Chemistry and Biology of Proteins - F. Haurowitz 1953

Hydrolytic Cleavage of Proteins
Fractionation and Isolation of Amino Acids and Peptides

Methods for the fractionation and isolation of all Amino acids have only recently begun to be successfully developed. Prior to this, it was only possible to separate individual groups of amino acids with varying degrees of precision. For instance, basic diamino acids—Arginine, Lysine, and Histidine—were precipitated with phosphotungstic acid and subsequently separated from one another by precipitation with silver sulfate at various pH levels [30].

The principal method for separating monoamino acid mixtures was Fischer's method, which is based on the fractional distillation of monoamino acid esters [31].

According to Fischer's Procedure, the protein is first hydrolyzed with Hydrochloric acid. Upon cooling the hydrolyzate, insoluble glutamic acid hydrochloride crystallizes out first. After removing this salt, the excess hydrochloric acid is distilled off under reduced pressure, and the remaining Amino acids are converted into esters by boiling with absolute ethanol while passing gaseous hydrogen chloride through the mixture. Upon cooling the solution, insoluble Glycine ester crystallizes out and is separated by centrifugation. The amino acid ester hydrochlorides remaining in the solution are neutralized with bases (e.g., sodium or potassium hydroxide, sodium carbonate, or sodium ethoxide). Fractional distillation of the amino acid ester mixture under reduced pressure yields the following fractions:

Fraction I (60°, 10 mm) — glycine, Alanine, leucine, Proline; Fraction II (100°, 10 mm) — valine, leucine, proline; Fraction III (100°, 0.5 mm) — leucine, proline; Fraction IV (180°, 0.5 mm) — phenylalanine, glutamic acid, aspartic acid, Serine.

Distillation entails substantial losses, rendering this approach unsuitable as a quantitative method for amino acid determination. It should be noted, however, that it was precisely this method that enabled researchers to establish the presence of peptide bonds in Proteins and determine their Amino Acid Composition. The difficulties associated with the distillation of amino acid esters prompted a search for alternative, simpler Separation techniques. Valuable results were achieved through the fractional extraction of amino acids from hydrolyzates using butanol [32].

Over the past few years, new methods for fractionating protein hydrolyzates have been developed. Among these, chromatographic analysis holds the greatest significance, being based on the differing adsorption capacities of amino acids On the surface of various adsorbents. For example, Diaminodicarboxylic Acids (aspartic and glutamic acids) are adsorbed by basic adsorbents such as aluminum oxide [33, 34] or Amberlite IR-4 [35]. Glutamic acid can subsequently be eluted from the adsorbent with dilute acetic acid, under which conditions aspartic acid remains in solution. The latter amino acid can, however, be readily eluted with a 0.5 N sodium hydroxide solution [33]. Basic diamino acids are adsorbed onto The surface of acidic adsorbents, such as acidic aluminum oxide [34], Amberlite IR 100-H, phenol-formaldehyde resin [36], or sulfonated phenolic ion-exchange resins [37]. During the elution of bases from adsorbents with acids, the resin is reactivated and can be reused for adsorption [38]. From the filtrate containing monoamino acids, Tyrosine and phenylalanine can be removed by exploiting their ability to adsorb onto animal charcoal [39]. Glycine, serine, Threonine, and Cysteine are adsorbed by acidic aluminum oxide following The addition of formaldehyde to the solution [40].

Synge [41, 42] utilized the partition principle—distributing amino acids between Water and water-immiscible (yet partially water-miscible) organic Solvents such as butanol, phenol, or collidine—to separate amino acids. Amino acid mixtures are adsorbed onto starch [43, 44], silica gel [45], or strips of filter paper [46, 47], after which water-saturated organic solvents are passed through the adsorbent. Separation occurs due to the differing rates at which the solvents extract the amino acids from the adsorbent. Further separation can be achieved by subsequently passing a second solvent through the adsorbent. When filter paper serves as the solid phase, passing two different solvents in mutually perpendicular directions distributes the amino acids across the paper as distinct "spots" after visualization with ninhydrin (Fig. 2). Since each spot corresponds to a single amino acid, this method makes it possible to separate all amino acids. The ratio of the migration rate of an amino acid to that of the pure solvent characterizes a specific Amino Acid and remains constant under identical conditions, denoted by the symbol R_f. The color intensity of the spot following the ninhydrin reaction can be measured spectrophotometrically by determining the light transmittance of the filter paper; this approach allows for the Quantitative determination of various amino acids [19].

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Fig. 2. Two-dimensional chromatogram of a wool hydrolyzate [29]. First solvent: collidine; second solvent: phenol. 300 µg of protein was used.

British authors allow the solvent to flow downward along suspended paper when developing chromatograms. Recently, a simplified modification of this method—so-called "ascending" Chromatography—has been proposed [48]. If radioactive Amino Acid Derivatives are employed in partition chromatography (e.g., p-iodophenylsulfonyl derivatives containing J¹³¹ or S³⁵), the quantities of individual amino acids can be determined from radiation intensity [149].

Partition chromatography is applied not only to the separation of amino acids, but also to their derivatives [45, 50] and Peptides [51, 52]. For instance, this method has yielded valuable results in separating the incomplete Hydrolysis products of Insulin [53] and gramicidin [54]. Partition chromatography demonstrated that norvaline and norleucine are not Structural components of the protein molecule [29]. It was revealed that "norleucine" is actually a mixture of d- and l-leucine [55]. The absence of norvaline in gelatin hydrolyzates was further confirmed by Raman spectroscopy [56]. Hydroxyglutamic acid must likewise be excluded from the list of Natural Amino Acids, as its presence in casein was not substantiated by chromatographic analysis [57]. It is possible that the so-called hydroxyglutamic acid fraction is simply a mixture of aspartic acid and other substances [58].

Presently, As a result of applying new investigative techniques, it has been established that protein molecules are composed of the following amino acids: glycine, alanine, valine, leucine, isoleucine, serine, threonine, cystine, cysteine, Methionine, aspartic acid, glutamic acid, arginine, lysine, hydroxylysine, phenylalanine, tyrosine, proline, hydroxyproline, histidine, and Tryptophan. Given that the nitrogen from these amino acids accounts for over 99% of the total nitrogen content in certain studied proteins, there is no reason to assume the presence of significant amounts of other, as-yet-unknown compounds in these proteins. However, these data cannot be generalized and applied to all other proteins. This is evidenced by the discovery of such compounds as aminoethanol in gramicidin hydrolyzates (see Chapter XV), as well as diiodotyrosine and dibromotyrosine in coral [59] and spongin [60] hydrolyzates.