Questions and Answers of Biochemistry Concepts And Connections

Referring to Question 23, how would you purify protein X using ion-exchange chromatography if it turns out the protein is only stable at a pH between 6 and 6.5?Question 23Design an experiment to
An amino acid mixture consisting of phenylalanine, glycine, and glutamic acid is to be separated by HPLC.The stationary phase is aqueous and the mobile phase is a solvent less polar than water. Which
In reverse-phase HPLC, the stationary phase is nonpolar and the mobile phase is a polar solvent at neutral pH.Which of the three amino acids in Question 28 will move fastest on a reverse-phase HPLC
Gel-filtration chromatography is a useful method for removing salts, such as ammonium sulfate, from protein solutions. Describe how such a separation is accomplished.
What physical parameters of a protein control its migration on electrophoresis?
What types of compounds make up the gels used in electrophoresis?
Of the two principal polymers used in column chromatography and electrophoresis, which one would be most immune to contamination by bacteria and other organisms?
What types of macromolecules are usually separated on agarose electrophoresis gels?
What does SDS–PAGE stand for? What is the benefit of doing SDS–PAGE?
The accompanying figure is from an electrophoresis experiment using SDS–PAGE. The left lane has the following standards: bovine serum albumin (MW 66,000), ovalbumin (MW 45,000), glyceraldehyde
How does the addition of sodium dodecylsulfate to proteins affect the basis of separation on electrophoresis?
Why is it no longer considered necessary to determine the N-terminal amino acid of a protein as a separate step?
A sample of an unknown peptide was divided into two aliquots. One aliquot was treated with trypsin; the other was treated with cyanogen bromide. Given the following sequences (N-terminal to
What useful information might you get if you did determine the N-terminal amino acid as a separate step?
A sample of a peptide of unknown sequence was treated with trypsin; another sample of the same peptide was treated with chymotrypsin. The sequences (N-terminal to C- terminal) of the smaller
What would happen during an amino acid sequencing experiment using the Edman degradation if you accidentally added twice as much Edman reagent (on a per-mole basis) as the peptide you were sequencing?
You are in the process of determining the amino acid sequence of a protein and must reconcile contradictory results. In one trial, you determine a sequence with glycine as the N-terminal amino acid
What are the two principal types of mass spectrometry?
What is the advantage of MALDITOF MS?
What is the difference between a primary antibody and a secondary antibody?
When deoxyhemoglobin was first isolated in crystalline form, the researcher who did so noted that the crystals changed color from purple to red and also changed shape as he observed them under a
Draw (using Haworth projections) the fragments of xylan and glucomannan structures shown on page 295
In the mitochondrial disease MNGIE a deficiency of thymidine phosphorylase causes dTTP to accumulate in mitochondria. Describe the mechanism by which this occurs. A secondary effect is depletion of
If the same polynucleotide described in Problem 2 is used with a mitochondria-derived cell-free protein-synthesizing system, the product is …Met–Tyr–Met–Tyr–Met–Tyr… What does this
Consider the –10 region sequences for the bacterial promoters in Figure 24.6. Why is the consensus sequence more active in promoting transcription than any of the sequences associated with actual
Of the four DNA bases, why is guanine most likely to be modified after exposure to polycyclic hydrocarbons such as benzo(a)pyrene, as shown in Figure 23.8? Figure 23.8.
