Food Industrial Process

amount of each acid in the original fat and can be predicted by simple calculation, as already discussed under the 1,2,3-random distribution hypothesis. 5.6.3.2 Industrial Process Interesterification can be accomplished by heating the fat at relatively high temperatures (<200°C) for a long period. However, catalysts are commonly used that allow the reaction to be completed in a short time (e.g., 30 min) at temperatures as low as 50°C. Alkali metals and alkali metal alkylates are effective low-temperature catalysts, with sodium methoxide being the most popular. Approximately 0.1% catalyst is required. Higher concentrations may cause excessive losses of oil resulting from the formation of soap and methyl esters. The oil to be esterified must be extremely dry and low in free fatty acids, peroxides, and any other material that may react with sodium methoxide. Minutes after the catalyst is added the oil acquires a reddish brown color due to the formation of a complex between the sodium and the glycerides. This complex is believed to be the “true catalyst.” After esterification, the catalyst is inactivated by addition of water or acid and removed. 5.6.3.3 Mechanisms Two mechanisms for interesterification have been proposed [135]. Enolate Ion Formation According to this mechanism, an enolate ion (II), typical of the action of a base on an ester, is formed. The enolate ion reacts with another ester group in the triacylglycerol molecule to produce a b-keto ester (III), which in turn reacts further to give another b-keto ester (IV). Intermediate IV yields the intramolecularly esterified product V. The same mode of action applies to ester interchange between two or more triacylglycerol molecules. The intra-ester-ester interchange is believed to predominate in the initial stages of the reaction. Carbonyl Addition In this proposed mechanism, the alkylate ion adds onto a polarized ester carboxyl, producing a diglycerinate intermediate: Pag e 306 This intermediate reacts with another glyceride by abstracting a fatty acid, thus forming a new triacylglycerol and regenerating a diglycerinate for further reaction. Ester interchange between fully saturated S3 and unsaturated U3 molecules is shown here: 5.6.3.4 Directed Interesterification A random distribution, such as that produced by intersterification, is not always the most desirable. Interesterification can be directed away from randomness if the fat is maintained at a temperature below its melting point. This results in selective crystallization of the trisaturated glycerides, which has the effect of removing them from the reaction mixture and changing the fatty acid equilibrium in the liquid phase. Interesterification proceeds with the formation of more trisaturated glycerides than would have otherwise occurred. The newly formed trisaturated acylglycerols crystallize and precipitate, thus allowing the formation of still more trisaturated glycerides, and the process continues until most of the saturated fatty acids in the fat have precipitated. If the original fat is a liquid oil containing a substantial amount of saturated acids, it is possible, by this method, to convert the oil into a product with the consistency of shortening without resorting to hydrogenation or blending with a hard fat. The procedure is relatively slow due to the low temperature used, the time required for crystallization, and the tendency of the catalyst to become coated. A dispersion of liquid sodium-potassium alloy is commonly used to slough off the coating as it forms. Rearrangement can also be selectively controlled during interesterification by adding excess fatty acids and continuously distilling out the liberated acids that are highly volatile. This impoverishes the fat of its acids of lower molecular weight. The content of certain acids in a fat also can be reduced by using suitable solvents to extract appropriate acids during the interesterification process. Pag e 307 FIGURE 38 Effect of interesterification on solid content index. (From Ref. 135.) 5.6.3.5 Applications Interesterification finds its greatest application in the manufacture of shortenings. Lard, due to its high proportion of disaturated triacylglycerols with palmitic acid in the 2 position, forms relatively large and coarse crystals, even when rapidly solidified in commercial chilling machines. Shortenings made from natural lard possess a grainy consistency and exhibit poor performance in baking. Randomization of lard improves its plastic range and makes it a better shortening. Directed interesterification, however, produces a product with a higher solids content at high temperatures (Fig. 38) and thus extends its plastic range. Salad oil of a relatively low cloud point can be made from palm oil by fractionation after directed interesterification. The use of interesterification has also been applied to the production of high-stability margarine blends and hard butters that have highly desirable melting qualities. Using a column with countercurrent flow of dimethylformamide, a process has been developed to selectively reduce the content of linolenic acid in soybean oil by direct interesterification. 5.7 Role of Food Lipids in Flavor 5.7.1 Physical Effects Pure food lipids are nearly odorless. However, apart from their major contributions as precursors of flavor compounds, they modify the overall flavor of many foods through their effect on mouth feel (e.g., the richness of whole milk and the smooth or creamy nature of ice cream) and on the volatility and threshold value of the flavor components present. Pag e 308 5.7.2 Lipids as Flavor Precursors In the preceding discussion we have seen that lipids can undergo a variety of reactions and give rise to a multitude of intermediates and decomposition end products. These compounds vary widely in their chemical and physical properties. They also vary in their impact on flavor. Some are responsible for pleasant aromas, as is typical in fresh fruits and vegetables; others produce offensive odors and flavors, often causing major problems in the storage and processing of foods. A review by Forss [38] provides detailed information regarding the characteristics of volatile flavor compounds derived from lipids. In the following, some typical off flavors of lipid origin are discussed (see also Chap. 11). 