Garrett R.H., Grisham C.M. - Biochemistry (1999)(2nd ed.)(en)

(From Suprenant, H., et al., 1980.

4.6 ● Separation and Analysis of Amino Acid Mixtures

101

Relative intensity

L-Alanine

COOH

+

H3N C H

CH3

Relative intensity

	L-Tyrosine	COOH
		+
		H3N C H
		CH2
		OH

10	9	8	7	6	5	4	3	2	1	0	10	9	8	7	6	5	4	3	2	1	0
					ppm											ppm

FIGURE 4.16 ● Proton NMR spectra of several amino acids. Zero on the chemical shift scale is defined by the resonance of tetramethylsilane (TMS). (Adapted from Aldrich Library

of NMR Spectra.)

teins. More sophisticated NMR measurements at very high magnetic fields are also used to determine the three-dimensional structures of peptides and even small proteins.

4.6 ● Separation and Analysis of Amino Acid Mixtures

Chromatographic Methods

The purification and analysis of individual amino acids from complex mixtures was once a very difficult process. Today, however, the biochemist has a wide variety of methods available for the separation and analysis of amino acids, or for that matter, any of the other biological molecules and macromolecules we

	14
	12		pK3
	10	pK2
pH	8
	6			carboxyl	α	ε	β	δ	γ
				C
	4
	2	pK1
	4700		4500	4300	1400	1200	1000	800	600
				Chemical shift in Hz (vs. TMS)

FIGURE 4.17 ● A plot of chemical shifts versus pH for the carbons of lysine. Changes in chemical shift are most pronounced for atoms near the titrating groups. Note the correspondence between the pKa values and the particular chemical shift changes. All chemical shifts are defined relative to tetramethylsilane (TMS).

Journal of Magnetic Resonance 40:231–243.)

102 Chapter 4 ● Amino Acids

encounter. All of these methods take advantage of the relative differences in the physical and chemical characteristics of amino acids, particularly ionization behavior and solubility characteristics. The methods important for amino acids include separations based on partition properties (the tendency to associate with one solvent or phase over another) and separations based on electrical charge. In all of the partition methods discussed here, the molecules of interest are allowed (or forced) to flow through a medium consisting of two phases—solid–liquid, liquid–liquid, or gas–liquid. In all of these methods, the molecules must show a preference for associating with one or the other phase. In this manner, the molecules partition, or distribute themselves, between the two phases in a manner based on their particular properties. The ratio of the concentrations of the amino acid (or other species) in the two phases is designated the partition coefficient.

In 1903, a separation technique based on repeated partitioning between phases was developed by Mikhail Tswett for the separation of plant pigments (carotenes and chlorophylls). Tswett, a Russian botanist, poured solutions of the pigments through columns of finely divided alumina and other solid media, allowing the pigments to partition between the liquid solvent and the solid support. Owing to the colorful nature of the pigments thus separated, Tswett called his technique chromatography. This term is now applied to a wide variety of separation methods, regardless of whether the products are colored or not. The success of all chromatography techniques depends on the repeated microscopic partitioning of a solute mixture between the available phases. The more frequently this partitioning can be made to occur within a given time span or over a given volume, the more efficient is the resulting separation. Chromatographic methods have advanced rapidly in recent years, due in part to the development of sophisticated new solid-phase materials. Methods important for amino acid separations include ion exchange chromatography, gas chromatography (GC), and high-performance liquid chromatography (HPLC).

