Biochemistry - The Chemical Reactions of Living Cells, Volume 1 - D. Metzler 1980

The Molecules We Are Made Of
How We Study Molecular Structure
Conformation of Macromolecules

Many of the physical Methods mentioned in the previous section boil down to measuring quantities that depend on both molecular weight and molecular shape. Consequently, these same methods can also be used to determine conformation. However, two other methods are far more valuable in this regard: X-ray crystallography of crystalline compounds and nuclear magnetic Resonance (NMR) spectroscopy of compounds in solution.

a. X-ray Crystallography

Shortly after the discovery of X-rays, diffraction patterns obtained by passing an X-ray beam through a crystal began to be used to determine interatomic distances and elucidate crystal Structure. This method was refined to the point where it became possible to determine the structures of quite complex Organic compounds with only a few days of experimentation. At the same time, the most serious challenge hindering the application of X-ray crystallography has often been the preparation of sufficiently large and well-ordered crystals.

In recent years, X-ray analysis has been widely applied to determine the structures of PROTEIN AND NUCLEIC acid molecules. Bond Lengths and angles precisely determined for small molecules are used as standard values on the assumption that they remain the same in more complex polymeric structures. One of the steps in determining The structure of Proteins and Nucleic Acids is the Construction of Molecular Models of polymers that are consistent with X-ray data while preserving standard values for bond lengths and valence angles (Fig. 4-19, B) [71].

b. Nuclear Magnetic Resonance (NMR) [157—162]

NMR spectroscopy is based on the absorption of radio-frequency electromagnetic waves by nuclei possessing a magnetic moment. All nuclei with odd mass numbers (e.g., ¹Н, ¹³С, ¹⁵N, ¹⁷O, ¹⁹F, and ³¹Р), as well as nuclei with an even mass number but an odd atomic number, exhibit magnetic properties, whereas ¹²С and ¹⁶О are non-magnetic. The absorption of a quantum of energy E = hv occurs only when the nuclei are placed in a strong

magnetic field of an NMR spectrometer. In a spectrometer with an operating frequency of 100 MHz (v = 100 MHz), the quantum energy is merely E = 6,625∙10^-34∙10⁸ = 0,04 Дж∙моль^-1, which is five orders of magnitude lower than the average thermal motion energy of molecules (3,7 кДж∙моль^-1). Thus, the spin transition induced in an NMR spectrometer does not significantly affect The chemical properties of molecules.

The resonance frequency v at which energy absorption occurs in the spectrometer is

Class="center">v = μH₀/h,       (2-32)

where Н₀ is the strength of the external magnetic field, μ is the magnetic moment of the nuclei under study, and h is Planck's constant.

In principle, an NMR spectrum can be obtained by keeping the magnetic field Н₀ constant and determining at what frequency absorption occurs, much like recording ultraviolet, visible, and Infrared Spectra (Chapter 13). However, NMR spectrometers operate at a fixed frequency (most commonly 60, 100, and 220 MHz), with the most powerful ones operating at frequencies exceeding 300 MHz. When recording a spectrum, the magnetic field produced by the spectrometer's powerful electromagnet is varied at a constant rate. Nevertheless, NMR spectra are always expressed in terms of frequency shifts in hertz, as if the generator frequency were being varied.

FIG. 2-40. PMR spectrum of pyridoxal 5'-phosphate; recorded at 60 MHz; pD = 7.05. Thiers' salt serves as the internal standard. The chemical shift value in hertz is indicated above each peak, with the scale shown at the bottom. To average out magnetic field inhomogeneities, the sample tube is rapidly rotated about its axis during measurements. Incomplete averaging results in so-called spinning sidebands; in our figure, these are the two peaks labeled SSB. (Courtesy of J. Likos.)

