Chemical Modifications that Affect Nutritional and Functional Properties of Proteins T. Richardson and J. J. Kester Department of Food Science, University of Wisconsin-Madison. Madison, WI 53706 The foremost goals of the food protein chemist are to understand and to minimize reactions leading to the chemical dearadation of the essential amino acids in food proteins. ~ s s e n t i aaminoacids l are those that must be acquired in the diet of the consumer. The two principal essential i~minoacids that are often nutritionally limiting f i r humans in plant-based foods are lvsine and methionine. During processing and storage of f&d products, lysine, methionin;, nnd other amino acids can be modified chemically so that thry are no longer available to the individual. ~ e s t r u c t i o nof the limiting essential amino acids in food proteins should he avoided since this will limit the nutritional quality of the proteins. This remains true although the other 18 or so major amino acids in the protein may be in excess. The situation is analogousto that in which a reagent is the limiting component in a chemical reaction. Of secondary importance to the nutritional quality of a nrotein. hut crucial to the develonment of new food nroducts is an understanding of the physicorhemical fact*)rsthat affect the functionalitv of food oroleins. The food chemist uses the term "function&ty7' to indicate the functions proteins serve in food oroducts. For examnle. the uniaue structures of the caseins in milk that contribute to their ~hysicochemicalbehavior orovide the basis for manufacturing cheese. Gluten in wheat flour contain substantialquantities of the thiol-containing amino acid cysteine in addition to its oxidation product cystine. The physicochemical properties of the gluten proteins coupled with thiol-disulfide interchange reactions involving the cysteine-cystine residues in the proteins are important factors in dough formation during breadmaking. ~ d d i t i o n d ythe , excellent foaming properties of egg white proteins are important in the production of stable foams essential for the preparation of meringues and angel food cakes. From experience, we have catalogued large numbers of gross, empirical relationships hetween protein structures and their functions in foods that have proven useful over the years. Unfortunately, little is known ahout the molecular basis for nrotein functionalitv in foods. Thus. a fundamental understanding of how prhteins contribute to the characteris~ic orownies of a food is essential to dweluo food oroducts from underutilized sources of microbial, plant, and animal proteins. During the processing and storage of foods enzymatic, .ohvsical. - . and chemical factors mav lead to changes in nroteins. as well as other constituents, which can he detrimentai to the& nutritional and functional qualities. Sometimes these changes are intentional and other times unintentional. Often, a desired effect can he accompanied by adventitious, undesired consequences. In this context, it h a y become necessary to optimize the process and/or storage condition to accentuate desired effects and to minimbe ;he derrimrntal changes. We would like ta discuss some intentional and unintentional chemical modifications of amino acids and amino acid residues which can alter the nutritional quality of food proteins and which may result in change of their functional characteristics. We will briefly discuss a few representative chemical alterations of selected ammo aclds that can occur as a result of environmental effects (e.g., changes that can result from
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photooxidations, from extremes in pH, and from thermally induced effects). This will he followed hv a presentation on the use of intentional chemical derivatizitions of the various functional groups in the side chains of amino acid residue in proteins to study changes in their functionality. Finally, we want to mention how recombinant DNA techniques or genetic engineering might he useful in structure/function studies of food proteins and enzvmes. Information generated from these studiks could prove-useful in eventuilly designing more functional proteins and enzymes for food processing. Environmental Effects The environment of a food protein can exert profound changes in its nutritional quality and/or functional characteristics. Factors such as heat, light, extremes in pH, presence of oxygen, etc., can lead to many undesirable changes in food proteins. In addition, other food constituents including photosensitizer~and reducing sugars can catalyze chemical alterations of proteins or can enter into reactions with the functional groups of amino acid residues of proteins. Photooxidations Two of the potent photosensitizers in foods are riboflavin and chlorophyll. In general, two types of reactions can occur in the presence of a photosensitizer, light of an appropriate wavelength, and oxygen (I). In a Type I reaction, the photoexcited sensitizer in the triplet state (3Sens*) can univalently oxidize an amino acid or other suhstrate thereby initiatina a varietv of free radical reactions leading to destruction ofthe affected amino acid. %ns*
+ RH
-'
SensH + R -
02
Complex Reactions
other magents
In a Type I1 reaction, the photoexcited sensitizer can excite ground state (triplet state) oxygen resulting in the formation of highly reactive singlet-state oxygen.