5.7.2.1 Rancidity Hydrolytic rancidity results from the release of free fatty acids by lipolysis. Since only the shortchain fatty acids have unpleasant odors, the problem is typically encountered in milk and dairy products. The word rancid, however, is a general term that also commonly refers to off flavors resulting from lipid oxidation. The qualitative nature of oxidation flavors, however, varies significantly from product to product and even in the same food item. For example, the rancid off flavors developed from oxidation of fat in meat, walnuts, or butter are not the same. Fresh milk develops various off flavors described as rancid, cardboard, fishy, metallic, stale, or chalky; all are believed to be oxidative. Although considerable advances have been made in understanding the basic mechanisms of lipid oxidation and the flavor significance of many individual compounds, progress in correlating specific descriptions of rancid flavors with individual compounds or combinations of selected compounds has been slow. This is not surprising if one considers (a) the vast number of oxidative decomposition products identified so far (literally in the thousands), (b) their wide range of concentrations, volatiles, and flavor potencies, (c) the many possible interactions among the lipid decomposition products or between these products and other nonlipid food components, (d) the different food environments in which these flavor compounds reside, (e) the subjective nature of flavor description, (f) the complex influence of oxidative conditions on the multitude of possible reaction pathways and reaction products, and (g) the persisting suspicion that some trace but important flavor components may still escape detection by the most elegant analysis. Unfortunately, much more extensive research has been done on identification of volatiles in oxidized fats than on correlating these volatiles with flavor. With the continuous improvements of sensitivity and resolution in analytical instrumentation the detection of whole series of new compounds is made possible, and interpretations regarding flavor become more complex. Perhaps new approaches are needed to establish more precise relationships between volatile (and pertinent nonvolatile) lipid-derived components and specific oxidized flavors. An interesting technique, aimed at identifying the most important volatile oxidation products, is one in which a flavor dilution factor (FD factor) is determined by gas chromatographic analysis and effluent sniffing of a dilution series of the original aroma extract [138]. 5.7.2.2 Flavor Reversion This problem is unique to soybean oil and other linolenate-containing oils. The off flavor has been described as beany and grassy and usually develops at low peroxide values (about 5 mEq/kg). Several compounds have been suggested as responsible for or contributing to reversion flavors. Smouse and Chang [134] postulated that 2-n-pentylfuran, one of the compounds identified in reverted soybean oil and found to produce reversion-like flavors in other oils if added at 2 ppm levels, is formed from the autoxidation of linoleate by the following mechanism: Pag e 309 The cis- and trans-2-(1-pentenyl) furans were also reported as possible contributors to the reversion flavor. Presence of the 18:3 acid catalyzes this reaction. It should be pointed out that the hydroperoxide intermediate involved here is the 10-hydroperoxide, which is not typical of linoleate autoxidation. It can arise, however, from singlet oxygen oxidation. An alternative mechanism involving singlet oxygen and the formation of a hydroperoxy cyclic peroxide [40] is given: Other compounds reported by various workers to be significant in soybean reversion are 3-cis- and 3-trans-hexenals, phosphatides, and nonglyceride components. Pag e 310 5.7.2.3 Hardening Flavor This off flavor develops in hydrogenated soybean and marine oils during storage. Compounds reported to contribute to this defect include 6-cis- and 6-trans-nonenal, 2-trans-6-trans-octadecadienal, ketones, alcohols, and lactones. These compounds are presumed to arise from autoxidation of the isomeric dienes, known as isolinoleates, formed during the course of hydrogenation. 5.8 Physiological Effects of Lipids 5.8.1 Composition and Trends Intakes of lipids in the United States have been estimated in several national surveys. The 1985 and 1986 USDA Continuing Survey of Food Intakes of Individuals [140, 141] indicated that fat provided an average of 36–37% of total calories for men and women 19–50 years of age. A mean of 13% of total calories were consumed from saturated fatty acids, 14% from monounsaturated fatty acids, and 7% from polyunsaturated fatty acids. The daily intake of cholesterol averaged 280 mg for women and 439 mg for men. Dietary cholesterol came mainly from meat, eggs, and dairy products. Recent trends in the United States include greater consumption of vegetable fats, especially salad and cooking oils; decreased consumption of animal fats; increased consumption of seafood; and a shift from lard to shortenings, from butter to margarine, and from whole milk to skim and low-fat milks. Low-fat and fat-free desserts and snack-food items are becoming more and more available. 5.8.2 Nutritional Functions Fats are a concentrated source of energy. As compared with proteins and carbohydrates, they supply more than twice as many calories per gram, that is, 9 kcal/g (37.7 kJ/g). They confer a feeling of satiety and contribute to the palatability of food. The essential fatty acids linoleic and arachidonic, as well as fat-soluble vitamins A, D, E, and K, are obtained from the lipid fraction of the diet. Only recently have the biological functions of the n-3 fatty acids been recognized [85, 92]. Linoleic acid (18:2, n-6) and a-linolenic acid (18:3, n-3) are recognized as the parent fatty acids of the n-6 and the n-3 series, respectively. They undergo successive desaturations and elongations, giving rise to various metabolites with important biological functions (Fig. 39). A nutritional strategy to provide optimum amounts and ratios of the n-6 and n-3 fatty acid series is being vigorously pursued. 