_________________ is a chemical reagent that is often used to detect the presence of amino acids.
ninhydrin
In the Edman procedure for peptide sequence, phenyl isothiocyanate is used to selectively remove the ________________ residue as a PTH-derivative
N-terminal
Disulfide bonds in peptides and proteins are readily oxidized to cysteic scid residues by treatment with _____________________.
performic acid
Automated peptide synthesis involves the activation of the carboxyl group of the incoming amino acid by ________________ and then reaction w/ the amino group of the growing peptide chain.
DCC
Polypeptides can be fragmented into smaller peptides by cleavage with chymotrypsin, which hydrolyzes the peptide bond at the C-terminal side of ___________ residues
Leu
Met
Tyr
Try
Phe
______________ gels are often used as the media for electrophoretic techniques such as SDS-PAGE and isoelectric focusing.
Polyacrylamide
The mobility of proteins in SDS-PAGE is inversely proportional to the ________________.
mass
What is the advantage of adding SDS to gel electrophoresis?
a) SDS colors the proteins for visualization
b) SDS reduces disulfide bonds
c) SDS allows proteins to be separated on the basis of approximate mass
d) none of the above
e) all of the above
c) SDS allows proteins to be separated on the basis of approximate mass
2D electrophoresis is a combination of what 2 techniques?
a) isoelectric focusing & affinity chromatography
B) ion-exchange chromatography and SDS-PAGE
C) affinity chromatography and SDS-PAGE
D) isoelectric focusing and SDS-PAGE
E) isoelectric focusing an
d) isoelectric focusing & SDS-PAGE
Cyanogen bromide cleaves the peptide bond at
A) the carboxyl side of Arg and Lys residues.
B) the carboxyl side of Met residues.
C) the amino terminus.
D) None of the above.
E) All of the above.
b) the carboxyl side of Met residues
Trypsin cleaves the peptide bond at
A) the carboxyl side of Arg and Lys residues.
B) the carboxyl side of Met residues.
C) the amino terminus.
D) None of the above.
E) All of the above.
a) the carboxyl side of Arg & Lys residues
Which of the following techniques can be used to determine the site of a disulfide bond?
A) Edman degradation
B) affinity chromatography
C) diagonal electrophoresis
D) MALDI-TOF
E) SDS-PAGE
c) diagonal electrophoresis
What types of molecules can serve as antigens?
A) proteins
B) polysaccharides
C) metal ions
D) All of the above
E) a and b
e) a & b
An ELISA can be used for
A) quantitative analysis.
B) size analysis.
C) absorbance measurements.
D) All of the above.
E) None of the above.
a) quantitative analysis
A technique used to identify proteins after gel electrophoresis, which employs antibodies in the detection process.
A) None of these.
B) Southwestern Blot
C) Western Blot
D) Southern Blot
E) Northern Blot
c) western blot
Which technique cannot be used for quantitative analysis?
A) X-ray crystallography
B) ELISA
C) absorbance spectroscopy
D) All of the above.
E) None of the above.
a) x-ray chrystallography
Techniques that can be used to obtain information about protein shape are
A) X-ray crystallography.
B) NMR spectroscopy.
C) SDS-PAGE.
D) a and b.
E) a, b, and c.
d) a & b
How is lactic acid dehydrogenase assayed?
--assayed by the inc in NADH present
--NADH has unique absorbance at 340 nm --> rxn can be monitored by the inc in absorbance at this wavelength
How can a protein's isolelectric point be used in protein purification?
--isoelectric focusing is an electrophoretic technique where gradient charge is applied
--proteins migrate thru gradient field until point reached where pH is same as protein's pI
What is the purpose of determining the specific activity, yield, and purification level of a protein purification protocol?
--measurements allow for determining if individual steps were effective at selectively isolating protein while maintaining its presence & activity
--to do this successfully, both yield & purification level must stay high
Describe the Edman degradation method for protein-sequence analysis.
--pure protein is reacted w/ phenylisothiocyanate --> binds free amino terminus
--in mildly acidic conditions --> derivatized amino acid is liberated & can be identified by chromatography
--steps are repeated to identify next amino acid exposed at amino t
How can the amino acid sequences be used to design a DNA probe?
--DNA sequence can be designed using amino acid sequence & genetic code
What is 1 advantage of using recombinant DNA methods to determine protein sequences?