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Electrophilic singlet oxygen readily reacts with double bonds eventually to form destructive peroxides.
-CH=CH-CH2-
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+ '0,
OOH
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Amino acids containine susceotible douhle honds. such as tryptophan, are subject to uxidat~vcdestruction ( 1 J. I t is often difficult tudistinauish hrtween Tsoe I and Tvoe I1 reactions based on the identity of products that are formed since both types of reactions can lead to identical products. Indeed, both Type I and Type I1 reactions can occur simultaneously. Factors that determine which type of reaction will be favored include: (1)the type of sensitizer and suhstrate, (2) the relative concentrations of oxygen, suhstrate (e.g., protein) and, (3) whether the phase in which the reactions occur is aqueous or non-aqueous (1).At the risk of oversimplification hydrophilic riboflavin tends to favor Type I reactions at atVolume 61 Number 4
April 1984
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In the utilization of proteins that have been denatured or of novel proteins, exposure of the proteins to extremes of pH
may be necessary. Denatured proteins resulting from thermal treatments or from treatment with organic solvents are usually insoluble in the pH range of 4-8. Thus, these proteins lose their functionality in a food. As a case in point, the utilization of fish protein concentrate (FPC), in which the fish muscle proteins were denatured during solvent extraction of accompanying lipids, was greatly hampered by the insolubility of the FPC. Often it is possible to dissolve denatured proteins at high nH. sav above 9. However. suhseauent reduction of the DHto values appruximnring those in food usually result in the preciuitation of the orotein. Nevertheless, the solubilization of a t high pH provides opportunities for denatured incorporatina these proteins into conventional or novel food prod&s. Fukhermore, certain undenatured, native proteins mav be soluble only under extremes in pH requiring addition of acid or base to prepare a solution of the protein. The eventual utilization of single-cell protein from microorganisms such as yeast will require removal of the nucleic acids that accompany single-cell protein concentrates. Purine bases in these nucleic acids are metabolized hy humans to relatively insoluble uric acid which is excreted in the urine. However, excessive purine bases in the human diet can lead to ahnormally high concentrations of uric acid in the blood (8).As the solubility of the uric acid in the blood is exceeded, it crystallizes in the joints and tissues of the individual leading to painful, gout-like symptoms. Single-cell proteins contain high levels of nucleic acids; and the treatment of the single-cell protein with acid or base to hydrolyze selectively and solubilize the nucleic acids associated with the single-cell proteins has been proposed to remove the offending purines from the proteins (9). Although treatment of food proteins a t highly basic pH values has been prooosed as Dart of numerous techno~o~iral processes, we shallsee that edposure of proteins to alkaline conditions can be detrimental to the nutritional quality of these proteins. In Fiaure 3 are summarized some of the reactions that amino acid residues of proteins can undergo at high pH (10). Racemization of amino acid residues in a orotein from the normal L-enantiomer to the ahnormal D-enantiomer is detrimental to the nutritional quality of the protein since Disomers of affected amino ac& are generally not utilized by humans (11). This racemization requires ionization of the protein a t the a-carbon of L-amino acid residues (Fig. 3). Subsequent reduction in pH and addition of a proton to the carbanion from the opposite side yields the D-aminoacid and leads to a mixture of D- and L-amino acid enantiomers. I t is crucial to realize that the racemization can occur under basic conditions in the intact proteins and that the racemization reactions affect essential and nonessential amino acid residues. Digestive, proteolytic enzymes and those enzymes involved in amino acid metabolism in the body usually react specifically with the L-enantiomer of an amino acid or its l v o.e ~ t i d(11). e s Thus. one can readilv understand that the .~ o .. nutritional quality of affected proteins will he greatly impaired.