Ion Exchange Chromatography

The separation of amino acids and other solutes is often achieved by means of ion exchange chromatography, in which the molecule of interest is exchanged for another ion onto and off of a charged solid support. In a typical procedure, solutes in a liquid phase, usually water, are passed through columns filled with a porous solid phase, usually a bed of synthetic resin particles, containing charged groups. Resins containing positive charges attract negatively charged solutes and are referred to as anion exchangers. Solid supports possessing negative charges attract positively charged species and are referred to as cation exchangers. Several typical cation and anion exchange resins with different types of charged groups are shown in Figure 4.18. The strength of the acidity or basicity of these groups and their number per unit volume of resin determine the type and strength of binding of an exchanger. Fully ionized acidic groups such as sulfonic acids result in an exchanger with a negative charge which binds cations very strongly. Weakly acidic or basic groups yield resins whose charge (and binding capacity) depends on the pH of the eluting solvent. The choice of the appropriate resin depends on the strength of binding desired. The bare charges on such solid phases must be counterbalanced by oppositely charged ions in solution (“counterions”). Washing a cation exchange resin, such as Dowex-50, which has strongly acidic phenyl-SO3 groups, with a NaCl solution results in the formation of the so-called sodium form of the resin (see Figure 4.19). When the mixture whose separation is desired is added to the column, the positively charged solute molecules displace the Na ions and bind to the

FIGURE 4.19

4.6 ● Separation and Analysis of Amino Acid Mixtures

103

(a) Cation Exchange Media

Structure

O

Strongly acidic, polystyrene resin (Dowex–50)

S

O–

O

O

Weakly acidic, carboxymethyl (CM) cellulose

O

CH2

C

O–

O

O–

Weakly acidic, chelating, polystyrene resin

CH2

N

CH2C

(Chelex–100)

CH2C

O–

O

FIGURE 4.18 ● Cation (a) and anion

(b) exchange resins commonly used for biochemical separations.

(b) Anion Exchange Media			Structure
									CH3
										+
	Strongly basic, polystyrene resin (Dowex–1)		CH2					N			CH3
								CH3
						CH2CH3
									+
	Weakly basic, diethylaminoethyl (DEAE)		OCH2CH2			N					H
	cellulose

CH2CH3

Cation exchange bead		Add mixture of	Add Na+ (NaCl)	Increase [Na+]	Increase [Na+]
before adding sample		Asp, Ser, Lys
Bead		Asp
		Lys
Na+ —SO	–	Ser
	3
(a)		(b)	(c) Asp, the least	(d) Serine is eluted next	(e) Lysine, the most
			positively charged		positively charged
			amino acid, is		amino acid, is
			eluted first		eluted last

● Operation of a cation exchange column, separating a mixture of Asp, Ser, and Lys. (a) The cation exchange resin in the beginning, Na form. (b) A mixture of Asp, Ser, and Lys is added to the column containing the resin. (c) A gradient of the eluting salt (e.g., NaCl) is added to the column. Asp, the least positively charged amino acid, is eluted first. (d) As the salt concentration increases, Ser is eluted. (e) As the salt concentration is increased further, Lys, the most positively charged of the three amino acids, is eluted last.

104 Chapter 4 ● Amino Acids

resin. A gradient of an appropriate salt is then applied to the column, and the solute molecules are competitively (and sequentially) displaced (eluted) from the column by the rising concentration of cations in the gradient, in an order that is inversely related to their affinities for the column. The separation of a mixture of amino acids on such a column is shown in Figures 4.19 and 4.20. Figure 4.21, taken from a now-classic 1958 paper by Stanford Moore, Darrel Spackman, and William Stein, shows a typical separation of the common amino acids. The events occurring in this separation are essentially those depicted in Figures 4.19 and 4.20. The amino acids are applied to the column at low pH (4.25), under which conditions the acidic amino acids (aspartate and glutamate, among others) are weakly bound and the basic amino acids, such as arginine and lysine, are tightly bound. Sodium citrate solutions, at two different concentrations and three different values of pH, are used to elute the amino acids gradually from the column.

A typical HPLC chromatogram using precolumn derivatization of amino acids with o-phthaldialdehyde (OPA) is shown in Figure 4.22. HPLC has rapidly become the chromatographic technique of choice for most modern biochemists. The very high resolution, excellent sensitivity, and high speed of this technique usually outweigh the disadvantage of relatively low capacity.

Sample containing several

amino acids

Elution column containing cationexchange resin beads

The elution process separates amino acids into discrete bands

Eluant emerging from the column is collected

Asp	Ser	Lys
Some fractions
do not contain
amino acids
Amino acid	concentration
	Elution time

FIGURE 4.20 ● The separation of amino acids on a cation exchange column.