5.8.3 Safety of Lipids Exposed to Frying Conditions The various changes induced in fats by heating and oxidation, and the factors that influence such changes were discussed earlier in this chapter. The possibility that consumption of heated and/or oxidized fats may produce adverse effects has been a major concern and has stimulated extensive research. Several reports and reviews on this subject are available [4, 7, 20, 68, 111, 117, 121, 137]. Nutritional and toxicity studies have been conducted with animals fed heated or oxidized fats, used frying oils, or certain fractions or pure compounds typical of those identified in such oils. Unfortunately, some information reported from laboratory experiments has been misleading because oils were heated continuously at abusive temperatures in the absence of food, or because fractions of heated oils were fed to animals at unreasonably high levels. For example, cyclic monomers that form in thermally abused oils cause acute toxicity when Pag e 311 FIGURE 39 Metabolic pathways of the n-6 and n-3 fatty acids. administered to rats in large doses. Results from studies of these kinds are inapplicable to “real-life” frying situations. The current consensus is that toxic compounds can be generated in fat by abusive heating and/or oxidation, but that moderate ingestion of foods fried in high quality oils using recommended practices is unlikely to pose a significant hazard to health. 5.8.4 Safety of Hydrogenated Fats [135, 51, 65, 71, 86, 101, 119, 126, 146] As pointed out earlier, some double-bond migration occurs during hydrogenation of oils, and this results in the formation of positional and geometric isomers. Trans fatty acids constitute 20–40% of the total acids of some margarines and shortenings. Trans fatty acids are not biologically equivalent to their cis isomers, and precise knowledge of their physiological properties, metabolism, and long-term effects on health remains controversial. Pag e 312 5.8.5 Safety of Irradiated Food Irradiation results in partial destruction of fat-soluble vitamins, with tocopherol being particularly sensitive. Extensive research, conducted over several decades, indicates that radiation pasteurization of food, conducted under proper conditions, is safe and wholesome [144]. However, widespread acceptance of this novel process has been hampered by fears, misconceptions, politics, false association with nuclear reactors or weapons, and lack of education. In November 1980 the Joint FAO/WHO/IAEA Expert Committee on Wholesomeness of Irradiated Food concluded that “the irradiation of any food commodity up to an overall average dose of 10 kGy causes no toxicological hazard and hence toxicological testing of foods so treated is no longer required.” In 1986 the U.S. Food and Drug Administration approved irradiation of fresh food at up to a maximum dose of 1 kGy to inhibit growth and maturation. Also, irradiation of spices was approved to a maximum dose of 30 kGy for disinfestation. Irradiation of raw poultry was approved by the Food and Drug Administration to control foodborne pathogens in 1990, and by the U.S. Department of Agriculture to control salmonellosis in 1992. 5.8.6 Dietary Lipids, Coronary Heart Disease, and Cancer [18, 19, 52, 57, 61, 70, 73, 78–80, 87, 91, 92, 105, 112, 116, 140, 141] The type and amount of lipids in the diet is one of several factors believed to have an influence on the incidence of coronary heart disease. Lipid intake can, in some individuals, have a moderately adverse influence on serum cholesterol concentration and on the ratios of low- and high-density lipoproteins, both factors affecting the likelihood of coronary heart disease (CHD). It is generally agreed that persons in the U.S. population consume greater amounts of lipids, especially saturated lipids and cholesterol, than is desirable. The fatty acid composition of lipids has thus received considerable attention with respect to its influence on risk of CHD. Fatty acid attributes that have received particular attention are chain length, degree and position (n-3, n-6, n-9) of unsaturation, geometric configuration (cis, trans), and sn position. The effects of these attributes on human health are frequently the subject of considerable controversy, and the reader is referred to the many sources of information on this subject. With respect to cancer, epidemiologic and animal data support a relationship between total fat intake and risk [101]. Some studies on humans suggest that high intake of saturated fatty acids is associated with increased risk of colon, prostate, and breast cancers. Results of some animal studies indicated increased risk caused by injection of n-3 fatty acids, whereas others suggest that diets with a high content of n-3 fatty acids may decrease the risk of cancers of the mammary gland, intestine, and pancreas. 5.8.7 Concluding Thoughts on Dietary Lipids and Health The relationship between dietary fats and health and disease is one of the most active areas of biochemical, nutritional, and medical research. This field is advancing rapidly, and the consumer is bombarded daily with information and advice that are often contradictory and confusing. Exceedingly complex metabolic interrelationships are involved, and therefore it is quite understandable that experimental work in this field is complicated and difficult. I do not, however, wish to leave the impression that published material should be dismissed, or that all recommendations should be ignored. One cannot quarrel with dietary modifications directed at correcting dietary excesses or nutritional imbalances, or with appropriate adjustments in total caloric intake. A proper balance among mono-unsaturated, n-3, and n-6 unsaturated fatty acids is justified. Pag e 313 But it should be understood that the role of dietary fat in health and disease remains one of considerable controversy. Thus, the general public would be advised not to engage in dietary practices without proper consideration of potential adverse consequences. Probably the best advice for the majority of human subjects remains as it has been for years: a well-balanced diet that includes ample amounts of all essential nutrients, fresh fruits and vegetables, and regular exercise. 