Figure 1. Phatasensitized oxidation of methionine to yield melhionai via a T y p I reanion ( I).
(I).
mospheric oxygen concentrations, whereas lipophilic chlorophyll . . tends to favor Type I1 reactions. As shown in Figures l a n d 2, the essential amino acids methionine and tryptophan are susceptible to photooxidative destruction. In the presence of flavins such as riboflavin, methionine is readily converted to methional via a Type I reaction. By the same token, rihoflavin and other photosensitizer~catalyze the photooxidative conversion of tryptophan to N-formyl-kynurenine and other complex products ( I , 2). The pathways of these photooxidations are complicated and may involve intermediate formation of various peroxides. In addition to destroying essential amino acids, and probahlv of more immediate imnortance. nhotocatalvzed oxidat i & +of amino acids (and otier food ccktituentsjcan lead to the formation of noxious and ~otentiallvtoxic comoounds. Methional, generaced in reactions photocatalyzed by r i b flavin in milk. mav rontrihute to undesirahle flavor changes in milk exposed 6 fluorescent lights and to sunlight ( 3 1 . ~ formyl-kynurenine, a common oxidation product of tryptophan, has been implicated in the formation of certain types of bladder tumors (4). Oxidative destruction of aminoacids secondary to the primaw oxidation can readilv occur. Traces of rhlorophvll soluble in vegetable oils or othe; plant lipids are thought initiate oxidation of unsaturated fatty acids via a Type I1 reaction involving singlet oxygen (5). ~ e r o m ~ o s i t i oofnthe resultant lipid peroxides can lead to free-radical-catalyzeddestruction or amino acid residues of proteins in juxtaposition with the peroxidized lipids (6). In the univalent oxidation of food constituents ohotocatalvzed bv rihoflavin (F). fullv reduced riboflavin (FH?) eventukly is produced upon acceptance of two electrons. However, the reduced rihoflavin (FHd can undergo a univalent oxidation to yield superoxide anion radical (02') and the stable riboflavin semiquinone radical (FH.) (7). The superoxide anion radical is not very reactive hut can undergo subsequent reactions leading to the formation of strong oxidizing agents such as hydrogen peroxide and hydroxyl radical (7). These latter agents can initiate anumber of oxidative changes in food constituents. Although the preceding discussion focuses on photacatalyzed oxidative reactions, there are other prooxidant constituents present in foods including.enzymes and transition metal icms such as those of copper and iron that can also ratalyee oxidative destruction of tnod constituents (7). Oxidative reactions that occur in foods are prohably the most i~rr\.asivegroup of reactions leading to rejection of foods by the consumer because of resultant poor organoleptic qualities. In addition, as we have learned, the numerous oxidative reactions can also result in destruction of nutrients and in the possible formation of toxicogenic compounds. ~
High pH
326
Journal of Chemical Education
Figure 2. Photosensitized oxidation of tryptophan to yield N-formylkynurenine
As shown in Firmre 3, additional reactions oecur under basic conditions that Fan adversely alter nutritional quality oi'a protein (10.11). Heta-elimination reactionu involving serine and cysteini residues in proteins can result from has; treatments. Note that gwd leaving groups such as phosphate favor the formation of dehydroalanine which can undergo suhsequent cross-linkingreactions. Consequently, phosphoproteins, such as the caseins, which contain relatively high concentrations of serine phosphate ester residues are particularly susceptible to @-eliminationreactions. Nucleophiles readily add to the &carbon of dehydroalanine (Fii.3). Thus,lysinoalanine or lanthionine result from addition of the c-amino group of lysine or the thiol group of cysteine, respectively. These cross-linkine reactions render these essential amino acids unavailable-for digestion and utilization hy the enzymes in animals, including those in humans (10,Zl). Thermal Effects Depending upon the severity of the thermal treatments, the reactions of proteins and their amino acid components will vary and may he affected by other c ~ n s t i t u e n t ~the i n food. The response may range from simple physical denaturation of the protein, which would not involve disruption or formation of covalent bonds, to extensive pvrolvsis of amino acid residues creating toxic.mutagens (12): It
