Biochem Test 2
pyranose: furanose:
-6 membered ring -5 membered ring
Hemoglobin is a protein in red blood cells that binds to oxygen. Which physiological changes that naturally occur in the body reduce hemoglobin's affinity for oxygen?
-Accumulation of carbon dioxide -Decrease in pH
BPG binds tighter to_____, which ____ its affinity for O2.
-HbA -Lowers
Which statement that best explains the role of BPG in O2 transport from mother to fetus?
-HbA-bound O2 will tend to move to HbF because HbF has a lower affinity for BPG, an allosteric inhibitor of O2 binding.
Which five statements about hemoglobin and myoglobin structure are true?
-Molecular oxygen binds reversibly to Fe2+ in heme -Heme is composed of an organic protoprphyrin component and a metal atom. -By itself, heme is not a good oxygen carrier. It must be part of a larger protein to prevent oxidation of the iron atom. -Each hemoglobin molecule can bind four oxygen molecules; each myoglobin can bind oxygen molecule. - Both hemoglobin and myoglobin contain a prosthetic group called heme, which contains a central iron atom.
How does training at high altitudes benefit athletes?
-THe body acclimatizes to the lower oxygen concentration found at high altitudes by producing more red blood cells, making oxygen uptake and transport more efficient upon return to lower altitude.
Select all the statements that correctly describe the cooperative binding of oxygen to hemoglobin.
-The hemoglobin-oxygen binding curve is sigmoidal -Hemoglobin consists of four heme-bound subunits.
Which statements about reducing sugars are true?
-The oxidation of a reducing sugar forms a carboxylic acid sugar -D-Mannose (an aldose) is a reducing sugar -A disaccharide with its anomeric carbons joined by the glycosidic linkages cannot be a reducing sugar.
Hemoglobin
-The oxygen dissociation curve is sigmoidal in shape (S shaped) -As oxygen binds to this molecule the shape of the molecule changes, enhancing further oxygen binding -the binding patterns for this molecule is considered cooperative
What characteristics determine the position of a protein on an IPG strip at the end of isoelectric focusing?
-The pI of the protein -Local pH in the medium
Which amino acids would be expected to produce a similar sickling effect if substituted for Val at position 6?
-alanine -leucine
b-L-galactose and b-L-glucose are:
-diastereomers and epimers
a-D-galactose and b-D-galactose
-epimers; diastereomers and anomers
Select all the monosaccharides that are correctly paired with their class.
-glyceraldehyde: triose aldose -fructose: hexose ketose -dihydroxyacetone: triose ketose
Choose two amino acids that would be reasonable candidates for the pocket-Val 66 interaction.
-leucine -phenylalanine
Neither
-oxygen binds irreversibly to this molecule -carbon monoxide binds at an allosteric site, lowering oxygen binding affinity
Myoglobin
-oxygen dissociation curve is hyperbolic -this molecule has a greater affinity for oxygen
The central atom has_____ bonds:_____ to nitrogen atoms in the porphyrin, one to a______ residue, and one to oxygen.
-six -four -histidine
The Hill plot (Graph B) indicates that lamprey hemoglobin is
-slightly cooperative
What are the roles of sodium dodecyl sulfate (SDS) in two‑dimensional electrophoresis?
-to denature proteins -to cause bound proteins to have a large negative charge
Consider a mixture of four proteins with various molecular weights. A histone molecule weighs 15 kDa, a p53 molecule weighs 53 kDa, an actin molecule weighs 42 kDa, and an IgG molecule weighs 150 kDa. Arrange the molecules in order of their elution from a gel filtration column.
1gG (150 kDA) p53 (53 kDA) actin (42) Histone (15 kDA)
Which of the following statements best describes how 2,3‑bisphosphoglycerate (2,3‑BPG) reduces hemoglobin's affinity for oxygen?
2,3‑BPG binds to positively charged Lys and His residues in the center of the hemoglobin tetramer, which stabilizes the T state.
Complete this statement: The furanose form of fructose is generated by formation of a hemiketal involving the attack of the C‑__ hydroxyl group on the C‑__ ketone.
6;1
Identify the best description of the cause of altitude sicckness.
An organisms' inability to absorb and transport enough oxygen throughout the body at higher altitudes.
In addition to transporting oxygen from the lungs to the tissues, hemoglobin also plays a minor role in transporting carbon dioxide from the tissues to the lungs. How is this accomplished?
Carbon dioxide reacts with the amino termini of globin chains to form carbamate.
Assign each perturbation or the lack of a perturbation to the curve it represents.
Curve 1: -a loss of quaternary structure Curve 2: -a decrease in CO2 -An increase in pH Curve 3: -No perturbation Curve 4: -an increase in 2,3-BPG
Compare and contrast ELISA and western blotting (immunoblotting) by placing the phrases to the technique that they describe. If a phrase describes both ELISA and western blotting, place it under Both.
