Hipótesis

Proteins are linear condensation products of various α-L-amino acids (a.a.) that differ in molecular weight, charge, and nonpolar character (Table 7.1), bound by trans-peptide linkages. They differ in number and distribution of various a.a. residues in the molecule. The chemical properties, size of the side chain, and sequence of the a.a. affect the conformation of the molecule, i.e., the secondary structure con-taining helical regions, β-pleated sheets, and β-turns; the tertiary structure or the spatial arrangement of the chain; and the quaternary structure — the assembly of several polypeptide chains.

The conformation affects the biological activity, nutritional value, and functional role of proteins as food components.

7.1.2 A^MINO A^CID C^OMPOSITION

In most proteins the proportion of each of the different a.a. residues, calculated as a percent of the total number of residues, ranges from 0 to about 30%. In extreme cases it may even reach 50%. Cereal proteins are generally very poor in Lys. Several major grains are deficient in Thr, Leu, Met, Val, and Trp. In most collagens there are no Cys and Trp residues, while the content of Gly, Pro, and Ala is 328, 118, and 104 residues/1000 residues, respectively. Paramyosin, abundant in the muscles of marine invertebrates, is rich in Glu (20–24%), Asp (12%), Arg (12%), and Lys (9%). The antifreeze fish serum glycoproteins contain several a.a. sequences of Thr-X₂-Y-X₇, where X is predominantly Ala and Y a polar residue. The antifreeze proteins of type I usually contain more than 60 mol% of Ala. Thr and Y, and in various antifreeze

proteins, other polar residues, form hydrogen bonds with ice crystals, inhibiting thereby the crystal growth. The β-caseins contain 14% of Pro residues. The molecule has a polar N-terminal region (1–43) with a charge of –12 and an apolar fragment, containing most of the Pro residues. Such sequence favors the temperature-, concen-tration-, and pH-dependent associations into threadlike polymers, stabilized mainly by hydrophobic adherences. Lysozyme, a basic protein of egg whites and other organisms, containing four -S-S- cross-links in a single polypeptide, retains its enzy-matic activity in acidic solution even after heating to 100°C. The Bowman–Birk trypsin inhibitor consists of 71 a.a. residues in one polypeptide chain with loops due to seven -S-S- bonds. The bovine serum albumin has 1 SH group and 17 intramolec-ular -S-S- bridges per molecule. Grain prolamines are very rich in Glu (up to 55%) and Pro (up to 30%). Among the 225 residues of phosvitin of egg yolk, there are 122 Ser, most of them phosphorylated (SerP). The typical sequences of phosvitin are: … Asp-(SerP)₆-Arg-Asp … and … His-Arg-(SerP)₆-Arg-His-Lys … .

Most food proteins, however, do not differ very much in a.a. composition.

Generally the contents of acidic residues are the highest, and those of His, Trp, and TABLE 7.1

Selected Properties of Proteinogenic Amino Acids

Amino Acid Abbreviation pK_a1 pK_a2 pK_R

Isoelectric Point pI

Side Chain Hydrophobicity

(Ethanol → Water) kJ/mol

Glycine Gly 2.34 9.60 — 6.0 0

Alanine Ala 2.34 9.69 — 6.0 3.1

Valine Val 2.32 9.62 — 6.0 7.0

Leucine Leu 2.36 9.60 — 6.0 10.1

Isoleucine Ile 3.26 9.68 — 6.0 12.4

Proline Pro 1.99 10.60 — 6.3 10.8

Phenylalanine Phe 1.83 9.13 — 5.5 11.1

Tyrosine Tyr 2.20 9.11 10.07 5.7 12.0

Tryptophan Trp 2.38 9.39 — 5.9 12.5

Serine Ser 2.21 9.15 13.60 5.7 0.2

Threonine Thr 2.15 9.12 13.60 5.6 1.8

Cysteine Cys 1.71 8.35 10.28 5.0 4.2

Methionine Met 2.28 9.21 — 5.7 5.4

Asparagine Asn 2.02 8.80 — 5.4 –0.04

Glutamine Gln 2.17 9.13 — 5.7 –0.4

Aspartic acid Asp 1.88 3.65 3.65 2.8 2.2

Glutamic acid Glu 2.19 4.25 4.24 3.2 2.3

Lysine Lys 2.20 8.90 10.56 9.6 6.2

Arginine Arg 2.18 9.09 12.48 10.8 3.1

Histidine His 1.80 5.99 6.00 7.5 2.1

Note: pK_a1 =–log [H⁺] [a.a.^+/–]/[a.a.⁺], pK_a2 = –log [H⁺] [a.a.–]/[a.a.⁺], pK_R = negative logarithm of dissociation constant of a.a. group in aqueous solution.

sulfur containing a.a. are the lowest. However, the number of residues capable of accepting a positive charge is often higher, especially in plant proteins, since about 50% of side chain carboxyl groups are amidated.