--large proteins difficult to sequence w/ traditional methods --> only short peptides can be sequenced
--long sections of DNA can be cloned & sequenced
--genetic code used to determine the amino acid sequence
What is an epitope?
--specific group/part of molecule that antibody recognizes
Why are monoclonal antibodies more useful than polyclonal antibodies?
--monoclonal are more specific --> have 1 type of antibody w/ specific binding site & affinity
--polyclonal have several different antibodies w/ slightly different binding affinities & specificities
--monoclonal most suitable for examining protein structu
Briefly describe how an ELISA works.
--in ELISA, antigen of interest is complexed w/ specific antibody under appropriate assay conditions --> excess antibody removed
--antibody is complexed to enzyme --> can be measured quantitatively w/ appropriate substrate & assay tool --> usually colorim
How can you identify the C-terminal amino acid?
--is the end of the amino acid chain --> terminated by free carboxyl group (-COOH)
How can you identify the N-terminal amino acid?
--is the front of the protein/polypeptide attached to a free amine group (-NH2)
Describe the Sanger's method and the Edman's method.
Sanger: identify n-terminal residue of peptide --> cycle can't be repeated, uses reagent called FDB, end product identified by gel chromatography
Edman: involves sequential identification of amino acid from n to c terminal, uses reagent called PTC, end pr
What does Carboxypeptidase do w/ the polypeptide chain?
removes amino acid residue from C-terminal of peptide chain
The ratio of enzyme activity relative to total protein is called ____________.
specific activity
The first step in protein purification from a homogenate is usually _____________.
centrifugation
____________ A type of purification that is based on the attraction of the protein for a particular chemical group.
affinity chromatography
____________ can be added prior to gel electrophoresis to denature the proteins.
SDS
Proteins with different sedimentation coefficients can be separated by ___________________.
zonal centrifugation
Proteins can be separated from small molecules and ions through a semi-permeable membrane by __________________.
dialysis
Exclusion gel or gel-filtration chromatography separates molecules on the basis of __________________.
size
When enzymes are purified, the assay is often based on
A) light absorbance.
B) catalytic activity.
C) pH.
D) temperature changes.
E) mRNA levels.
b) catalytic activity
What is the advantage of adding SDS to gel electrophoresis?
A) SDS colors the proteins for visualization.
B) SDS reduces disulfide bonds.
C) SDS allows proteins to be separated on the basis of approximate mass.
D) None of the above.
E) All of the above.
c) SDS allows proteins to be separated on the basis of approximate mass
Which of the following affect the sedimentation of a particle?
A) mass
B) shape
C) the density of the solution
D) All of the above.
E) a and b
d) all of the above
Why is an assay necessary for protein purification studies?
--allows researchers to accurately measure amount of desired protein
--important in determining if particular purification steps are effective in isolating proteins from other cellular material
How do gel-filtration and ion-exchange chromatography differ?
--properties of column material determine how separation is done
--gel filtration --> based on porous beads, molecules separated by size
--ion-exchange chromatography --> column material is charged w/ (+) or (-) charged molecules, separation based on prot
Differences between enzyme activity & specific activity?
--enzyme activity: measurement of quantity of active enzymes present, dep on specified conditions
--specific activity: amount of substrate converted thru enzymatic processes per unit of time & mass of total
List types of chromatographic separation methods for proteins (4)
--ion exchange chromatography
--chromatography based on hydrophobicity
--gel-filtration/size exclusion chromatography
--affinity chromatography
The net charge of a protein depends on:
# of charged amino acids it has & pH of its environment
The mobility of proteins in SDS-PAGE is inversely proportional to the _____________.
size of the molecule
This is a reactive oxygen species that is damaging to biological materials.
superoxide
This is the organic portion of the heme group in hemoglobin.
protoporphyrin
This is the chemical form in which most of the carbon dioxide is transported in the blood.
bicarbonate ion
This substance is produced when carbon dioxide reacts with water.
carbonic acid
This type of hemoglobin is composed of two ? chains and two ? chains.
fetal
This is the molecule whose function is to store oxygen is muscle cells.
myoglobin
This oxidized hemeprotein does not reversibly bind oxygen.
metmyoglobin
This type of binding is indicated by a sigmoidal-shaped binding curve.