Fig. 2-40 shows the proton NMR (PMR) spectrum of the coenzyme Pyridoxal phosphate in a ²Н₂О (deuterium oxide) solution; the spectrum was recorded on a 60 MHz spectrometer (see also [163]). Four parameters can be determined from such a spectrum. 1. Intensity (peak area). In proton NMR spectra, these areas are generally proportional to the number of chemically equivalent protons giving rise to the given peak. 2. Chemical shift — the difference in frequency between the peaks corresponding to a given proton and a reference compound. In Fig. 2-40, the reference peak is located in the right-hand part of the spectrum and corresponds to Thiers' salt (sodium 3-trimethylsilyl-1-propanesulfonate). 3. Peak width (in hertz) at half-height. For molecules moving rapidly in solution, the width of a proton peak is approximately 0.1 Hz. 4. Spin-spin coupling constants, which are a measure of the interaction between neighboring nuclei possessing a magnetic moment.

At Н₀ = 14 000 G and a frequency of 60 MHz, proton signals from organic compounds are spread over a range of approximately 700 Hz. THE POSITION OF the signals is always determined relative to the peak of a reference compound located in the high-frequency region. For protons, the most common reference compound is tetramethylsilane (TMS), an inert substance that can be added directly to the sample tube. Since biochemists typically use D₂О as a solvent, the Water-soluble Thiers' salt serves as the standard. The peak positions for the two compounds differ only slightly.

When recording an NMR spectrum in D₂О, it is often necessary to know the "pD" value of the medium. Typically, 0.40 is added to the pH meter reading for this purpose.

In molecules such as TMS, nuclei are shielded from the external magnetic field by their surrounding electrons, which is why the signals appear in the region of higher frequencies (higher energies). Protons bonded to a carbon atom or any other electron-deficient atom (due to attachment to electron-withdrawing atoms or groups) lack such shielding (i.e., they are deshielded). The greater the deshielding, the larger the downfield shift of the NMR peak relative to the TMS signal position. In the spectrum shown in Fig. 2-40, the peak corresponding to the 2-methyl protons is located 147 Hz to the left of the Thiers' salt peak, yet it still corresponds to a relatively high field. In aromatic rings, protons are strongly deshielded by ring currents caused by π-electrons. Thus, the peaks corresponding to the 5-methylene protons, which are located near the aromatic ring, are shifted by 303 and 310 Hz downfield. A proton directly attached to the ring is deshielded even further, with its peak shifted downfield by 463 Hz. The 4'-hydrogen of the aldehyde group is deshielded due to electron currents in the carbonyl group; its peak lies in an even lower field.

The magnitude of such a chemical shift—that is, the shift in resonance frequencies of chemically non-equivalent nuclei relative to the frequency of a standard substance—can be expressed directly in hertz, but in that case, the chemical shift will increase with increasing magnetic field strength (it will be larger on a 100 MHz spectrometer than on a 60 MHz one). To make this parameter independent of the instrument type, the δ value is introduced, measured in parts per million (ppm):

δ represents the relative frequency shift and is independent of field strength. However, the dependence on the reference compound remains; therefore, when citing a δ value, the reference compound used for its determination must be indicated. The τ scale is used somewhat less frequently

т = 10—δ (relative to TMS).       (2-34)

The energy of a proton spin transition is strongly influenced by local fields generated by other magnetic nuclei, particularly other protons. Such spin-spin coupling (interaction) leads to the splitting of proton NMR signals into several closely spaced lines. For instance, an ethyl group most commonly appears as four equally spaced lines corresponding to the СН₂ group and three lines from the СН₃ protons. Protons attached to the same carbon atom generally do not cause splitting of their own lines, but they do split the signals from protons attached to neighboring carbon atoms. These neighboring nuclei can reside in either of two spin states, which manifests as a readily measurable difference in the energy of the NMR transitions under consideration [157—161].

The spin-spin coupling constant J represents the distance in hertz between adjacent peaks of a multiplet. This value is independent of field strength and, consequently, of the spectrometer frequency. The signal from the 5'-methylene protons shown in Fig. 2-40 is split due to ¹Н-³¹Р coupling with J ≃ 7 Hz. The closer nuclei are to one another, the stronger the spin-spin interaction, although it can sometimes be observed across as many as five covalent bonds.