Recent data indicate, as mentioned in the text, that the signal for eukaryotic mismatch repair, for identifying the strand to be repaired, is incorporated ribonucleotides that have not yet been
As discussed in Chapter 13, calcium is an important regulator of the citric acid cycle. Calcium is transported across the mitochondrial inner membrane by a Ca2+ uniporter that is driven by the
Which of the pairs in Problem 15 are diastereoisomers?Data in Problem 15Are mannose and galactose epimers? Allose and altrose? Gulose and talose? Ribose and arabinose? Consider only d-sugars. Explain
If you want to purify a DNA-binding protein from a crude mixture of proteins at pH 7, should you use a DEAE-cellulose or a CM-cellulose column (see Figure 5.A5)? Briefly explain your reasoning.Figure
A biochemical reaction takes place in a 1.00 ml solution of 0.0250 M phosphate buffer initially at pH = 7.20 (see Table 2.6 for pKas of phosphate species). (a) Are the concentrations of any of the
Fill in the blanks to identify each substance as(a) A nucleobase(b) A ribonucleoside(c) A ribonucleotide(d) A deoxyribonucleoside(e) A deoxyribonucleotide(f) A dinucleotide Adenosine _Cytidine
(a) The plasmid pBR322 (4362 base pairs) was isolated and the circular DNA was found to be underwound, with a superhelix density (σ) of -0.05. How many superhelical turns does this molecule
On page 351 we showed that the oxidation of glucose and palmitic acid yields 15.94 kJ/g and 38.90 kJ/g, respectively. Calculate these values in terms of kJ/mol and kJ per carbon atom oxidized for
The muscle isozyme of lactate dehydrogenase is inhibited by lactate. Steady-state kinetic analysis yielded the following data, with lactate either absent or present at a fixed concentration.(a)
How many high-energy phosphates are generated or consumed in converting (a) 1 mole of glucose to lactate? (b) 2 moles of lactate to glucose
14CO2 was bubbled through a suspension of liver cells that was undergoing gluconeogenesis from lactate to glucose. Which carbons in the glucose molecule would become radioactive?
Ribose-5-phosphate is incubated with a mixture of purified transketolase, transaldolase, phosphopentose isomerase, phosphopentose epimerase, and glyceraldehyde-3-phosphate. Predict the distribution
Pyruvate was administered to isolated liver cells in the presence of sufficient malonate to block succinate dehydrogenase completely. After a time, isocitrate was isolated and found to contain label
β-adrenergic receptors are subject to phosphorylation at several serine residues by β-adrenergic receptor kinase, and this is followed by binding to the phosphorylated sites by a protein called
Cis-Vaccenate is an 18-carbon unsaturated fatty acid abundant in E. coli membrane lipids. Propose a metabolic route for synthesis of this fatty acid, in light of the fact that stearic acid, the C18
You need to make a buffer whose pH is 7.0, and you can choose from the weak acids shown in Table 2.6. Briefly explain your choice. TABLE 2.6 Some weak acids and their conjugate bases Acid (Proton
Is RNA-binding enzyme RNase A more likely to have a pI of 9.2 or 5.0? Briefly explain your reasoning.
When a hydrophobic substance like a hydrocarbon is dissolved in water, a clathrate cage of ordered water molecules is formed about it (see Figure 2.14). If we consider only the effects on water, what
Bovine ribonuclease folds with ∆H° = -280 kJ mol-1 and ∆S° = -0.79 kJ mol-1 K-1. Assume ∆H° and ∆S° are independent of temperature. What fraction of bovine ribonuclease is unfolded at 42
What is the difference between a nucleoside triphosphate and a trinucleotide?
Suppose that you centrifuged a transfer RNA molecule to equilibrium in an alkaline CsCl gradient, as described in Problem 7. What result would you expect?Problem 7.For some DNAs, it is possible to
Would you expect Neurospora crassa DNA to have a higher or lower Tm than human DNA? Explain.
A circular double-stranded DNA molecule contains 4200 base pairs. In solution, the molecule is in a B-form helix, with about 10.5 base pairs per turn. The DNA circle has 12 superhelical turns. What
(a) What two enthalpic factors stabilize DNA in double-helical form at low temperature? (b) What entropic factor destabilizes helical DNA at high temperature? (c) Why is the double-helical
In studying the mechanism of a particular enzyme, for which the cloned gene is available, you wish to change a putative active site histidine residue to a proline by site-directed mutagenesis. The
Identify each amino shown below in Fischer projection. Indicate whether the d- or l-enantiomer is shown. H T (H₂C)₂HC-C-NH3 coo (a) H H₂N-C-COO CH(CH3)OH (b) oocc(CH, CONH2 NH3 (c)
What DNA sequence feature is required for a G-quadruplex to form?
