Amino-Acid Composition

The amino acid composition, i.e. the quantity of each amino acid residue in the peptide chain, is a characteristic parameter for each protein. Often, an unknown protein can be identified by measuring the relative percentage of the amino acid residues and comparing these to a database. 

Measurement of the amino acid composition can be achieved in two steps. First, all the peptide bonds in the protein are cleaved by either acidic, basic or enzymatic hydrolysis. Subsequently, the free amino acids are separated from each other and quantified. 

In acid catalysed hydrolysis, polypeptides are treated with 6M HCl under vacuum to prevent oxidation of the sulfur containing amino acids by air. The reaction mixture is heated to 100-120 for about 24 h. Longer reaction times may be required for complete hydrolysis of the aliphatic amino acids Leu, Val and He. However, some other amino acid residues are degraded under these harsh conditions. The rate of degradation of certain residues such as Thr, Tyr and Ser can be measured and a correction factor can be included to account for their loss as a function of the hydrolysis time. Trp, however, is extensively degraded. Furthermore, acidic hydrolysis converts Asn to Asp and Gin to Glu, respectively NHJ is eliminated in both reactions. To determine the amount of these amino acids, the total content of Asx (Asn + Asp), Glx (Gin + Glu) and NH (Asn + Gin) must be measured and compared. Optimum hydrolysis conditions are difficult to 

Base-catalysed hydrolysis is even more problematic and only used in special cases. The polypeptides are reacted with 4 M NaOH at 100 for 4 to 8 h. Under these conditions Arg, Cys, Ser, and Thr are decomposed and other amino acids are de-aminated and racemised. This limits the use of basic hydrolysis to the determination of the Trp content. 

Enzymatic methods are most often used for determining the Asn, Gin and Trp content. Exo- and endopeptidases are enzymes that catalyse the hydrolysis of specific peptide bonds (section 7.6.1). As these enzymes exhibit a high specificity, it is essential to use a mixture of them to ensure hydrolysis of all peptide bonds. Enzymes should be used at low concentrations ( 1 %) as they are proteins themselves that can degrade and thus contaminate the reaction mixture. 

The amino acid composition of some )S-lg variants is shown in Table 4.4. It is rich in sulphur amino acids which give it a high biological value of 110. It contains 2 moles of cystine and 1 mole of cysteine per monomer of 18 kDa. The cysteine is especially important since it reacts, following heat denatura-tion, with the disulphide of K-casein and significantly affects rennet coagulation and the heat stability properties of milk it is also responsible for the cooked flavour of heated milk. Some /S-lgs, e.g. porcine, do not contain a free sulphydryl group. The isoionic point of bovine j3-lgs is c. pH 5.2. 

The amino acid composition of draglines was analyzed by a Hitachi High Speed Amino Acid Analyzer (835-50 type). Before the amino acid analysis, the silk sample was purified with water containing Triton XIOO. 

The amino-acid composition of whole thynnine was reported to change with the age of the fish, becoming much simpler in older individuals than in younger ones. Thus, whole thynnine from 5-year-old fish contained only seven kinds of amino acids (Arg, Val, Ala, Ser, Pro, Tyr and Glu), whereas that from 2 to 3-year-old fish had small or negligible amounts of seven more kinds (Thr, Lys, Gly, Asp, Phe, Leu and He) in addition to those found in the older fish (Waldschmidt-Leitz and Guter-MANN, 1966). 

The amino acid composition of homogeneous components of some protamines is described in Chaps. VII and VIII. 

Protamine Species (Name or origin of fish) Ex- pres- sion Gly Ala Val He Ser Thr Pro Arg 

Protamines of trout family Iridine Salmo irideus 

The determination of amino acid composition has been performed in hydrolysates of adrenodoxin in 6 N-HC1 at 110° C for various periods after the removal of iron from adrenodoxin by trichloroacetic acid precipitation or by a, a -dipyridyl treatment. The tentative amino acid composition is proposed to be as shown in Table 3. Adrenodoxin contains about 100 amino acid residues with an abundance of aspartic and glutamic acids, which may account for the strong affinity of adrenodoxin for 

DEAE-cellulose. A paucity of tyrosine residue may reflect the unusual low absorbance of adrenodoxin in the region of 280 mp. In general, the amino acid composition of adrenodoxin is similar to that of spinach ferredoxin as compared in Table 3, but it considerably differs from that of Cl. pasteurianum ferredoxin which lacks amino acid residues of methionine, tryptophan, histidine, arginine, and leucine. 