When interaction is suspected, double-resonance or spin-decoupling techniques are frequently employed. The sample is irradiated at the resonance frequency of one of the nuclei involved in spin-spin coupling, while the spectrum is recorded over the resonance frequency range of the other Nucleus in the pair. Under these conditions, the multiplet collapses into a singlet, which serves as Evidence of the mutual coupling between the two nuclei.

The spin-spin coupling constant between two protons attached to adjacent carbon atoms (or other atoms) depends on the torsion angle <р [equation (2-35)]1.

The Karplus equation (2-35) was derived theoretically, but constants A, B, and C are determined empirically [164]. In the approximate equations (2-36) through (2-38) [164] given below, the middle term of equation (2-35) is omitted:

Since the second term in these equations is 1 Hz or less, the single-term Karplus equation is frequently used.

NMR spectra make it possible to determine the torsional angle between the hydrogen atoms at the a-carbon of The amino acid residue and the adjacent nitrogen atom of the peptide bond (φ', but not φ)²; this helps establish whether the peptide group adopts a cis or trans configuration.

¹ The equation can also be written as follows: J ≈ A'+B'cos φ+C'cos 2φ (M. Karplus, JACS, 85, 2870–2871, 1963). Since cos 2φ = 2cos²φ–1, this is equivalent to equation (2-35), although the constants A', B', and C' turn out differently. Formulations using sin²φ are also encountered.

² If φ (Sec. B.3) = 180°, φ'≃+120.

The NMR method has been used to determine the conformation of small Peptides and polyesters [165–167], as well as the Structure of CARBOHYDRATES [168] and NUCLEOTIDES.

Very valuable information is provided by NMR spectra recorded in the presence of paramagnetic lanthanide ions, such as europium (Eu) and praseodymium (Pr) [169, 169a]. These ions induce substantial shifts in many NMR signals, which, given certain empirical correlations, helps determine the structure of a given compound.

Protein PMR spectra are extremely complex, yet significant progress has been achieved in their interpretation [170–175]. Fig. 2-41 shows the PMR spectra of the enzyme Ribonuclease obtained at 60 and 220 MHz. As can be readily seen, resolution is higher at the higher frequency. It is also noteworthy that following thermal Denaturation of the enzyme (up to 72.5°C), many signals in the spectrum recorded at 220 MHz become sharper. This indicates that As a result of denaturation, all identical side chains of the protein find themselves in an approximately equivalent environment. Furthermore, it has been demonstrated that the PMR spectra of proteins in a random coil conformation correspond well to spectra that can be predicted from the standard chemical shifts of individual Amino Acids [171], which is consistent with the Discussion above.

As a rule, PMR spectra are recorded in D₂O because the H₂O signal obscures a large portion of the spectrum. Nevertheless, when spectra are recorded in H₂O, weak peaks originating from the NH protons of imidazole side chains are visible at the far low-field end of the spectrum (δ > 10) (Fig. 2-42). Spectra recorded at three different pH values show that as the pH is raised from 4.4 to 8.9, the lowest-energy signal shifts from 12.9 to 11.1 ppm. At an intermediate pH value (5.31), the peak occupies an intermediate position quite close to its position at pH 4.4. By plotting chemical shift as a function of pH, an S-shaped titration curve is obtained (Chap. 4, Sec. C), from which the pKa values of dissociating groups can be estimated. For Histidine (which is considered a functional group of the enzyme's Active Site; see Chap. 7, Sec. D.2), the pKa thus obtained is 5.8.

FIG. 2-41. PMR spectrum of native ribonuclease recorded at 60 MHz (A) and 220 MHz (B), and of the denatured enzyme recorded at 220 MHz (C). The Enzyme Concentration in ²H2O was 11%, pD = 7.5 (A) and 6.8 (B). Standard: Thierry's salt (MacDonald C. C., Phillips W. D., J. Am. Chem. Soc., 89, 6333, 1967).