Many a.a. residues undergo posttranslational enzymatic amidation, hydroxyla-tion, oxidahydroxyla-tion, esterificahydroxyla-tion, glycosylahydroxyla-tion, methylahydroxyla-tion, or cross-linking. Some segments of the polypeptide chains may be removed (Figure 7.1). Modified residues in a given protein can be used for analytical purposes, e.g., hydroxyproline (ProOH), which is characteristic for collagens.

Posttranslational modifications may result in covalent attachment of various groups to the proteins. They may change the ionic character of the molecule, e.g., the phosphoric acid residues or saccharides. The residues involved in phosphoryla-tion and binding of saccharide moieties are Ser, Thr, LysOH, ProOH, His, Arg, and Lys. Among the proteins containing many phosphorylated a.a. residues is αS-casein.

FIGURE 7.1 Posttranslational modifications in collagen. (From Sikorski, Z.E., Proteins, in Chemical and Functional Properties of Food Components, Sikorski, Z.E., Ed., Technomic Publishing Co. Inc., PA, 1997. With permission.)

Polyrybosome

Hydroxylases

Procollagen Glycosyltransferases Endopeptidases Pro - α1

Pro - α1

Pro - α2

Tropocollagen

Collagen fibers

In the central region of αS1-casein SerP occurs in sequences: … SerP-Ala-Glu … ,

… SerP-Val-Glu … , … SerP-Glu-SerP … , and … SerP-Ile-SerP-SerP-SerP-Glu

… . Such distribution favors oligomer formation, due to hydrophobic interactions of the apolar fragments of the molecules, with the charged sequences exposed to the solvent. High contents of saccharides are characteristic for the allergenic glyco-proteins of soybeans (up to about 40%), several egg white glyco-proteins (up to 30%), albumins of cereal grains (up to 15%), whey immunoglobulins (up to 12%), and collagens of marine invertebrates (up to 10%). In κ-casein there is a hydrophobic N-terminal part (1–105) and a hydrophilic macropeptide (106–169), or a glycomac-ropeptide, with a saccharide moiety (0.5%) of N-acetylneuraminic acid, D-galactose, N-acetylgalactosamine, and D-mannose residues.

7.1.3 HYDROPHOBICITY

7.1.3.1 Average Hydrophobicity

The nonpolar character of an a.a. can be expressed by hydrophobicity, i.e., change of the free energy (F_ta) accompanying the transfer of the a.a. from a less polar solvent to water. Exposure of an a.a. with a large hydrocarbon side chain to the aqueous phase results in a corresponding decrease in entropy due to structuring of water around the chain. The hydrophobicity of the side chain of an a.a. is: F_tr = F_ta – F_tGly, where F_tGly is the hydrophobicity of Gly.

The average hydrophobicity (F_tav) of a protein can be estimated as F_tav = ΣFta/n, where n is the number of a.a. residues in the protein molecule.

It is not possible to predict the conformation and behavior of a protein in solution on the basis of F_tav. However, proteins of high F_tav yield bitter hydrolysates.

7.1.3.2 Surface Hydrophobicity

Most hydrophobic a.a. residues of a globular protein are burried in the interior of the native molecule. However, some of them form hydrophobic clefts or occur on the surface as individual residues or patches of residues.

Phe, Tyr, and Try residues in food proteins can be monitored by measuring the intrinsic fluorescence. They absorb ultraviolet radiation and emit fluorescence in the order:

The intensity of fluorescence and the wavelength of maximum intensity depend on the polarity of the environment. Thus a Try residue located in a nonpolar region emits fluorescence at 330–332 nm, and in complete exposure to water at 350–353 nm. Furthermore, electron withdrawing groups, like carboxyl, azo, and nitro groups, as well as different salt ions, have a quenching effect on fluorescence. However, measurements of intrinsic fluorescence and of fluorescence quenching have not found wide application in hydrophobicity determinations, because they are restricted to the effect of aromatic a.a. residues.

Phe 260 nm 283 nm

Tyr 275 nm 303 nm

Trp 283 nm 343 nm

The simplest and most commonly used are hydrophobic probes, based on the phenomenon that the quantum yield of the fluorescence of compounds containing some conjugated double bond systems is about 100 times higher in a nonpolar environment than in water. Thus hydrophobic groups can be monitored by aromatic or aliphatic probes and fluorescence measurements. Most often used are 1-anili-nonaphthalene-8-sulfonate (ANS) (Formula 7.1) and cis-parinaric acid (CPA) (For-mula 7.2). The binding of triacylglycerols or sodium dodecylsulfate may also be determined.

7.2 CONFORMATION