FIGURE 4.22

(Adapted from Moore, S., Spackman, D., and Stein, W., 1958.

FIGURE 4.21

4.6 ● Separation and Analysis of Amino Acid Mixtures

105

pH 3.25	pH 4.25
0.2N Na citrate	0.2N Na citrate

	0.30												Valine		Methionine
	0.25				Threonine											Isoleucine
							Serine										Leucine
solute
	0.20			Aspartic				Glutamic			Glycine
					acid			acid
of	0.15											Alanine								Phenyl-
Amount
																		Tyrosine		alanine
	0.10
	0.05								Proline				Cystine
	0	25	50	75	100	125	150	175	200	225	250	275	300	325	350	375	400	425	450	475
										Volume of eluant

pH 5.28 0.35N Na citrate

	0.30
		Phenylalanine
	0.25
solute		Tyrosine
	0.20
			Lysine
of
	0.15
Amount				Histidine
	0.10			NH4+
					Arginine
	0.05
	0	25	50	75	100	125

Volume of eluant

● Chromatographic fractionation of a synthetic mixture of amino acids on ion exchange columns using Amberlite IR-120, a sulfonated polystyrene resin similar to Dowex-50. A second column with different buffer conditions is used to resolve the basic amino acids.

Chromatography of amino acids on sulfonated polystyrene resins. Analytical Chemistry

30 :1185–1190.)

								100
								B
					β -Ala Tyr		Ile	50
				Arg			Val
					Ala
				Thr		Met	Phe	0
			Gln	Gly
fluorescence		Asn						solvent%
	Asp		Ser			Trp	Lys
Relative		Glu
0	5	10		15	20	25	30	35
				Time (minutes)

● HPLC chromatogram of amino acids employing precolumn derivatization with OPA. Chromatography was carried out on an Ultrasphere ODS column using a complex tetrahydrofuran:methanol:0.05 M sodium acetate (pH 5.9) 1:19:80 to methanol:0.05 M sodium acetate (pH 5.9) 4:1 gradient at a flow rate of 1.7 mL/min.

(Adapted from Jones, B. N., Pääbo, S., and Stein, S., 1981. Amino acid analysis and enzymic sequence determination of peptides by an improved o-phthaldialdehyde precolumn labeling procedure. Journal of Liquid Chromatography 4:56–586.)

106 Chapter 4 ● Amino Acids

PROBLEMS

1.Without consulting chapter figures, draw Fischer projection formulas for glycine, aspartate, leucine, isoleucine, methionine, and threonine.

2.Without reference to the text, give the one-letter and threeletter abbreviations for asparagine, arginine, cysteine, lysine, proline, tyrosine, and tryptophan.

3.Write equations for the ionic dissociations of alanine, glutamate, histidine, lysine, and phenylalanine.

4.How is the pK a of the -NH3 group affected by the presence on an amino acid of the -COO ?

5.Draw an appropriate titration curve for aspartic acid, labeling the axes and indicating the equivalence points and the pKa values.

6.Calculate the concentrations of all ionic species in a 0.25 M solution of histidine at pH 2, pH 6.4, and pH 9.3.

7.Calculate the pH at which the -carboxyl group of glutamic acid is two-thirds dissociated.

8.Calculate the pH at which the -amino group of lysine is 20% dissociated.

9.Calculate the pH of a 0.3 M solution of (a) leucine hydrochloride, (b) sodium leucinate, and (c) isoelectric leucine.

10.Quantitative measurements of optical activity are usually expressed in terms of the specific rotation, [ ]D25, defined as

	[ ]D25		Measured rotation in degrees 100
			(Optical path in dm) (conc. in g mL)

FURTHER READING

Barker, R., 1971. Organic Chemistry of Biological Compounds, Chap. 4. Englewood Cliffs, NJ: Prentice-Hall.

Barrett, G. C., ed., 1985. Chemistry and Biochemistry of the Amino Acids. New York: Chapman and Hall.