A protein has been sequenced after cleavage of disulfide bonds. The protein is known to contain 3 Cys residues, located as shown here. Only one of the Cys has a free —SH group, and the other two
Despite the fact that many peptides have critical physiological functions (e.g., as hormones, neurotransmitters, antibacterial toxins), they are not considered to be ideal as drugs. The following
Apamine is a small protein toxin present in the venom of the honeybee. It has the sequence CNCKAPETALCARRCQQH (a) If apamine does not react with iodoacetate (see Tools of Biochemistry 5B), then how
Based on the information in Figure 5.17, which single-nucleotide mutation event is more likely: Arg-to-His, or Arg-to-Ser? Explain.Figure 5.17 Base in first position (5'
Assume the following portion of an mRNA. Find a start signal, and write the amino acid sequence that is coded for.5′...GCCAUGUUUCCGAGUUAUCCCAAAGAUAAAAAAGAG...3′
Bovine pancreatic trypsin inhibitor (BPTI; Figure 6.25) contains six cysteine residues that form three disulfide bonds in the native structure of BPTI. Suppose BPTI is reduced and unfolded in urea
In the protein adenylate kinase, the C-terminal region has the sequence The hydrophobic residues in this sequence are presented in boldface type. Suggest a possible reason for the periodicity in
Shown below are two cartoon views of the small globular protein StrepG in which an a helix is packed against a four-strand β sheet. The sheet is made up of two “β-hairpins” (a b-hairpin is a
The following sequence is part of a globular protein. Predict the secondary structure in this region. . . . RRPVVLMAACLRPVVFITYGDGGTYYHWYH . . .
Why does it make biochemical sense that chaperones recognize hydrophobic surface area? What catastrophic event are chaperones meant to prevent in cells?
In most cases, mutations in the core of a protein that replace a smaller nonpolar side chain in the wild-type (e.g., Ala, Val) with a larger nonpolar side chain (e.g., Leu, Ile, Phe, Trp) in the
Cartoon renderings of the proteins Top7 and adaH2 are shown below. Both are soluble, densely packed proteins of roughly 96 residues, and each has a topology of 2 a helices packed onto a 5-stranded β
Disulfide bonds have been shown to stabilize proteins (i.e., make them less likely to unfold). Consider the cases shown schematically below for two variants of the same protein. In case #1 the
A Leu → Ala mutation at a site buried in the core of the enzyme lysozyme is found to be destabilizing. Explain the observed effect of this mutation on lysozyme stability by predicting how enthalpy
Antibodies raised against a macromolecular antigen (e.g., a protein) generally form an antigen-Ab precipitate when mixed with the antigen at roughly equimolar concentrations. However, little to no
What qualitative effect would you expect each of the following to have on the P50 of hemoglobin? (a) Increase in pH from 7.2 to 7.4 (b) Increase in PCO2 from 20 to 40 mm Hg (c) Dissociation into
Suppose your biking partner claims that hyperventilating at the bottom of steep hill climbs is a good idea because it will “increase the O2 saturation of the blood” and thereby provide “more O2
Hemoglobin Rainier (HbR) is a mutant in which Tyr 145 of the β-globin is replaced by Cys. This Cys forms an intramolecular disulfide with β Cys93 in the oxy state but cannot form the intramolecular
An antibody has been isolated that binds to F-actin but not to G-actin. What structural feature(s) of F-actin do you suppose the antibody binds (i.e., how is the antibody able to distinguish between
The enzyme urease catalyzes the hydrolysis of urea to ammonia plus carbon dioxide. At 21 °C the uncatalyzed reaction has an activation energy of about 125 kJ>mol, whereas in the presence of urease
TPCK and TLCK are irreversible inhibitors of serine proteases. One of these inhibits trypsin and the other chymotrypsin. Which is which? Explain your reasoning. ZI TPCK IZ TLCK NH₂
Would you expect an “enzyme” designed to bind to its target substrate as tightly as it binds the reaction transition state to show a rate enhancement over the uncatalyzed reaction? In other
ATP is a (+) allosteric effector, and CTP is a (-) allosteric effector of the enzyme ATCase. Both of these heterotropic effectors bind to the regulatory subunits on ATCase. The substrates of ATCase,
A reducing sugar will undergo the Fehling reaction, which requires a (potential) free aldehyde group. Which of the disaccharides shown in Figure 9.14 are reducing and which are nonreducing? Figure
Shown below is a proposed mechanism for the cleavage of sialic acid by the viral enzyme neuraminidase. The kcat for the wild-type enzyme at pH = 6.15, 37 °C is 26.8 s-1. (a) Describe the roles of
In kinetics experiments, the hydrolysis of the substrate sialic acid by neuraminidase appears to obey Michaelis–Menten kinetics. Neuraminidase activity is critical for viral infectivity; thus, this
Indicate whether the structures shown are R or S in the absolute system. I H₂N (a) CH3 COOH CH₂ [² CH3 (b) CH3 0-CH3
What is the natural polysaccharide whose repeating structure can be symbolized by GlcUAβ(1 → 3)GlcNAc, with these units connected by β (1 → 4) links?