Our study on the distribution of electron transferring proteins in animal sources is still in progress. From present knowledge, adrenodoxin can be found in adrenal cortexes from pig, beef, and rat. Further, a similar protein was isolated from pig testis (see II-A-2), and it was also found in the ovary. However, brain, heart, liver, kidney, and pancreas appear to lack adrenodoxin-like protein. If this is correct, the proteins of the ferredoxin family are located solely in the glands which directly act in the biosynthesis of steroid hormones. It is of interest that adrenodoxin-like protein does not participate in the steroid hydroxylation involved in cholesterol and cholic acid biosyntheses. All of these reactions without the participation of adrenodoxin are similar to enzymes responsible for microsomal non-specific hydroxylation, which consist of the following sequence of electron transfer  

It should be emphasized here that the electron transfer system consisting of adrenodoxin differs from that of the microsomal hydroxylating system. 

Determination of the amino acid sequence of a purified protein begins with determination of the amino acid composition, i.e., the number of moles of each amino acid residue present per mole of protein, as follows. 

The protein is completely hydrolyzed by acid (6 N HCl, 24 hours or longer at 110°C, under vacuum or inert gas) to its constituent amino acids and the resultant hydrolysate is evaporated to dryness. The amino acid composition is determined on protein hydrolysates obtained after 24,48, and 72 hours of acid treatment. The content of amino acids with bulky aliphatic side chains such as isoleucine, leucine, and valine, which undergo slow hydrolysis, is calculated from an extrapolation of the hydrolysate data to infinite time. The content of hydroxyl-containing amino acids, which are slowly destroyed during hydrolysis, is obtained by a corresponding extrapolation to zero time. Since cysteine, cystine, and methionine residues are somewhat unstable to hydrolysis, these residues are oxidized to cysteic acid and methionine sulfone, respectively, with performic acid before quantitative analysis. Cysteine, or half-cystine, is quantitated as a derivative such as carboxymethyl cysteine after reduction and alkylation, a necessary prerequisite to subsequent sequence analysis. Tryptophan 

Amino acids are weakly attracted by hydrophobic and van der Waals interactions through their side chains. The strength of these attractions is determined by the adsorption characteristics of the resin and leads to different 

Separation and quantitation of amino acids by ion exchange chromatography. (Courtesy of Dr. N.S. Reimer.) 

None of the three licheniformins possesses a terminal-NHj group, according to the ffuorodinitrobenzene method. Therefore, they are probably cyclic peptides. The presence of at least one D-amino acid in the licheniformins A and B, was revealed by the use of D-amino acid oxidase (108). 

Cleavage of polypeptide chain into smaller manageable fragments, specifically and reproducibly. 

Purification of the peptide fragments and analysis of their amino acid compositions. 

Determination of amino acid sequences of the peptide fragments from the N-terminus and/or the C-terminus. 

Repeat steps 5-7, using a different cleavage procedure to generate a different and therefore overlapping set of peptide fragments. 

Construction of the overall amino acid sequence of the protein from the sequences in overlapping fragments. 

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Weber, P. L. Buck, D. R. Capillary Electrophoresis A Past and Simple Method for the Determination of the Amino Acid Composition of Proteins, /. Chem. Educ. 1994, 71, 609-612. This experiment describes a method for determining the amino acid composition of cyctochrome c and lysozyme. The proteins are hydrolyzed in acid, and an internal standard of a-aminoadipic acid is added. Derivatization with naphthalene-2,3-dicarboxaldehyde gives derivatives that absorb at 420 nm. Separation is by MEKC using a buffer solution of 50 mM SDS in 20 mM sodium borate. 

Legume forages, such as alfalfa or clover, are considered high quaHty, readily available protein sources. Animal sources of supplemental protein include meat and bone meal blood meal, 80% CP fish meal other marine products and hydroly2ed feathermeal, 85—90% CP. Additionally, synthetic amino acids are available commercially. Several sources (3,9,19) provide information about the protein or amino acid composition of feedstuffs. 

Table 3. Amino Acid Composition of Photosynthetically Grown Algae, g/16 g Nitrogen...

The nutrient sparing effect of antibiotics may result from reduction or elimination of bacteria competing for consumed and available nutrients. It is also recognized that certain bacteria synthesize vitamins (qv), amino acids (qv), or proteins that may be utilized by the host animal. Support of this mode of action is found in the observed nutritional interactions with subtherapeutic use of antibiotics in animal feeds. Protein concentration and digestibiHty, and amino acid composition of consumed proteins may all influence the magnitude of response to feeding antibiotics. Positive effects appear to be largest... 