FIG. 2-42. PMR spectrum of ribonuclease A in 0.1 M NaCl solution at 22°. The three spectra correspond to three pH values: 4.4, 5.3, and 8.9. Each spectrum represents the average of several measurements; averaging was performed on a Varian C1024 computer (Griffen J. H. et al., Biochemistry, 12, 2097, 1973).

In proton NMR spectra (up to 360 MHz) of tRNA molecules, it was possible to identify 26 resonance lines corresponding to twenty protons involved in hydrogen bonding between Base Pairs (N—H—N, Fig. 2-24) and Hydrogen Bonds stabilizing tertiary structure (in particular, hydrogen bonds in Hoogsteen base triplets; Sec. F.6) [176].

C. ¹³C Magnetic Resonance (¹³C NMR)

The Use of ¹³C nuclei in NMR spectroscopy is rather limited due to the low natural Abundance of this isotope. Another difficulty stems from spin-spin coupling between ¹³C and ⁴H, as A large number of protons participate in it within organic compounds. Striking advances in ¹³C NMR spectroscopy were achieved through the use of broadband ¹³C—⁴H decoupling (noise decoupling). Since the natural abundance of ¹³C is only 1.1%, this isotope rarely occupies adjacent positions in a molecule. Thus, (¹³C—¹³C) coupling introduces no complications, and in noise-decoupled ¹³C NMR spectra, each carbon atom gives a single peak. However, even in this form, ¹³C NMR spectroscopy did not find widespread Practical Application until the advent of pulse spectrometers based on Fourier transformation [181]. In such instruments, the sample is irradiated with a strong radiofrequency pulse lasting a few microseconds. Each subsequent pulse arrives after 1–2 s, so that the equivalent of a conventional NMR spectrum is acquired within 1–2 s. The data are fed into a computer, where spectra accumulated over multiple scans spanning several minutes, hours, or even days are stored. This makes it possible to obtain high-precision ¹³C NMR spectra.

Chemical shifts in ¹³C NMR spectra frequently reach 100 ppm or more relative to TMS. The presence of multiple substituent groups attached to a carbon atom most often produces an additive effect [177].

FIG. 2-43. Noise-decoupled ¹³C—H ¹³C NMR spectrum. A. Enterobactin monomer [N-(2,3-dihydroxybenzoyl)-L-Serine]. B. Free enterobactin. C. Ga³⁺-enterobactin. Samples were dissolved in (CD₃)₂SO at ~50°. Chemical shift was measured relative to an internal standard, p-dioxane. The presented results are the average of 17,000 to 49,000 scans (Llinas M. et al., Biochemistry, 12, 3840, 1973).

Fig. 2-43 shows the ¹³C NMR spectrum of enterobactin, an iron-chelating compound synthesized by the bacterium E. coli. It is a cyclic trimer of 2,3-dihydroxy-N-benzoyl-L-serine (Fig. 2-44). Paper [167], from which Fig. 2-43 is taken, besides illustrating the application of NMR spectra for structure elucidation, is of great biochemical interest. Complexation with trivalent ions induces profound Conformational Changes in the enterobactin molecule. Comparison of free enterobactin with its gallium chelate reveals that the ψ angle remains virtually unchanged; φ changes from 60° to −150°, while the side-chain angle χ takes a value of 60° (instead of the previous 162°). In free enterobactin, the amide group is nearly planar, whereas in the chelate, the torsional angle ω shifts to −133°.

FIG. 2-44. Structure of enterobactin from E. coli capable of forming an iron complex, as well as the structure of its complex with the Ga³⁺ ion [167].