One or more of the compounds shown below will satisfy each of the following statements. Not all compounds may be used; some may be used twice. Put the number(s) in the blank. (1) Found in
Write the structure of UDP-N-acetylglucosamine and UDP-galactosamine
Indicate whether each of the following disaccharides is a reducing (R) or nonreducing (NR) sugar by the criterion of reaction with Fehling’s solution. (a) Glcα(1 → 2)Frucβ (b) Galβ(1 →
Briefly describe the function of uridine triphosphate (UTP) in carbohydrate metabolism.
Explain how oseltamivir (Tamiflu) interferes with influenza virus replication.
Why is a person with type AB blood able to receive a blood transfusion from a donor with any of the major blood types (A, B, AB, and O) but is able to donate blood only to another type AB individual?
Given these molecular components—glycerol, fatty acid, phosphate, longchain alcohol, and carbohydrate—answer the following: (a) Which two are present in both waxes and sphingomyelin? (b) Which
Shown below is a schematic diagram of the E. coli leader peptidase (Lep), which has several basic amino acids in a cytoplasmic loop. Propose a mutant of Lep that would be a test of the “inside
In each of a, b, and c of Problem 10, will any appreciable transport of material take place in establishing the membrane potential? Briefly explain each answer.Problem 10.Calculate the equilibrium
One of the curves in this graph describes nonmediated diffusion, and the other describes facilitated transport. Which is which? Explain your choices. Rate of transport Concentration
The transport of aspirin (pKa = 3.5, structure shown here) from the digestive tract to the circulation occurs by nonmediated absorption into cells lining the stomach (where pH = 0.8) and the small
The sarcoplasmic reticulum Ca2+-ATPase, pumps 2 mol Ca2+ out of sarcomeres per mol ATP hydrolyzed. (a) Given the following steady-state concentrations and a membrane potential of 65 mV (inside
Consider the simple bimolecular reaction A + B C + D, where k+1 and k-1 are the rate constants for the forward and reverse reactions. ΔG is related to the concentration of the reactants and products
(a) NAD+ kinase catalyzes the ATP-dependent conversion of NAD+ to NADP+. How many reducing equivalents are involved in this reaction? (b) How many reducing equivalents are involved in the conversion
The Nutrition Facts label on a full-fat yogurt lists the following amounts per 200 g serving: 10 g fat; 8 g sugars; 18 g protein. Calculate the calories (Cal) from each nutrient, and the total
Assume that you have a solution of 0.1 M glucose 6-phosphate. To this solution you add the enzyme phosphoglucomutase, which catalyzes the reaction:(a) Does this reaction proceed at all as written at
Multiprotein complexes are formed by weak noncovalent interactions between the proteins. Suppose proteins A and B form a heterodimer with a Kd = 10-6 M:(a) Calculate the fraction of monomers that
(a) A mitochondrion can be modeled as a cylinder 1.5 mm (microns) in length and 0.6 μm in diameter. Calculate the number of citrate synthase enzyme molecules contained in a single mitochondrion,
Suppose it were possible to label glucose with 14C at any position or combination of positions. For yeast fermenting glucose to ethanol, which form or forms of labeled glucose would give the most

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