cooperative
Under normal conditions, the heme iron in myoglobin and hemoglobin is in the ____________ oxidation state.
ferrous/Fe2+
The ability of myoglobin to bind oxygen depends on the presence of a bound prosthetic group called _____________.
heme
In hemoglobin, the iron of the heme is bonded to the four nitrogens of porphyrin and to the proximal ______________ residue of the globin chain.
histidine
The binding of 2-3-bisphosphogycerate to hemoglobin ____________ (increases,/decreases) its affinity of oxygen binding.
decreases
The effect of pH on oxygen-binding of hemoglobin is referred to as the _____________.
bohr effect
Carbon dioxide reacts with the amino terminal groups of hemoglobin to form carbamate groups, which carry a ______________ charge.
negative
The T-state of hemoglobin is stabilized by a salt bridge between ?1 Asp 94 and the C-terminal ___________________ of the ?1 chain.
histidine
As the pressure of carbon increases, the affinity of oxygen binding to hemoglobin ______________.
decreases
2,3-Bisphosphoglycerate binds only to the __________________ form of hemoglobin.
T- / deoxy
What factor(s) influence(s) the binding of oxygen to myoglobin?
A) The concentration of bicarbonate ion, HCO3-
B) The partial pressure of oxygen, pO2
C) The concentration of hemoglobin present
D) The concentration of 2,3-BPG
E) Responses b and d
b) the partial pressure of oxygen, pO2
Which of the following is correct concerning the differences between hemoglobin and myoglobin?
A) Both hemoglobin and myoglobin are tetrameric proteins.
B) Hemoglobin exhibits a hyperbolic O2 saturation curve while myoglobin exhibits a sigmoid shaped curv
c) hemoglobin exhibits cooperative binding of O2 while myoglobin does not
Which of the following is not correct concerning myoglobin?
A) The globin chain contains an extensive ?-helix structure.
B) The heme group is bound to the globin chain by two disulfide bonds to cysteine residues
C) The iron of the heme group is in the Fe+
b) the heme group is bound to the globin chain by 2 disulfide bonds to cysteine residues
The structure of normal adult hemoglobin can be described as
A) a tetramer composed of four myoglobin molecules.
B) a tetramer composed of two ?? dimmers.
C) a tetramer composed of two ?2 and two ?2 dimers.
D) a tetramer composed of two ?2 and two ?2 dime
b) a tetramer composed of 2 ?? dimers
Which of the following is correct concerning fetal hemoglobin?
A) Fetal hemoglobin is composed of two ? and two ? subunits.
B) Fetal hemoglobin binds 2,3-BPG more tightly than normal adult hemoglobin.
C) Fetal hemoglobin binds oxygen less than HbA at all
a) fetal hemoglobin is composed of 2 ? and 2 ? subunits
Hemoglobin-binding of oxygen is best described as a
A) concerted model.
B) Michaelis-Menten model.
C) sequential model.
D) combination of sequential and concerted models.
E) None of the above.
d) combination of sequential and concerted models
2-3 Bisphosphoglycerate
A) binds in the central cavity in the T-form of hemoglobin.
B) preferentially binds to deoxyhemoglobin and stabilizes it.
C) is present in the red blood cells.
D) All of the above.
E) None of the above.
d) all of the above
What is the Bohr effect?
A) the ability of hemoglobin to retain oxygen when in competition with myoglobin
B) the regulation of hemoglobin-binding by hydrogen ions and carbon dioxide
C) the alteration of hemoglobin conformation during low oxygen stress
D)
b) the regulation of hemoglobin-binding by hydrogen ions and carbon dioxide
Which of the following statements is correct for hemoglobin and oxygen transport?
A) The oxygen binds to the proximal histidine residue of the globin chain.
B) Bonding of carbon dioxide to hemoglobin molecules increases the binding of oxygen.
C) Hemoglobi
e) the binding of each O2 molecule to hemoglobin increases its affinity for the next O2
Which of the following describes the Bohr Effect?
A) Lowering the pH results in the release of O2 from oxyhemoglobin.
B) Increasing the pressure of CO2 results in the release of O2 from oxyhemoglobin.
C) Increasing the pH increases the T-form of hemoglobi
e) a & b
Which of the following is correct concerning the following equilibria?
CO2 + H2O H2CO3
A) An increase in the pressure of CO2 will result in a decrease of pH.