¹³C-NMR spectroscopy is also used to study Cell/13.html">Protein Structure. Fig. 2-45 shows a part of the ¹³C-NMR spectrum of cytochrome c (Chapter 10, Section B.5). Many resonance lines have been successfully identified; they belong to carbon atoms that are part of aromatic amino acids or the porphyrin ring. Note the striking Changes in the spectrum when iron is reduced from the ferric to the ferrous state.

FIG. 2-45. Part of the ¹³C-NMR spectrum of horse Heart cytochrome c, showing the resonance absorption bands of aromatic carbons and Arginine residues. The spectrum was recorded using broadband proton decoupling at pH 6.7; Temperature 41 °C, frequency 15.18 MHz. A. Ferricitrochrome c, 14.4 mM (average of 46,000 spectra recorded over 14 h). B. Ferrocytochrome c, 11.5 mM (average of 16,384 spectra) [178].

g. ³¹P Magnetic Resonance

The sensitivity of phosphorus NMR is only about 1/15 that of proton NMR, yet such NMR spectra are quite widely used in biochemistry. Of particular interest is the possibility of measuring intracellular pH from the chemical shift of the orthophosphate (inorganic phosphate, P_i) NMR signal [182]. ³¹P NMR makes it possible to monitor Chemical Reactions Involving phosphorylated substrates occurring inside Cells [183–185]. Recently, natural-abundance ¹⁵N NMR has begun to be used in The Study of proteins and other biochemical compounds [186].

d. Optical methods for Conformational Studies

Conformational changes in polymers can often be inferred from changes in the absorption spectra of aromatic amino acid side chains, as well as purine and pyrimidine bases (Fig. 2-28). Other valuable methods include Infrared Spectroscopy, Raman spectroscopy, fluorescence analysis, and CD spectroscopy; all of these methods are discussed in Chapter 13.

Questions and Problems

1. Draw the following hydrogen-bonded structures:

a. acetic acid dimer;

b. Tyrosine-carboxylate group in a protein molecule;

c. ionic phosphate-guanidinium pair in an enzyme-substrate complex;

d. GC and GU base pairs (recall that the GU pair does not fit the Watson-Crick scheme).

2. Draw the structures of the possible tautomeric forms of the cation formed by protonation of 9-methyladenine.

3. Fill in the following table:

4. Try to predict what conformation the peptide fragments listed below are most likely to adopt in a protein: an a-helix or a ß-Structure.

a. poly-β-leucine;

b. poly-L-valine;

c. Pro-Glu-Met-Val-Phe-Asp-Ile;

d. Pro-Glu-Ala-Leu-Phe-Ala-Ala.

5. Compare the water and ether Solubility of Amino acids and saturated Fatty acids, as well as their physical state. How do these differences relate to the structure of these compounds?

6. What functional groups are found in protein side chains? What is the structural and Functional Significance of (a) hydrophobic groups; (b) acidic and basic groups; (c) sulfhydryl groups?

7. Compile a table of pK_a values for the acidic and basic groups present in proteins. Which of these groups have the greatest effect on protein titration curves?

8. The pK_a values for the tripeptide L-Ala—L-His—L-Gln are as follows: 3.0 (a-COOH), 9.1 (a-NH₃), 6.7 (imidazole).

a. What is the isoelectric point of the tripeptide—that is, the pH value at which its net charge is zero? Hint: For amino acids, pI is generally the arithmetic mean of two pK_a values (see E. J. Cohn and J. T. Edsall, Proteins, Amino Acids and Peptides, pp. 90–93, Reinhold, New York, 1943).

b. Draw the structures of the ionic forms that the peptide adopts at pH 5 and pH 9. Calculate the percentage of each ionic form at these respective pH values. Hint: Definitions of pH and pK_a can be found in Chapter 4, which provides a detailed explanation of their significance and discusses buffer properties.