ELISA -Proteins are in their native state -Can be used to detect antigen or antibody in a sample, depending on procedure Western blot with SDS-PAGE -Proteins have no higher order structure -Proteins transferred to a member or sheet Both -Requires formation of an antigen-antibody complex
Epimers are monosaccharide diastereoisomers that have a single asymmetric carbon. Anomers are diastereoisomers in which the hemiacetal carbon of a cyclic monosaccharide is asymmetric. Determine whether each pair of monosaccharides consists of epimers, anomers, or an aldose‑ketose pair.
Epimers: D-glucose and D-mannose D-galactose and D-glucose Anomers: a-D-glucose and B-D-glucose Aldose-ketose pair: -D-glyceraldehyde and dihydroxyacetone -D-glucose and D-fructose -D-ribose and D-ribulose
Under normal conditions, the central atom of heme is
Fe2+
Which hemoglobin has a higher affinity for O2 at the tissue pO2 of around 4 kPa?
HbF
Match the characteristics of the two steps of two‑dimensional electrophoresis.
Isoelectric focusing: -proteins are separated by charge -carried out in a pH gradient -final net protein charge is zero SDS-PAGE: -Proteins are separated by mass -carried out in a detergent solution -final net protein charge is negative
The binding of oxygen to myoglobin and hemoglobin has what effect on the heme‑bound iron ion?
It causes the iron to move closer to the approximate plane of the porphyrin ring.
A researcher resolves a mixture of peptides using isoelectric focusing. Order the peptides based on their relative positions in the immobilized pH gradient strip at the end of the experiment.
Low pH -glu-gly-glu-asp -Asp-Ala-Leu-Asp -Arg-Gly-Glu-Lys -Arg-Leu-Ala-Arg -Arg-Ala-Lys-Lys High pH
Many scientific methods are used to determine protein mass. Which of the following methods would yield the most precise estimate of protein mass?
Mass spectrometry
How do monoclonal antibodies differ from polyclonal antibodies?
Monoclonal antibodies are derived from multiple cell populations, whereas polyclonal antibodies are derived from one cell population. Monoclonal antibodies differ slightly in their sequences, whereas polyclonal antibodies are all identical. Monoclonal antibodies are raised against a single antigen molecule, whereas polyclonal antibodies are raised against a mixture of antigen molecules. Monoclonal antibodies recognize only a single portion of an antigen molecule, whereas polyclonal antibodies recognize different parts of the antigen molecule.
A protein sample complex consists of two proteins, a smaller protein, X, and a larger protein, Y. Protein X is composed of two polypeptide chains linked by disulfide bonds. Protein Y is composed of three polypeptide chains linked by disulfide bonds. The complex is analyzed by native PAGE, reducing SDS‑PAGE and non‑reducing SDS‑PAGE. Native PAGE does not include sodium dodecyl sulfate, or SDS. Reducing SDS‑PAGE uses both SDS and a reducing agent in the buffer. Non‑reducing SDS‑PAGE uses SDS, but no reducing agent. Match each gel pattern to an experimental method. The first lane in each gel is a molecular weight ladder.
Native Page: 1 Bar on right side Non-reducing SDS-PAGE: 2 Bars on right side Reducing SDS-PAGE: 3 Bars on right side Molecular Weights of protein X polypeptides: 50 kDA each Molecular Weights of the Protein Y Polypeptides: 60 kDa and 30 kDA
How does HbS aggregation occur in sickle‑cell anemia? Place the steps in the correct order. Note that deoxyhemoglobin is in the T state; oxyhemoglobin is in the R state.
No aggregation -O2 decreases due to vigorous exercise or high altitude. -R state Hb shifts to T state Hb. -Val interacts with the pocket of bet chain on another Hbs. -Additional T state HbS interact with the growing aggregate to form an insoluble fiber. Sickled red blood cell
Each chain of hemoglobin can be viewed as existing in one of two states: the R (relaxed, high‑affinity) state and the T (tense, low‑affinity) state.
Oxygen binding converts hemoglobin from the T state to the R state.
Consider an experiment where the researcher used Western blotting to detect protein X, which weighs 50 kDa in the whole cell lysates of normal cells and cancer cells. Why are there two bands present in one of the lanes?
Protein X is post-translationally modified in cancer cells.
Select the true statements about SDS‑PAGE, a method of separating proteins. Assume that SDS‑PAGE is performed under reducing conditions.
Proteins are separated in a polyacrylamide gel matrix. Protein‑SDS complexes migrate toward the negative electrode. Smaller proteins migrate faster through the polyacrylamide gel. Sodium dodecyl sulfate binds proteins, resulting in protein‑SDS complexes that are similar in size. Proteins are visualized using a dye that binds to the gel matrix, but not to proteins. A protein binds roughly 1.4 times its mass of SDS, resulting in a large overall negative charge.