Bovey, F. A., and Tiers, G. V. D., 1959. Proton N.S.R. spectroscopy. V. Studies of amino acids and peptides in trifluoroacetic acid. Journal of the American Chemical Society 81:2870–2878.

Cahn, R. S., 1964. An introduction to the sequence rule. Journal of Chemical Education 41:116–125.

Greenstein, J. P., and Winitz, M., 1961. Chemistry of the Amino Acids. New York: John Wiley & Sons.

Heiser, T., 1990. Amino acid chromatography: The “best” technique for student labs. Journal of Chemical Education 67:964–966.

Herod, D. W., and Menzel, E. R., 1982. Laser detection of latent fingerprints: Ninhydrin. Journal of Forensic Science 27:200–204.

Iizuka, E., and Yang, J. T., 1964. Optical rotatory dispersion of L-amino acids in acid solution. Biochemistry 3:1519–1524.

For any measurement of optical rotation, the wavelength of the light used and the temperature must both be specified. In this case, D refers to the “D line” of sodium at 589 nm and 25 refers to a measurement temperature of 25°C. Calculate the concentration of a solution of L-arginine that rotates the incident light by 0.35° in an optical path length of 1 dm (decimeter).

11.Absolute configurations of the amino acids are referenced to D- and L-glyceraldehyde on the basis of chemical transformations that can convert the molecule of interest to either of these reference isomeric structures. In such reactions, the stereochemical consequences for the asymmetric centers must be understood for each reaction step. Propose a sequence of reactions that would demonstrate that L( )-serine is stereochemically related to L( )- glyceraldehyde.

12.Describe the stereochemical aspects of the structure of cystine, the structure that is a disulfide-linked pair of cysteines.

13.Draw a simple mechanism for the reaction of a cysteine sulfhydryl group with iodoacetamide.

14.Describe the expected elution pattern for a mixture of aspartate, histidine, isoleucine, valine, and arginine on a column of Dowex-50.

15.Assign (R,S ) nomenclature to the threonine isomers of Figure

Kauffman, G. B., and Priebe, P. M., 1990. The Emil Fischer–William Ramsey friendship. Journal of Chemical Education 67:93–101.

Mabbott, G., 1990. Qualitative amino acid analysis of small peptides by GC/MS. Journal of Chemical Education 67:441–445.

Meister, A., 1965. Biochemistry of the Amino Acids, 2nd ed., Vol. 1. New York: Academic Press.

Moore, S., Spackman, D., and Stein, W. H., 1958. Chromatography of amino acids on sulfonated polystyrene resins. Analytical Chemistry 30:1185– 1190.

Roberts, G. C. K., and Jardetzky, O., 1970. Nuclear magnetic resonance spectroscopy of amino acids, peptides and proteins. Advances in Protein Chemistry 24:447–545.

Segel, I. H., 1976. Biochemical Calculations, 2nd ed. New York: John Wiley & Sons.

Suprenant, H. L., Sarneski, J. E., Key, R. R., Byrd, J. T., and Reilley, C. N., 1980. Carbon-13 NMR studies of amino acids: Chemical shifts, protonation shifts, microscopic protonation behavior. Journal of Magnetic Resonance 40:231–243.

C	hapter 5	. . . by small and simple things are great
		things brought to pass.
		ALMA 37.6, The Book of Mormon

Proteins: Their Biological

Functions and Primary

Structure

OUTLINE

5.1 ● Proteins Are Linear Polymers

of Amino Acids

5.2 ● Architecture of Protein Molecules

5.3 ● The Many Biological Functions

of Proteins

5.4 ● Some Proteins Have Chemical Groups

Other Than Amino Acids

5.5 ● Reactions of Peptides and Proteins

5.6 ● Purification of Protein Mixtures

5.7 ● The Primary Structure of a Protein:

Determining the Amino Acid Sequence

5.8 ● Nature of Amino Acid Sequences

5.9 ● Synthesis of Polypeptides

in the Laboratory

Although helices are uncommon in manmade architecture, they are a common structural theme in biological macromole- cules—proteins, nucleic acids, and even polysaccharides.