9. List the names of all isomeric tripeptides containing one residue each of tyrosine, Alanine, and valine.

10. a. Write the structural formula of glycyl-L-tryptophanyl-L-prolyl-L-seryl-L-Lysine.

b. What Amino acids can be obtained from this peptide via acid Hydrolysis?

c. Via alkaline hydrolysis?

d. Upon Treatment with nitrous acid followed by acid hydrolysis?

e. How will this peptide migrate in an electrolytic cell at pH 7.0—toward the cathode or the anode? What is its approximate isoelectric point?

f. If a solution of this peptide is adjusted to pH 7 and then titrated with sodium hydroxide in the presence of 10% formaldehyde, how many equivalents of base per mole of peptide are required to raise the pH to 10?

11. Compare the structural features and Properties of the following proteins: Silk Fibroin, α-keratin, Collagen, and bovine serum albumin.

12. How does Protein solubility typically change with variations in pH? Why?

13. A peptide is known to contain only L-lysine and L-Methionine. Titration data indicate that there are 3 free amino groups for every free carboxyl group in the peptide. Treatment of the peptide with nitrous acid (HNO₂) in a Van Slyke apparatus releases 1 mole of N₂ per amino group. Complete acid hydrolysis of the deaminated peptide followed by treatment of the hydrolyzate with HNO₂ releases the same amount of N₂ as obtained from the original peptide. Treatment of the original peptide with an excess of dinitrofluorobenzene yields a dinitrophenyl (DNP) peptide which, according to spectrophotometric analysis, contains three DNP groups per free carboxyl group. Complete hydrolysis of this DNP peptide reveals the following products: a colorless S-containing compound (A₁); a yellow S-containing compound (A₂); and a yellow S-free compound (A₃). Partial hydrolysis of the DNP peptide yields A₁, A₂, and A₃ along with four yellow compounds designated B₁, B₂, B₃, and B₄. Complete hydrolysis of B₁ yields A₁, A₂, and A₃; B₂ yields A₁ and A₂; B₃ yields A₁ and A₃; and B₄ yields only A₃. What is the most probable STRUCTURE OF THE original peptide?

14. A non-reducing disaccharide yields an octamethyl derivative upon treatment with dimethyl sulfate and alkali. Acid hydrolysis of this derivative produces 1 mole of 2,3,4,6-tetramethyl-D-glucose and 1 mole of 2,3,4,6-tetramethyl-D-galactose. The disaccharide is rapidly hydrolyzed by maltase or lactase (β-galactosidase).

Provide an appropriate descriptive name for this disaccharide and draw its Haworth projection formula.

15. An aldopentose (A) of the D-configuration upon oxidation with concentrated nitric acid yields 2,3,4-trihydroxypentanedioic acid (trihydroxyglutaric acid, B), which is optically inactive. Addition of HCN to A followed by hydrolysis, lactonization, and reduction yields two stereoisomeric aldohexoses (C and D). Oxidation of D yields the optically inactive 2,3,4,5-tetrahydroxyhexanedioic acid (saccharic acid, E). Write the structural formulas for compounds A through E.

16. What products are formed from the reaction of periodic acid with sorbitol?

17. Methylation and acid hydrolysis of 10.0 g of Glycogen yield 6.0 millimoles of 2,3-di-O-methylglucose.

a. What percentage of glucose residues in glycogen are substituted at the α-(1→6) position?

b. What is the average chain length (number of glucose residues per unbranched segment)?

c. How many millimoles of 2,3,6-tri-O-methylglucose could be formed from this polymer?

d. How many glucose residues does the polysaccharide contain if its molecular weight is 2·10⁶?

e. How many non-reducing ends are present per molecule—in other words, how many chains does the molecule contain?

18. What is the melting temperature (T_m) of a DNA preparation? How does T_m depend on nucleotide composition, and how can this be explained?

19. Draw a schematic diagram of the polynucleotide fragments of DNA and RNA molecules and indicate the sites of chain Cleavage upon the following treatments:

a) HCl, mild hydrolysis;

b) HCl, harsher hydrolysis;

c) NaOH, mild hydrolysis;

d) NaOH, harsher hydrolysis;

e) pancreatic ribonuclease;

f) pancreatic DNase;

g) Spleen DNase;

h) spleen phosphodiesterase;

i) snake venom phosphodiesterase;

j) micrococcal DNase.