For each of the six scenarios, determine whether the hemoglobin binding curve would shift left or shift right.
Shifts Left: - The adult hemoglobin is replaced by an infant's fetal hemoglobin -Hemoglobin is isolated from red blood cells and stripped of 2,3-biphosphoglycerate. -Tetrameric hemoglobin is dissociated into its subunits Shifts right: -The blood pH drops from 7.4 to 7.2 -The Co2 concentration in the blood increases. -The concentration of 2,3-biphosphoglycerate increases during acclimation to high altitude.
Each of the given statements describes a type of column chromatography. Match the statements to the type of chromatography they describe. If a statement can describe all of the types, place that statement in the All category. (Note that size‑exclusion chromatography may also be called gel filtration or molecular‑exclusion chromatography.)
Size-Exclusion Chromatography: Separates molecules by size; the stationary phase contains cross-linked polymers with different pore sizes. Affinity Chromatography: Can separate molecules based on protein-ligand binding; The stationary phase has a covalently bound group to which a protein in the mobile phase can bind. Ion-exchange chromatography: Separates molecules by charge; The stationary phase may contain negatively or positively charged groups. All: Uses a mobile phase and stationary phase to separate protein
Size-exclusion column chromatography and SDS-PAGE are methods used to separate mixtures of proteins. Match each statement with the appropriate category.
Size-exclusion chromatography: - Proteins are separated in their native state -Larger proteins travel faster during separation -Proteins of similar molecular weight but different shape can be separated SDS-PAGE -Proteins are separated in their denatured state -Smaller proteins travel faster during separationg. -Proteins move toward the positive electrode. Both -Proteins are separated by size
Classify the overall structure of hemoglobin in its two conformational states, based on images depicting the conformational changes in heme.
T State: -Stabilized by increased ion pairing at the alpha1beta2 and alpha2beta interfaces -iron ion protrudes from heme towards His F8 -Tense state of hemoglobin R state: -narrowed pocket between beta subunits -iron ion assumes a planar conformation -relaxed state of hemoglobin
Hemoglobin Titusville (described by Schneider et al. in 1975) is a hemoglobin variant in which an Asn residue is substituted for Asp at residue 94 of the α chain. This mutation destabilizes the R state. Which graph could represent hemoglobin Titusville's dissociation curve?
The Pink line drops significantly to the purple line
Consider an experiment where your goal is to isolate Bruton's tyrosine kinase (BTK) enzyme from a whole‑cell lysate. You have an affinity chromatography column with a tyrosine kinase inhibitor molecule covalently attached to the beads. The tyrosine kinase inhibitor binds and inhibits BTK. As a result of the experiment, you are able to elute BTK from the column, but in a mixture of other tyrosine kinases.
The kinase inhibitor has low binding affinity. BTK is rapidly degraded during cell lysis. The kinase inhibitor has low specificity. BTK is inactive in the cell.
Which of the following statements accurately represents characteristics of NMR and x‑ray crystallography?
The limitations of x‑ray crystallography and NMR are the same with respect to all proteins. X‑ray crystallography generally gives a more detailed (higher resolution) view than does NMR. NMR is preferable to x‑ray crystallography because it can be used with proteins in a wider range of molecular weights. X‑ray crystallography can be used only for larger proteins or complexes.
Label blood types O and B with the correct monosaccharides. The defining monosaccharide is noted by the gray and orange bond. Each monosaccharide may be used more than once.
Type O: N-acetyl-d-glucosamine-->D-galactose-->L-fucose Type B: N-acetyl-d-glucosamine-->D-galactose (d-galactose beneath this as well)-->L-fucose
The results of a separation using two-dimension gel electrophoresis are shown here.
Which protein or proteins have the highest pI value? -d and e Which protein has the highest molecular weight? -b
Lamprey hemoglobin is a dimeric protein. When the first oxygen atom binds to deoxygenated lamprey hemoglobin dimers, they dissociate into monomers. Based on the Hill plot, lamprey hemoglobin monomers have
a higher oxygen affinity than the dimers
Hemoglobin S (HbS), which is an abnormal form of hemoglobin responsible for sickle‑cell anemia, is the result of a mutation in the gene for the β subunit.The mutation found in HbS results in the change of
a negatively charged amino acid R group to a positively charged amino acid R group.
ribose
aldopentose
The processes of N-linked and O-linked glycosylation
both take place in the golgi apparatus
Which of the following is the LEAST useful for determining the three‑dimensional structure of a protein?
cryoelectron microscopy analytical ultracentrifugation x‑ray crystallography nuclear magnetic resonance spectroscopy
In_______, the central iron atom is displace 0.4 amstrums out of the plane of the porphyrin ring system.
deoxyhemoglobin
The use of phenyl isothiocyanate, the key reagent in the Edman degradation, makes it possible to
determine the number of disulfide bonds in the protein. maintain the protein in an unfolded state during sequencing. remove and identify one amino acid at a time. unambiguously identify only the N-terminal amino acid.
