide bonds
-dansyl has bright yellow fluoresecent shirt on detected by HPLA
Gene duplication
can occur through aberrant genetic recombination event (ortho, paralogs)
Orthologous
homologous proteins from different organisms
Paralogous
homologous proteins in same organism
phylogeny
-A phylogenetic tree can be constructed that indicates ancestral relationship between different species.
Neutral Drift
-random mutations occur which do not affect protein function
-random process is referred to as neutral drift
-Comparison of primary sequence of homologous proteins indicate which proteins are essential for their function
-ex: Pigeon cytochrome c can reduce wheat oxidase
malaria resistance
-SCA Mendelian genetics
-Heterozygotes have approx. 40% HbS. Such people have sickle cell trait and can lead normal life.
-heterozygote are resistant to malaria
SCA mutation
-Caused my mutant hemoglobin
-sickle cell hemoglobin HbS is two units more positive than HbA (normal).
-Caused by single mutation causing change of Glu (6th aa of B subunits) to Val (charge difference)
-under certain conditions (low O2 levels) HbS distort into sickle shapes
-block small capillaries causing necrosis of the tissue
-Conversion of charged glu to hydrophobic val forms patch that now can bind to hydrophobic portion of another deoxy Hb molecule. Polymerizes into fibers when deoxygenated. Causes rbc to become sickle shape
Sickle Cell anemia
-Hemoglobin has 4 subunits: a2b2
-in rbc's
-In Sickle cell anemia, rbc assume crescent-like shape in low oxygen tension. Are more rigid and hinders free passage
Hemolytic anemia caused by lysis of rbc
Determining site of disulfide bond
-Separate digested peptide with paper electrophoresis
-Treat samples with performic acid to cleave disulfides
-Run again on paper electrophoresis to which has two spots
-Remove bands and sequence to identify
tandem mass spec
-short polypeptides 9<25 aas) can be directly sequenced
-electrospray ionization
-2 MS's
-1st MS selects protein of interest
-peptide ion then crashes into chemically inert cell (He)
-peptide bonds break and MW of charged fragments measured
-computerized
MALDI-TOF
-Matrix-assited laser desorption/ionization-time of flight mass spec
Peptide is embedded in crystalline matrix of some low molecular weight compound.
Peptide is irradiated with laser pulse which ejects peptides into gas phase
electrospray ionization
-macromolecule H2O sol'n sprayed from high voltage
-fine, highly charged droplets remain
-H2O evaporates
-now have gas-phase macromolecule ions
-go through MS
Mass Spec
-Can be used to accurately measure molecular weight
-Use a spray of protein, remove water and then have proteins
-3 techniques to get gas-phase ions required for MS:
-electrospray ionization
-MALDI
-tandem
Peptide cleavage: Endopeptidases
Trypsin
Chymotrypsin
Elastase
Elastase
cleaves on carboxy side of small neutral residues: Ala, Gly, Ser, Val
Trypsin
cleaves on C-side of positively charged residues Arg or Lys
Chymotrypsin
cleaves on C-side of bulky hydrophobic residues Phe, Tyr, Trp
iodoacetate
-ICH2COO-
-after DTT to acetylate
to cleave SS bonds b/w cys residues...
-Can be directly oxidized by performic acid. But this will also oxidize methionine residues.
-Can be reduced and then acetylated
-DTT reduces
-performic acid hydrolyzes
C-terminus analysis
-Carboxypeptidase
-Hydrazinolysis
Carboxypeptidase
-exopeptidase that cleaves from C terminus
-Different enzymes have different specificities
Because of differences in specificities, cannot be used for sequencing.
-if 2nd aa cleaved same time as 1st, both aas releases simultaneously
-Carboxypeptidase also not active on Pro residues
N terminus methods
-dansyl chloride
-Edman degradation
Identification of N-terminus
-Dansyl chloride reacts with N terminus
-Acid hydrolysis to break all peptide bonds
-Dansyl amino acid has fluoresence and detected by HPLC
Cyanogen bromide
cleaves at Met
DTT
-reduces disulfide bonds
Oxidize dissulfide
with performic to cysteic acid
pH, area, guanidinium hydrochloride, or high salt
-ways to separate protein subunits if multimeric
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