(Loretto Chapel, Santa Fe, NM/ © Sarbo)

Proteins are a diverse and abundant class of biomolecules, constituting more than 50% of the dry weight of cells. This diversity and abundance reflect the central role of proteins in virtually all aspects of cell structure and function. An extraordinary diversity of cellular activity is possible only because of the versatility inherent in proteins, each of which is specifically tailored to its biological role. The pattern by which each is tailored resides within the genetic information of cells, encoded in a specific sequence of nucleotide bases in DNA.

107

FIGURE 5.1

108 Chapter 5 ● Proteins: Their Biological Functions and Primary Structure

Each such segment of encoded information defines a gene, and expression of the gene leads to synthesis of the specific protein encoded by it, endowing the cell with the functions unique to that particular protein. Proteins are the agents of biological function; they are also the expressions of genetic information.

5.1 ● Proteins Are Linear Polymers of Amino Acids

Chemically, proteins are unbranched polymers of amino acids linked head to tail, from carboxyl group to amino group, through formation of covalent peptide bonds, a type of amide linkage (Figure 5.1).

Peptide bond formation results in the release of H2O. The peptide “backbone” of a protein consists of the repeated sequence ONOC OCO, where the N represents the amide nitrogen, the C is the -carbon atom of an amino acid in the polymer chain, and the final C is the carbonyl carbon of the amino acid, which in turn is linked to the amide N of the next amino acid down the line. The geometry of the peptide backbone is shown in Figure 5.2. Note that the carbonyl oxygen and the amide hydrogen are trans to each other in this figure. This conformation is favored energetically because it results in less steric hindrance between nonbonded atoms in neighboring amino acids. Because the -carbon atom of the amino acid is a chiral center (in all amino acids except glycine), the polypeptide chain is inherently asymmetric. Only L-amino acids are found in proteins.

The Peptide Bond Has Partial Double Bond Character

The peptide linkage is usually portrayed by a single bond between the carbonyl carbon and the amide nitrogen (Figure 5.3a). Therefore, in principle, rotation may occur about any covalent bond in the polypeptide backbone because all three kinds of bonds (NOC , C OCo, and the CoON peptide bond) are single bonds. In this representation, the C and N atoms of the peptide grouping are both in planar sp2 hybridization and the C and O atoms are linked by a bond, leaving the nitrogen with a lone pair of electrons in a 2p orbital. However, another resonance form for the peptide bond is feasible in which the C and N atoms participate in a bond, leaving a lone e pair on the oxygen (Figure 5.3b). This structure prevents free rotation about the CoON peptide bond because it becomes a double bond. The real nature of the peptide bond lies somewhere between these extremes; that is, it has partial double bond character, as represented by the intermediate form shown in Figure 5.3c.

Peptide bond resonance has several important consequences. First, it restricts free rotation around the peptide bond and leaves the peptide backbone with only two degrees of freedom per amino acid group: rotation around

R1

O

R2

O

R1

O

R2

O

+

+

+

+

H3N

CH

C

O–

H3N

CH

C

O–

H3N CH

C

N

CH

C

O–

H2O

H

Amino acid 1

Amino acid 2

Dipeptide

● Peptide formation is the creation of an amide bond between the carboxyl group of one amino acid and the amino group of another amino acid. R1 and R2 represent the R groups of two different amino acids.

FIGURE 5.3

FIGURE 5.2

O

			0.123nm		H
	121.1		123.2
	nm	C	0.133nm
					.145nm
	.152
					0
	0			121
		115.6
	Cα
				N
			119.5		118.2
R	H				0.1nm

H

R

Cα

5.1 ● Proteins Are Linear Polymers of Amino Acids

109

● The peptide bond is shown in its usual trans conformation of carbonyl O and amide H. The C atoms are the -carbons of two adjacent amino acids joined in peptide linkage. The dimensions and angles are the average values observed by crystallographic analysis of amino acids and small peptides. The peptide bond is the light gray bond between C and N. (Adapted from Ramachandran, G. N., et al., 1974.