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Lipases: Their Structure, Biochemistry, and Application, Cambridge University Press, Cambridge.Proteins play a central role in biological systems. Although the information for evolution and biological organization of cells is contained in DNA, the chemical and biochemical processes that sustain the life of a cell/organism are performed exclusively by enzymes. Thousands of enzymes have been discovered. Each one of them catalyzes a highly specific biological reaction in cells. In addition to functioning as enzymes, proteins (such as collagen, keratin, elastin, etc.) also function as structural components of cells and complex organisms. The functional diversity of proteins essentially arises from their chemical makeup. Proteins are highly complex polymers, made up of 20 different amino acids. The constituents are linked via substituted amide bonds. Unlike the ether and phosphodiester bonds in polysaccharides and nucleic acids, the amide linkage in proteins is a partial double bond, which further underscores the structural complexity of protein polymers. The myriad of biological functions performed by proteins might not be possible but for the complexity in their composition, which gives rise to a multitude of three-dimensional structural forms with different biological functions. To signify their biological importance, these macromolecules were named proteins, derived from the Greek word proteois, which means of the first kind. At the elemental level, proteins contain 50–55% carbon, 6–7% hydrogen, 20–23% oxygen, 12–19% nitrogen, and 0.2–3.0% sulfur. Protein synthesis occurs in ribosomes. After the synthesis, some amino acid constituents are modified by cytoplasmic enzymes. This changes the elemental composition of some proteins. Proteins that are not enzymatically modified in cells are called homoproteins, and those that are modified or complexed with nonprotein components are called conjugated proteins or heteroproteins. The nonprotein components are often referred to as prosthetic groups. Examples of conjugated proteins include nucleoproteins (ribosomes), glycoproteins (ovalbumin, k-casein), phosphoproteins (a- and b-caseins, kinases, phosphory-lases), lipoproteins (proteins of egg yolk, several plasma proteins), and metalloproteins (hemoglobin, myoglobin, and several enzymes). Glyco- and phosphoproteins contain covalently linked carbohydrate and phosphate groups, respectively, whereas the other conjugated proteins are noncovalent complexes containing nucleic acids, lipids, or metal ions. These complexes can be dissociated under appropriate conditions. Proteins also can be classified according to their gross structural organization. Thus, globular proteins are those that exist in spherical or ellipsoidal shapes, resulting from folding of the polypeptide chain(s) on itself. On the other hand, fibrous proteins are rod-shaped molecules containing twisted linear polypeptide chains (e.g., tropomyosin, collagen, keratin, and elastin). Fibrous proteins also can be formed as a result of linear aggregation of small globular proteins, such as actin and fibrin. A majority of enzymes are globular proteins, and fibrous proteins invariably function as structural proteins. The various biological functions of proteins can be categorgized as enzyme catalysts, structural proteins, contractile proteins (myosin, actin, tubulin), hormones (insulin, growth hormone), transfer proteins (serum albumin, transferrin, hemoglobin), antibodies (immuno- Pag e 323 globulins), storage proteins (egg albumen, seed proteins), and protective proteins (toxins and allergens). Storage proteins are found mainly in eggs and plant seeds. These proteins act as sources of nitrogen and amino acids for germinating seeds and embryos. The protective proteins are a part of the defense mechanism for the survival of certain microorganisms and animals. All proteins are essentially made up of the same primary 20 amino acids; however, some proteins may not contain one or a few of the 20 amino acids. The differences in structure and function of these thousands of proteins arise from the sequence in which the amino acids are linked together via amide bonds. Literally, billions of proteins with unique properties can be synthesized by changing the amino acid sequence, the type and ratio of amino acids, and the chain length of polypeptides. All biologically produced proteins can be used as food proteins. However, for practical purposes, food proteins may be defined as those that are easily digestible, nontoxic, nutritionally adequate, functionally useable in food products, and available in abundance. Traditionally, milk, meats (including fish and poultry), eggs, cereals, legumes, and oilseeds have been the major sources of food proteins. However, because of the burgeoning world population, nontraditional sources of proteins for human nutrition need to be developed to meet the future demand. The suitability of such new protein sources for use in foods, however, depends on their cost and their ability to fulfill the normal role of protein ingredients in processed and home-cooked foods. The functional properties of proteins in foods are related to their structural and other physicochemical characteristics. A fundamental understanding of the physical, chemical, nutritional, and functional properties of proteins and the changes these properties undergo during processing is essential if the performance of proteins in foods is to be improved, and if new or less costly sources of proteins are to compete with traditional food proteins. 