20. Acid hydrolysis of a DNA sample yielded the following base composition (in %): adenine — 24.0; thymine — 33.0; guanine — 23.0; cytosine — 20.0. This is a rather unusual result. State two of its distinctive features and suggest possible explanations based on the structural properties of DNA.

21. The iodine number of a compound is defined as the number of grams of I₂ absorbed by one gram of fat upon saturation of C = C bonds (with The formation of diiodide derivatives). Note: halogenating Reagents such as iodine monochloride (ICl) and iodine monobromide (IBr) are typically used, yet the iodine number is expressed specifically in grams of I₂. The saponification number is the number of grams of KOH required for the complete saponification (hydrolysis and subsequent neutralization of fatty acids) of 1 g of fat. Given a pure triglyceride with a saponification number of 198 and an iodine number of 59.7,

a. What is its molecular weight?

b. What is the average chain length of its fatty acids?

c. How many double bonds are present in the triglyceride molecule?

22. Spermaceti (a wax extracted from the HEAD of the sperm whale), both in its physical properties and in its inertness toward reagents such as Br₂/CHCl₃ and KMnO₄, resembles high-molecular-weight Hydrocarbons; qualitative analysis indicates unequivocally only the presence of carbon and hydrogen in spermaceti. However, IR spectra reveal the presence of an ester bond, and quantitative analysis yields the empirical formula C₁₆H₃₂O. Titration of a sample of the solution obtained by prolonged stirring of the wax in an alcoholic KOH solution shows that there is one equivalent of base per 475 g of wax. Upon The addition of water and ether to the cooled mixture, it separates into two layers — aqueous and ethereal. Acidification of the aqueous layer yields a solid product A, whose neutralization equivalent is 260±5. Evaporation of the ether yields a solid, non-titratable substance B. Reduction of both spermaceti and substance A with lithium aluminum hydride yields substance B as the sole product.

What is the most probable structure of spermaceti?

23. a. Briefly explain two advantages of the isotope dilution method.

b. Based on the data provided below, calculate The amount of cAMP (cyclic AMP) present in 1 mL of human Skeletal Muscle cells. The cells were treated with ³²P-cAMP with a specific activity of 50 µCi·µmol^-1 for 0.2 h (during which all the cAMP was taken up by the cells). Subsequently, the cells were homogenized, and the soluble cAMP was isolated and purified. The specific activity of the isolated cAMP was found to be 10 µCi·µmol^-1. The total amount of added cAMP was 1.0·10^–7 moles per 1 mL of cells.

24. The figure in Appendix 2-B illustrates the high-performance Separation of soluble E. coli proteins. The proteins were radiolabeled by incorporation of ¹⁴C-amino acids.

a. How would you set up the labeling experiment?

b. What other isotopes could be used for protein labeling? What chemical forms of the isotope would you prefer? What limitations might arise here?

c. Which soluble components of sonicated E. coli cells might interfere with such a two-dimensional separation, and how can they be removed?

d. What other methods (besides the use of radioactive isotopes) can be employed to detect the localization of proteins?

e. If all soluble E. coli proteins could be detected, how many distinct proteins would you expect to see?

f. State at least two critical qualities a separation method must possess in order to be applicable to systems containing a very large number of proteins.

25. ³⁵S emits only ß-particles; the decay process is characterized by the following parameters: t¹/₂ = 86.7 days, ε_max = 0.168 MeV.

a. Write the equation for the radiochemical decay of ³⁵S.

b. Discuss the advantages and limitations associated with the use of 35S as an isotopic label.

26. Given a supply of agarose, Cyanogen bromide, 6-aminohexanoic acid, and other components required for Affinity Chromatography, outline the chemical reactions necessary to achieve the attachment of Tryptophan via its amino group.