Biochimica Biophysica Acta 359:298–302.)

the NOC bond and rotation around the C OCo bond.1 Second, the six atoms composing the peptide bond group tend to be coplanar, forming the so-called amide plane of the polypeptide backbone (Figure 5.4). Third, the CoON bond length is 0.133 nm, which is shorter than normal CON bond lengths (for example, the C ON bond of 0.145 nm) but longer than typical CPN bonds (0.125 nm). The peptide bond is estimated to have 40% double-bond character.

(a)

Cα		H	C	H
			α
			C	N
C	N
			O	α
				C
O		Cα
			A pure double bond between C
			and O would permit free rotation
			around the C	N bond.

(b)

Cα	H	Cα	H
+		C	N
C N		O	C
–O			α
	Cα

The other extreme would prohibit C N bond rotation but would place too great a charge on

O and N.

1The angle of rotation about the NOC bond is designated , phi, whereas the C OCo angle of rotation is designated , psi.

● The partial double bond character of the peptide bond. Resonance interactions among the carbon, oxygen, and nitrogen atoms of the peptide group can be represented by two resonance extremes (a and b). (a) The usual way the peptide atoms are drawn. (b) In an equally feasible form, the peptide bond is now a double bond; the amide N bears a positive charge and the carbonyl O has a negative charge. (c) The actual peptide bond is best described as a resonance hybrid of the forms in

(a) and (b). Significantly, all of the atoms associated with the peptide group are coplanar, rotation about CoON is restricted, and the peptide is distinctly polar. (Irving Geis)

Cα	H
C	N
O	Cα

(c) The true electron density is intermediate. The barrier to C N bond rotation of about 88 kJ/mol is enough to

keep the amide group planar.

110 Chapter 5 ● Proteins: Their Biological Functions and Primary Structure

FIGURE 5.4 ● The coplanar relationship of the atoms in the amide group is highlighted as

an imaginary shaded plane lying between two

O

successive -carbon atoms in the peptide backbone.

H R

C α -carbon

C

N

α -carbon	C	H
		H

R

The Polypeptide Backbone Is Relatively Polar

Peptide bond resonance also causes the peptide backbone to be relatively polar. As shown in Figure 5.3b, the amide nitrogen represents a protonated or positively charged form, and the carbonyl oxygen becomes a negatively charged atom in the double-bonded resonance state. In actuality, the hybrid state of the partially double-bonded peptide arrangement gives a net positive charge of 0.28 on the amide N and an equivalent net negative charge of 0.28 on the carbonyl O. The presence of these partial charges means that the peptide bond has a permanent dipole. Nevertheless, the peptide backbone is relatively unreactive chemically, and protons are gained or lost by the peptide groups only at extreme pH conditions.

Peptide Classification

Peptide is the name assigned to short polymers of amino acids. Peptides are classified by the number of amino acid units in the chain. Each unit is called an amino acid residue, the word residue denoting what is left after the release of H2O when an amino acid forms a peptide link upon joining the peptide chain. Dipeptides have two amino acid residues, tripeptides have three, tetrapeptides four, and so on. After about 12 residues, this terminology becomes cumbersome, so peptide chains of more than 12 and less than about 20 amino acid residues are usually referred to as oligopeptides, and, when the chain exceeds several dozen amino acids in length, the term polypeptide is used. The distinctions in this terminology are not precise.

Proteins Are Composed of One or More Polypeptide Chains

The terms polypeptide and protein are used interchangeably in discussing single polypeptide chains. The term protein broadly defines molecules composed of one or more polypeptide chains. Proteins having only one polypeptide chain are monomeric proteins. Proteins composed of more than one polypeptide chain are multimeric proteins. Multimeric proteins may contain only one kind of polypeptide, in which case they are homomultimeric, or they may be composed of several different kinds of polypeptide chains, in which instance they are heteromultimeric. Greek letters and subscripts are used to denote the polypeptide composition of multimeric proteins. Thus, an 2-type protein is a dimer of identical polypeptide subunits, or a homodimer. Hemoglobin (Table 5.1) consists of four polypeptides of two different kinds; it is an 2 2 heteromultimer.