6.2 Physicochemical Properties of Amino Acids 6.2.1 General Properties 6.2.1.1 Structure and Classification a-Amino acids are the basic structural units of proteins. These amino acids consist of an a-carbon atom covalently attached to a hydrogen atom, an amino group, a carboxyl group, and a side-chain R group. (1) Natural proteins contain up to 20 different primary amino acids linked together via amide bonds. These amino acids differ only in the chemical nature of the side chain R group (Table 1). The physicochemical properties, such as net charge, solubility, chemical reactivity, and hydrogen bonding potential, of the amino acids are dependent on the chemical nature of the R group. Amino acids can be classified into several categories based on the degree of interaction of the side chains with water. Amino acids with aliphatic (Ala, Ile, Leu, Met, Pro, and Val) and aromatic side chains (Phe, Trp, and Tyr) are hydrophobic, and hence they exhibit limited solubility in water (Table 2). Polar (hydrophilic) amino acids are quite soluble in water, and they are either charged (Arg, Asp, Glu, His, and Lys) or uncharged (Ser, Thr, Asn, Gln, and Cys). The side chains of Arg and Lys contain guanidino and amino groups, respectively, and thus are positively charged (basic) at neutral pH. The imidazole group of His is basic in nature. However, Pag e 324 Pag e 325 at neutral pH its net charge is only slightly positive. The side chains of Asp and Glu acids contain a carboxyl group. These amino acids carry a net negative charge at neutral pH. Both the basic and acidic amino acids are strongly hydrophilic. The net charge of a protein at physiological conditions is dependent on the relative numbers of basic and acidic amino acids residues in the protein. TABLE 2 Solubilities of Amino Acids in W ater at 25°C Amino acid Solubility (g /L) Amino acid Solubility (g /L) Alanine 167.2 Leucine 21.7 Arg inine 855.6 Lysine 739.0 Asparag ine 28.5 Methionine 56.2 Aspartic acid 5.0 Phenylalanine 27.6 Cysteine — Proline 1620.0 Glutamine 7.2 (37°C) Serine 422.0 Glutamic acid 8.5 Threonine 13.2 Glycine 249.9 Tryptophan 13.6 Histidine — Tyrosine 0.4 Isoleucine 34.5 Valine 58.1 Pag e 326 The polarities of uncharged neutral amino acids fall between those of hydrophobic and charged amino acids. The polar nature of Ser and Thr is attributed to the hydroxyl group, which is able to hydrogen bond with water. Since Tyr also contains an ionizable phenolic group that ionizes at alkaline pH, it is also considered to be a polar amino acid. However, based on its solubility characteristics at neutral pH, it should be regarded as a hydrophobic amino acid. The amide group of Asn and Gln is able to interact with water through hydrogen bonding. Upon acid or alkaline hydrolysis, the amide group of Asn and Gln is converted to a carboxyl group with release of ammonia. A majority of the Cys residues in proteins exists as cystine, which is a Cys dimer created by oxidation of thiol groups to form a disulfide cross-link. Proline is a unique amino acid because it is the only imino acid in proteins. In proline, the propyl side chain is covalently linked to both the a-carbon atom and the a-amino group, forming a pyrrolidine ring structure. The amino acids listed in Table 1 have genetic codes. That is, each one of these amino acids has a specific t-RNA that translates the genetic information on m-RNA into an amino acid sequence during protein synthesis. Apart from the 20 primary amino acids listed in Table 1, several proteins also contain other types of amino acids that are derivatives of the primary amino acids. These derived amino acids are either cross-linked amino acids or simple derivatives of single amino acids. Proteins that contain derived amino acids are called conjugated proteins. Cystine, which is found in most proteins, is a good example of a cross-linked amino acid. Other cross-linked amino acids, such as desmosine, isodesmosine, and di- and trityrosine, are found in structural proteins such as elastin and resilin. Several simple derivatives of amino acids are found in several proteins. For example, 4- hydroxyproline and 5-hydroxylysine are found in collagen. These are the result of posttranslational modification during maturation of collagen fiber. Phosphoserine and phosphothreonine are found in several proteins, including caseins. NMethyllysine is found in myosin, and g-carboxyglutamate is found in several blood clotting factors and calcium binding proteins. (2) 6.2.1.2 Stereochemistry of Amino Acids With the exception of Gly, the a-carbon atom of all amino acids is asymmetric, meaning that four different groups are attached to it. Because of this asymmetric center, amino acids exhibit optical activity; that is, they rotate the plane of linearly polarized light. In addition to the asym- Pag e 327 metric a-carbon atom, the b-carbon atoms of Ile and Thr are also asymmetric, and thus both Ile and Thr can exist in four enantiomeric forms. Among the derived amino acids, hydroxyproline and hydroxylysine also contain two asymmetric carbon centers. All proteins found in nature contain only L-amino acids. Conventionally, the L- and D-enantiomers are represented as (3) This nomenclature is based on D- and L-glyceraldehyde configurations, and not on the actual direction of rotation of linearly polarized light. That is, the L-configuration does not refer to levorotation as in the case of L-glyceraldehyde. In fact most of the L-amino acids are dextrorotatory, not levorotatory. 6.2.1.3 Acid-Base Properties of Amino Acids Since amino acids contain a carboxyl group (acidic) and an amino group (basic), they behave both as acids and bases; that is, they are ampholytes. For example, Gly, the simplest of all amino acids, can exist in three different ionized states, depending on the pH of the solution. (4) At around neutral pH, both the a-amino and a-carboxyl groups are ionized, and the molecule is a dipolar ion or a zwitterion. The pH at which the dipolar ion is electrically neutral is called the isoelectric point (pI). When the zwitterion is titrated with an acid, the COO- group becomes protonated. The pH at which the concentrations of COO- and COOH are equal is known as pKa1 (i.e., negative logarithm of the dissociation constant Ka1). Similarly, when the zwitterion is titrated with a base, the group becomes deprotonated. As before, the pH at which = [NH2] is known as pKa2. A typical electrometric titration curve for a dipolar ion is shown in Figure 1. In addition to the a-amino and a-carboxyl groups, the side chains of Lys, Arg, His, Asp, Glu, Cys, and Tyr also contain ionizable groups. The pKa values of all the ionizable groups in amino acids are given in Table 3. The isoelectric points of amino acids can be estimated from their pKa1, pKa2, and pKa3 values, using the following expressions: For amino acids with no charged side chain, pI=(pKa1 + pKa2)/2. For acidic amino acids, pI = (pKa1 + pKa3)/2. For basic amino acids, pI = (pKa2 + pKa3)/2. The subscripts 1,2, and 3 refer to a-carboxyl, a-amino, and side-chain ionizable groups, respectively. In proteins, the a-COOH of one amino acid is coupled to the a-NH2 of the next amino acid through an amide bond; thus the only ionizable groups are the N-terminus amino group, the C-terminus carboxyl group, and ionizable groups on side chains. The pKa of these ionizable groups in proteins are different from those of free amino acids (Table 3). In protein, the pKa3 Pag e 328 FIGURE 1 Titration curve of a typical amino acid. TABLE 3 pKa and pl Values of Ionizable Groups in Free Amino Acids and Proteins at 25°C pKaR Amino acid pKa1 (a-COOH) pKa2 aAA Side chaina pl Alanine 2.34 9.69 — 6.00 Arg inine 2.17 9.04 12.48 > 12.00 10.76 Asparag ine 2.02 8.80 — 5.41 Aspartic acid 1.88 9.60 3.65 4.60 2.77 Cysteine 1.96 10.28 8.18 8.80 5.07 Glutamine 2.17 9.13 — 5.65 Glutamic acid 2.19 9.67 4.25 4.60 3.22 Glycine 2.34 9.60 5.98 Histidine 1.82 9.17 6.00 7.00 7.59 Isoleucine 2.36 9.68 — 6.02 Leucine 2.30 9.60 — 5.98 Lysine 2.18 8.95 10.53 10.20 9.74 Methionine 2.28 9.21 — 5.74 Phenylalanine 1.83 9.13 — 5.48 Proline 1.94 10.60 — 6.30 Serine 2.20 9.15 — 5.68 Threonine 2.21 9.15 — 5.68 Tryptophan 2.38 9.39 — 5.89 Tyrosine 2.20 9.11 10.07 9.60 5.66 Valine 2.32 9.62 — 5.96 a pKa values in proteins. Pag e 329 values of acidic side chains (Glu and Asp) are larger and those of basic side chains are lower than those of the corresponding free amino acids. The degree of ionization of a group at any given solution pH can be determined by using the Henderson-Hasselbach equation: (5) The net charge of a protein at a given pH can be estimated by determining the degree of ionization of individual ionizable groups using this equation, and then adding up the total number of negative and positive charges. 6.2.1.4 Hydrophobic Properties of Amino Acids One of the major factors affecting physicochemical properties, such as structure, solubility, fat-binding properties, etc., of proteins and peptides is the hydrophobicity of the constituent amino acid residues. Hydrophobicity can be defined as the excess free energy of a solute dissolved in water compared to that in an organic solvent under similar conditions. The most direct and simplest way to estimate relative hydrophobicities of amino acid side chains involves experimental determination of free energy changes for dissolution of amino acid side chains in water and in an organic solvent, such as ethanol. The chemical potential of an amino acid dissolved in water can be expressed by the equation (6) where is the standard chemical potential of the amino acid, g AA is the activity coefficient, CAA is concentration, T is absolute temperature, and R is the gas constant. Similarly, the chemical potential of an amino acid dissolved in ethanol can be expressed as (7) In saturated solutions, in which CAA,W and CAA,Et represent solubilities in water and ethanol, respectively, the chemical potentials of the amino acid in water and in ethanol are the same, that is, (8) Thus (9) The quantity which represents the difference between the chemical potentials arising from the interaction of the amino acid with ethanol and with water, can be defined as the free energy change (DGt,Et w) of transfer of the amino acid from ethanol to water. Thus, assuming that the ratio of activity coefficients is one, the preceding equation can be expressed as (10) where SAA,Et and SAA,w represent solubilities of the amino acid in ethanol and water, respectively. As is true of all other thermodynamic parameters, DGt is an additive function. That is, if a molecule has two groups, A and B, covalently attached, the DGt for transfer from one solvent to another solvent is the sum of the free energy changes for transfer of group A and group B. That is, Pag e 330 DGt,AB = DGt,A + DGt,B (11) The same logic can be applied to the transfer of an amino acid from water to ethanol. For example, Val can be considered as a derivative of Gly with an isopropyl side chain at the a-carbon atom. (12) The free energy change of transfer of valine from ethanol to water can then be considered as DGt,val = DGt,glycine + DGt,side chain (13) or DGt,side chain = DGt,val – DGt,glycine (14) In other words, the hydrophobicities of amino acid side chains can be determined by subtracting DGt,gly from DGt,AA. The hydrophobicity values of amino acid side chains obtained in this manner are given in Table 4. Amino acid side chains with large positive DGt values are hydrophobic; they would prefer to be in an organic phase rather than in an aqueous phase. In proteins, these residues tend to locate themselves in the protein interior. Amino acid residues with negative DGt values are hydrophilic, and these residues tend to locate themselves on the surface of protein molecules. It should be noted that although Lys is considered to be a hydrophilic amino acid residue, it has a positive DGt; this is due to the four -CH2- groups, which prefer to be in an organic environment. In fact, with proteins, part of this chain is usually buried with the e-amino group protruding at the protein surface. TABLE 4 Hydrophobicity of Amino Acid Side Chains at 25°C Amino acid DG t (ethanol water) (kJ/mol) Amino acid DG t (ethanol water) (kJ/mol) Alanine 2.09 Leucine 9.61 Arg inine — Lysine — Asparag ine 0 Methionine 5.43 Aspartic acid 2.09 Phenylalanine 10.45 Cysteine 4.18 Proline 10.87 Glutamine -0.42 Serine -1.25 Glutamic acid 2.09 Threonine 1.67 Glycine 0 Tryptophan 14.21 Histidine 2.09 Tyrosine 9.61 Isoleucine 12.54 Valine 6.27 Source: Refs. 80 and 104. Pag e 331 TABLE 5 Ultraviolet Absorbance and Fluorescence of Aromatic Amino Acids Amino acid lmax of absorbance (nm) Molar extinction coefficient (1 cm-1 mol1 ) lmax of fluorescence (nm) Phenylalanine 260 190 282ª Tryptophan 278 5500 348b Tyrosine 275 1340 304b ªExcitation at 260 nm. bExcitation at 280 nm. 6.2.1.5 Optical Properties of Amino Acids The aromatic amino acids Trp, Tyr, and Phe absorb light in the near-ultraviolet region (250–300 nm). In addition, Trp and Tyr also exhibit fluorescence in the ultraviolet region. The maximum wavelengths of absorption and fluorescence emission of the aromatic amino acids are given in Table 5. Since both absorption and fluorescence properties of these amino acids are influenced by the polarity of their environment, changes in their optical properties are often used as a means to monitor conformational changes in proteins. 