B) This reaction is catalyzed by carbonic anhydrase.
C) The H2CO3 dissociates to H+ and bicarbona
e) all of the above
Carbon dioxide forms carbamate groups in proteins by reaction with
A) aspartate residues.
B) cysteine residues.
C) N-terminal amino groups.
D) tyrosine residues.
E) heme groups.
c) n-terminal amino groups
Which of the following is correct concerning the oxygenation plot of proteins X and Y shown below?
[oxygen binding plot]
A) Protein Y exhibits tighter oxygen-binding than protein X.
B) Protein Y corresponds to fetal hemoglobin, and protein X corresponds t
c) protein X corresponds to fetal hemoglobin, and protein Y corresponds to normal adult hemoglobin
Which of the following is not correct concerning the oxygenation plot of proteins X and Y shown below?
[oxygen binding plot]
A) Protein X exhibits tighter oxygen binding than protein Y.
B) Protein Y would function as a better transport protein than protei
c) protein X exhibits cooperative binding, whereas Y does not
Which is not correct concerning the models that are accepted to describe cooperative binding?
A) In the sequential model, the binding of a ligand changes the conformation of the subunit to which it binds, which in turn induces a change in neighboring subu
b) all known allosteric proteins exhibit either the concerted or sequential model exclusive of the other
Consider the oxygen-binding profile at three different pH values of 7.6, 7.4, and 7.2. Which statement is most correct?
[Hb oxygen binding plot]
A) Curve X most likely corresponds to pH 7.2.
B) Curve Z most likely corresponds to pH 7.6.
C) Hb has a higher
d) curve Y most likely corresponds to pH 7.4
What would be the expected result of a Lys residue being substituted with a Ser residue in the BPG binding site of hemoglobin?
A) BPG would bind tighter because of the loss of a positive charge.
B) BPG would bind tighter because of the gain of a positive
c) BPG would bind less tightly because of the loss of a positive charge
Why is it advantageous for hemoglobin to have allosteric properties?
--hemoglobin binds O in a (+) cooperative manner --> allows it to become saturated in the lungs where O pressure is high
--when hemoglobin moves to tissues --> lower O pressure induces release of O and deliver it where it's needed
What is fetal hemoglobin? How does it differ from adult hemoglobin?
--fetal --> has 2 ? and 2 ? chains, ? chain result of gene duplication & divergence, higher affinity for O
--adult --> has 2 ? and 2 ? chains
What is metmyoglobin?
-- formed when the heme iron ion is oxidized to the +3 oxidation state
--oxidized form of myoglobin does not bind dioxygen and is not functional
What functional role does the "distal histidine" play in the function of myoglobin and hemoglobin?
--bonding between Fe & O --> resonance structures
--distal histidine donates H bond to this complex & stabilizes complex & inhibits oxidation of Fe to the ferric state
Describe cooperative binding
--occurs in multi-subunit proteins that has multiple binding sites
--binding of ligand to 1 site causes conformational change that influences binding of ligand to next site
--binding sites not ind --> each binding event affects affinity of next binding ev
Describe concerted model to explain allosteric cooperative binding
--protein exists in 2 conformations --> T state (tense, lower affinity for ligand) & R state (relaxed, higher affinity for ligand)
--concerted model --> molecules exist either in T or R state
--at each ligand concentration --> equilibrium between the 2 st
Describe the role of 2,3-bisphosphoglycerate in the function of hemoglobin.
--small, highly anionic molecule found in RBC
--only binds to center cavity of deoxyhemoglobin (T state)
--size of center cavity dec upon change of R form
--presence of 2,3-BPG shifts equilibrium to T state --> T state unstable normally without BPG
Describe the chemical basis of the Bohr effect.
--observed by Bohr
--hemoglobin becomes deoxygenated as pH dec
--in deoxyhemoglobin --> 3 amino acid residues form 2 salt bridges to stabilize T state
--as pH inc, stabilizing salt bridge is broken
--pH dec, equilibrium shifted to T state --> release of O
Describe how carbon dioxide affects the oxygenation of hemoglobin.
--inc levels of CO2 cause hemoglobin to release O
--more active in tissue --> more fuel burned --> more CO2 made
--CO2 combines w/ N-terminal amino acid groups to make (-) charged carbamate groups --> form salt bridges to stabilize T state
--inc of CO2 ca