D-Glucose and L-glucose are
enantiomers
a-D-galactose and a-L-galactose
enantiomers
Select the most appropriate technique for separating the digestion products of AVGWRVKS if cleaved by trypsin.
gel filtration chromatography
Glucose yields a positive test with cupric ions, such as those of Fehling's solution, but sucrose does not because
glucose is in equilibrium with its free aldehyde form, whereas sucrose is not.
The prosthetic group of hemoglobin and myoglobin is
heme
Glycoproteins are proteins to which carbohydrates have been covalently attached.The amino acid R groups that serve as sites for O‑linkages in glycoproteins include
hydroxyl-containing R groups
Once formed, the α and β forms of D‑glucose are
interconvertible only through a linear, open-chain intermediate with which the cyclic forms are both in equilibrium.
Breaking disulfide bonds is a necessary step in preparing proteins for sequencing.Which of the following reagents is used to accomplish this?
iodoacetate mercaptoethanol sodium hydroxide guanidine hydrochloride
Suppose that you are given a mixture of proteins with their properties provided in the following chart.
ion exchange chromatography and gel filtration chromatography
Select the most appropriate technique for separating the digestion products of AVGWRVKS if cleaved by chymotrypsin.
ion-exchange chromatography
The log of the molecular weights of proteins can be plotted against the relative mobility on SDS‑PAGE. Using this plot, the relative mobility of an unknown protein can be used to estimate its molecular weight. The molecular weights are in daltons. Estimate the molecular weight (𝑀r)(Mr) of the unknown protein that is circled on the graph. Enter the weight as a whole number.
log(𝑀r)=4.2 10^4.2=𝑀r Mr=15848
Identify the best description of a heme group
one of the four iron-containing parts of hemoglobin that bind to oxygen molecules.
In the lungs, oxygen diffuses into the blood and is loaded onto hemoglobin for transport. In the tissues, oxygen is unloaded from hemoglobin and diffuses from the blood into nearby cells. What drives the diffusion of oxygen?
partial pressure of oxygen
A protein has been eluted from a diethylaminoethylcellulose (DEAE‑cellulose) column by washing the column with a buffered salt solution. Since salt can interfere with the next step of your protocol, which is protein quantification, this salt must be removed from your eluted sample.Which of the following will NOT efficiently remove salt from your sample?
passage through a carboxymethyl‑cellulose column dilution and concentration using a protein concentrator device passage through a gel filtration column dialysis
The organic ring component of heme is
prophyrin
Which of the following can SDS polyacrylamide electrophoresis be used to do?
purify a monomeric enzyme in its active form determine the folding state of a monomeric enzyme determine the molecular weights of subunits of an oligomeric protein determine the molecular weight of an oligomeric (multisubunit) protein
Rapidly metabolizing tissues generate protons and carbon dioxide in high concentrations. The result is that the the oxygen‑binding curve of hemoglobin (Y versus pO2)
shifts to the right, which reflects less saturation of hemoglobin at higher pO2 levels.
A nuclear magnetic resonance, or NMR, spectrometer is an instrument that can be used to characterize molecules based upon
the behavior of the nuclei of certain atoms in a magnetic field.
Formation of pyranose and furanose forms of a sugar generates a new asymmetric carbon.If the anomeric alcohol group in a cyclic sugar molecule is on the same side as the C‑6, the sugar is described as
the beta anomer
In fetal hemoglobin (HbF), the two β subunits are replaced with two γ subunits. The result is that HbF has a higher affinity for oxygen than the mother's adult hemoglobin (HbA). The greater oxygen affinity of HbF compared with HbA is due to
the decreased affinity of 2,3‑BPG to the γ subunits of HbF.
All of the cells in the body need oxygen. Hemoglobin molecules in red blood cells transport oxygen through the bloodstream. Oxygen is loaded onto hemoglobin molecules in the lungs and unloaded from the hemoglobin molecules in the tissues. What drives the loading of oxygen onto hemoglobin molecules in the lungs?
the high partial pressure of oxygen in the lungs
How would you classify the product of the reaction? Note that a hemiacetal formed from a ketone is also called a hemiketal; an acetal formed from a ketone is also called a ketal.
the product is a hemiketal
Human ABO blood groups are
the result of differing glycosyltransferase activities.
The difference between the concerted and sequential models of oxygen binding to hemoglobin is
whether the transition between T and R states is an "all‑or‑nothing" event or an intermediate state exists with a mixture of R and T states in the same molecule.
Identify the applications for gel filtration chromatography.
•Separation of components in a mixture by size •Estimation of molecular weights