6.2.2 Chemical Reactivity of Amino Acids The reactive groups, such as amino, carboxyl, sulfhydryl, phenolic, hydroxyl, thioether (Met), imidazole, and guanyl, in free amino acids and proteins are capable of undergoing chemical reactions that are similar to those that would occur if they were attached to other small organic molecules. Typical reactions for various side-chain groups are presented in Table 6. Several of these reactions can be used to alter the hydrophilic and hydrophobic properties and the functional properties of proteins and peptides. Some of these reactions also can be used to quantify amino acids and specific amino acid residues in proteins. For example, reaction of amino acids with ninhydrin, O-phthaldialdehyde, or fluorescamine is regularly used in the quantification of amino acids. Reaction with Ninhydrin The ninhydrin reaction is often used to quantify free amino acids. When an amino acid is reacted with an excess amount of ninhydrin, the following products are formed. For every mole of amino acid reacted with ninhydrin, one mole each of ammonia, aldehyde, CO2, and hydrindantin are formed as intermediates. The liberated ammonia subsequently reacts with one mole of ninhydrin and one mole of hydrindantin, forming a purple product known as Ruhemann’s purple, which has maximum absorbance at 570 nm. Proline and hydroxyproline give a yellow product that has a maximum absorbance at 440 nm. These color reactions are the bases for colorimetric determination of amino acids. (15) Pag e 332 Pag e 333 Pag e 334 Pag e 335 The ninhydrin reaction is usually used to help determine the amino acid composition of proteins. In this case, the protein is first acid hydrolyzed to the amino acid level. The freed amino acids are then separated and identified using ion exchange/hydrophobic chromatography. The column eluates are reacted with ninhydrin and quantified by measuring absorbance at 570 and 440 nm. Reaction with O-phthaldialdehyde Reaction of amino acids with O-phthaldialdehyde (1,2-benzene dicarbonal) in the presence of 2-mercaptoethanol yields a highly fluorescent derivative that has an excitation maximum at 380 nm and a fluorescence emission maximum at 450 nm. (16) Reaction with Fluorescamine Reaction of amino acids, peptides, and proteins containing primary amines with fluorescamine yields a highly fluorescent derivative with fluorescence emission maximum at 475 nm when excited at 390 nm. This method can be used to quantify amino acids as well as proteins and peptides. (17) 6.3 Protein Structure 6.3.1 Structural Hierarchy in Proteins Four levels of protein structure exist: primary, secondary, tertiary, and quaternary. 6.3.1.1 Primary Structure The primary structure of a protein refers to the linear sequence in which the constituent amino acids are covalently linked through amide bonds, also known as peptide bonds. The peptide linkage results from condensation of the a-carboxyl group of the ith amino acid and the a-amino group of the i + 1th amino acid with removal of a water molecule. In this linear sequence, all the amino acid residues are in the L-configuration. A protein with n amino acid residues Pag e 336 (18) contains n-1 peptide linkages. The terminus with the free a-amino group is known as the N-terminal, and that with the free aCOOH group is known as the C-terminal. By convention, N represents the beginning and C the end of the polypeptide chain. The chain length (n) and the sequence in which the n residues are linked determine the physicochemical, structural, and biological properties and functions of a protein. The amino acid sequence acts as the code for formation of secondary and tertiary structures, and ultimately determines the protein’s biological functionality. The molecular mass of proteins ranges from a few thousand daltons to over a million daltons. For example, titin, which is a single-chain protein found in muscle, has a molecular weight of over one million, whereas secretin has a molecular weight of about 2300. Many proteins have molecular masses in the range of 20,000 to 100,000 daltons. (19) The backbone of polypeptides can be depicted as repeating units of -N-C-C- or -C-C-N-. The expression -NH-CHR-COrelates to an amino acid residue, whereas -CHR-CO-NH- represents a peptide unit. Although the CO-NH bond is depicted as a single covalent bond, in reality it has a partial double bond character because of the resonance structure caused by delocalization of electrons. (20) This has several important structural implications in proteins. First, the resonance structure precludes protonation of the peptide N-H group. Second, because of the partial double bond character, the rotation of the CO-NH bond is restricted to a maximum of 6°, known as the w angle. Because of this restriction, each six-atom segment (-Ca-CO-NH-Ca-) of the peptide Pag e 337 backbone lies in a single plane. The polypeptide backbone, in essence, can be depicted as a series of -Ca-CO-NH-Ca- planes connected at the Ca atoms (Figure 2). Since peptide bonds constitute about one-third of the total covalent bonds of the backbone, their restricted rotational freedom drastically reduces backbone flexibility. Only the N-Ca and the Ca-C bonds have rotational freedoms, and these are termed f (phi) and y (psi) dihedral angles, respectively. These are also known as main-chain torsion angles. Third, delocalization of electrons also imparts a partial negative charge to the carbonyl oxygen atom and a partial positive charge to the hydrogen atom of the N-H group. Because of this, hydrogen bonding (dipole-dipole interaction) between the C=O and N-H groups of the peptide backbone is possible under appropriate conditions. Another consequence of the partial-bond nature of the peptide bond is that the four atoms attached to the peptide bond can exist either in cis or trans configuration. (21) However, almost all protein peptide bonds exist in the trans configuration. This is due to the fact that the trans configuration is thermodynamically more stable than the cis configuration. Since trans cis transformation increases the free energy of the peptide bond by 34.8 kJ/mol, isomerization of peptide bonds does not occur in proteins. One exception to this is peptide bonds FIGURE 2 Planar config uration of the atoms of peptide units of a polypeptide backbone. f and y are the dihedral (torsional) ang les of C a-N and Ca-C bonds. The side chains are located above or below the planes. Pag e 338 involving proline residues. Since the free energy change for trans cis transformation of peptide bonds involving proline residues is only about 7.8 kJ/mol, at high temperatures these peptide bonds sometimes do undergo trans-cis isomerization. Although the N-Ca and Ca-C bonds are truly single bonds, and thus the f and y dihedral angles can theoretically have 360° rotational freedom, in reality their rotational freedoms are restricted by steric hindrances from side chain atoms at the Ca atom. These restrictions further decrease flexibility of the polypeptide chain. 