The hide proteins differ in amino acid composition and physical stmcture. The principal amino acids (qv) of the hide proteins are hsted in Table 1. Of particular importance is the difference in the water solubiUty of the proteins. AH of the proteins are soluble in water when heated, and upon the addition of either strong acids or bases. Proteins (qv) are amphoteric, possessing both acid and base binding capacity. 

Amino acid compositions of the four oilseeds are given in Table 3 along with the amino acid (see Amino ACIDS) requirements for humans suggested by a Joint FAO/WHO/UNU Expert consultation. 

Proteins and Meals. Nutritional properties of the oilseed protein meals and their derived products are deterrnined by the amino acid compositions, content of biologically active proteins, and various nonprotein constituents found in the defatted meals. Phytic acid (3), present as salts in all four meals, is beheved to interfere with dietary absorption of minerals such as 2inc, calcium, and iron (67) (see Food toxicants, naturally occurring Mineral nutrients). ... 

The packing interactions between a helices and p strands are dominated by the residues Val (V), He (I), and Leu (L), which have branched hydrophobic side chains. This is reflected in the amino acid composition these three amino acids comprise approximately 40% of the residues of the P strands in parallel P sheets. The important role that these residues play in packing a helices against P sheets is particularly obvious in a/P-barrel structures, as shown in Table 4.1. 

Cleave the peptide into smaller fragments, separate these fragments, and deter-mine the amino acid composition of the fragments. 

FIGURE 5.5 (a) The hydroxy amino acids serine and threonine are slowly destroyed during the course of protein hydrolysis for amino acid composition analysis. Extrapolation of the data back to time zero allows an accurate estimation of the amonnt of these amino acids originally present in the protein sample, (b) Peptide bonds involving hydrophobic amino acid residues snch as valine and isolencine resist hydrolysis by HCl. With time, these amino acids are released and their free concentrations approach a limiting value that can be approximated with reliability. 

Amino Acid Composition of Some Selected Proteins ... 

ADH Horse liver alcohol dehydrogenase, an enzyme dimer of identical 374 amino acid polypeptide chains. The amino acid composition of ADH is reasonably representative of die norm for water-solnble proteins. 

The amino acid composition of each polypeptide chain is determined. 

Each polypeptide chain is cleaved into smaller fragments, and the amino acid composition and sequence of each fragment are determined. 

The standard protocol for analysis of the amino acid composition of proteins is discussed in Section 5.1. Results of such analyses allow the researcher to anticipate which methods of polypeptide fragmentation might be useful for the protein. 

With a knowledge of the methodology in hand, let s review the results of amino acid composition and sequence studies on proteins. Table 5.8 lists the relative frequencies of the amino acids in various proteins. It is very unusual for a globular protein to have an amino acid composition that deviates substantially from these values. Apparently, these abundances reflect a distribution of amino acid polarities that is optimal for protein stability in an aqueous milieu. Membrane proteins have relatively more hydrophobic and fewer ionic amino acids, a condition consistent with their location. Fibrous proteins may show compositions that are atypical with respect to these norms, indicating an underlying relationship between the composition and the structure of these proteins. 

Implicit in the presumption of folding pathways is the existence of intermediate, partially folded conformational states. The notion of intermediate states on the pathway to a tertiary structure raises the possibility that segments of a protein might independently adopt local and well-defined secondary structures (a-helices and /3-sheets). The tendency of a peptide segment to prefer a particular secondary structure depends in turn on its amino acid composition and sequence. 

Two polypeptides, A and B, have similar tertiary structures, but A normally exists as a monomer, whereas B exists as a tetramer, B4. What differences might be expected in the amino acid composition of A versus B ... 

The amino acid compositions and sequences of the /3-strands in porin proteins are novel. Polar and nonpolar residues alternate along the /3-strands, with polar residues facing the central pore or cavity of the barrel and nonpolar residues facing out from the barrel where they can interact with the hydrophobic lipid milieu of the membrane. The smallest diameter of the porin channel is about 5 A. Thus, a maltodextrin polymer (composed of two or more glucose units) must pass through the porin in an extended conformation (like a spaghetti strand). 

The method described above is applicable to a wide range of samples for the determination of amino acids in different matrices. For example, the amino acid composition and distribution of single enantiomers has been determined in protein hydrolysates, orange juice (Fig. 7-11), yogurt and seawater [23]. 

Most enzymes consist of several identical or different subunits. It is known that subunits of similar activity but different origin, which therefore differ in size and amino acid composition and sequence, replace each other in oligomeric enzymes, leading to the formation of enzyme chimeras of catalytic activity 47). The feasibility... 

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