6.3.1.2 Secondary Structure Secondary structure refers to the periodic spatial arrangement of amino acid residues at certain segments of the polypeptide chain. The periodic structures arise when consecutive amino acid residues in a segment assume the same set of f and y torsion angles. The twist of the f and y angles is driven by near-neighbor or short-range noncovalent interactions between amino acid side chains, which leads to a decrease in local free energy. The aperiodic or random structure refers to those regions of the polypeptide chain where successive amino acid residues have different sets of f and y torsion angles. In general, two forms of periodic (regular) secondary structures are found in proteins. These are helical structures and extended sheet-like structures. The geometric characteristics of various regular structures found in proteins are given in Table 7. Helical Structures Protein helical structures are formed when the f and y angles of consecutive amino acid residues are twisted to a same set of values. By selecting different combinations of f and y angles, it is theoretically possible to create several types of helical structures with different geometries. However, in proteins, only three types of helical structures, namely, a-helix, 310-helix, and p-helix, are found (Fig. 3). Among the three helical structures, the a-helix is the major form found in proteins and it is the most stable. The pitch of this helix, that is, the axial length occupied per rotation, is 5.4 Å. Each helical rotation involves 3.6 amino acid residues, with each residue extending the axial length by 1.5 Å. The angle of rotation per residue is 100° (i.e., 360°/3.6). The amino acid side chains are oriented perpendicular to the axis of the helix. TABLE 7 Geometric Characteristics of Reg ular Secondary Structures in Proteins Structure f y n r h(Å) t a-Rig ht-handed helix -58° -47° 3.6 13 1.5 100° a-Left-handed helix +58° +47° 3.6 13 1.5 100° p-Helix -57°.06 -69°.6 4.4 16 1.15 81° 310-Helix -75°.5 -4°.5 3 10 2 120° Fully extended chain 180° 180° 2 — 3.63 180° b-Parallel sheet -119° +113° 2 — 3.25 — b-Antiparallel sheet -139° +135° 2 — 3.5 — Polyproline I (cis) -83° +158° Polyproline II (trans) -78° +149° Note:f and y represent dihedral ang les of the N-C a and Ca-C bonds, respectively; n is number of residues per turn; r, number of backbone atoms within a hydrog en bonded loop of helix; h, rise of helix per amino acid residue; t=360°/n, twist of helix per residue. Source: Ref. 39. Pag e 339 FIGURE 3 Spatial arrang ement of polypeptides in (a) a-helix, (b) 3 10-helix, and (c) p-helix forms. (From Ref. 8; courtesy of Academic Press.) a-Helices are stabilized by hydrogen bonding. In this structure, each backbone N-H group is hydrogen bonded to the C=O group of the fourth preceding residue. Thirteen backbone atoms are in this hydrogen-bonded loop; thus the a-helix is sometimes called the 3.613 helix. The hydrogen bonds are oriented parallel to the helix axis, and the N, H, and O atoms of the hydrogen bond lie almost in a straight line; that is, the hydrogen bond angle is almost zero. The hydrogen bond length, that is, the N-H … O distance, is about 2.9 Å, and the strength of this bond is about 18.8 kJ/mol. The a-helix can exist in either a right- or lefthanded orientation. However, the right-handed orientation is the more stable than the two. The details for a-helix formation are embedded as a binary code in the amino acid sequence [52]. The binary code is related to the arrangement of polar and nonpolar residues in the sequence. Polypeptide segments with repeating heptet sequences of -PN-P-P-N-N-P-, where P and N are polar and nonpolar residues, respectively, readily form a-helices in aqueous solutions. It is the binary code, and not the precise identities of the polar and nonpolar residues in the heptet sequence, that dictates a-helix formation. Slight variations in the binary code of the heptet are tolerated, provided other inter- or intramolecular interactions are favorable for a-helix formation. For example, tropomyosin, a muscle protein, exists entirely in a coiled-coil a-helical rod form. The repeating heptet sequence in this protein is -N-P-P-N-P-P-P-, which is slightly different from the preceding sequence. In spite of this variation, tropo-myosin exists entirely in the a-helix form because of other stabilizing interactions in the coiled-coil rod [69]. Most of the a-helical structures found in proteins are amphiphilic in nature; that is, one side of the helical surface is occupied by hydrophobic side chains, and the other side by hydrophilic residues. This is schematically shown in the form of a helical wheel in Figure 4. In most proteins, the nonpolar surface of the helix faces the protein interior, and is generally engaged in hydrophobic interactions with other nonpolar surfaces. Other types of helical structures found in proteins are the p-helix and the 310-helix. The p- and 310-helices are about 2.1 kJ/mol and 4.2 kJ/mol, respectively, less stable than the a-helix. These helices exist only as short segments involving a few amino acid residues, and they are not of major importance to the structures of most proteins. In proline residues, because of the ring structure formed by covalent attachment of the

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