Protein hydrolysates analysis

Determination of the amount and distribution of amino acids is required for a variety of bioanalytical questions. In bioprocess monitoring, the amount of certain free amino acids has to be monitored in order to avoid media depletion. In the case of a protein or a protein hydrolysate, analysis or verification of the amino acid composition may be a first step toward identification or characterization. The early work on amino acid analysis has been dominated by Stein and Moore, who in 1972 were awarded the Nobel prize... 

Nokihara K, Gerhardt J Development of an improved automated gas-chromatographic chiral analysis system application to non-natural amino acids and natural protein hydrolysates. Chirality 2001 13 431. 

Table 9.2 Derivatisation methods for the GC analysis of protein hydrolysates from paint samples...

Two other reagents used in HPLC are 9-fluorenyl methoxycarbonyl chloride (FMOC) and phenylisothiocyanate (PITC). 9-fluorenyl methoxycarbonyl chloride is becoming increasingly popular in protein chemistry research because it reacts with secondary amines and also offers rapid analysis of protein hydrolysates. 

The analysis time for protein hydrolysates is 85 min using standard columns. For extra high resolution a high-resolution lithium cation exchange column is recommended which achieves baseline separation of virtually all 40 amino acids (Fig. 1.3). 

Block, R. J. Amino acid analysis of protein hydrolysates, in A laboratory manual of analytical methods of protein chemistry". Vol. 2, 1 —57. Ed. P. Alexander u. R. J. Block, Pergamon Press, 1960. 

Peptide composition can be used to characterize foods and protein hydrolysates by applying multivariate analysis to the peptide pattern [175,182]. Table 19.6 reports basic information on some of the most recent HPLC applications for peptide analysis. 

Unfortunately, there are numerous ways to circumvent the analysis of total amino acids, such as by the addition of ammonium salts, inexpensive amino acids, peptides and protein hydrolysates. Several approaches have been made to verify the authenticity of the total amino acid values. Rockland and Underwood (10) developed a paper chromatographic technique for quantitatively estimating the individual amino acids. 

Akiyama, H., Sakata, K., Yoshioka, Y., Murata, Y., Ishihara, Y., Teshima, R., Sawada, J., Maitani, T. 2006. Profile analysis and immunoglobulin E reactivity of wheat protein hydrolysates. Int Arch Allergy Immunol 40 36 4-2. 

The amino acids in a protein hydrolysate can be conveniently separated for qualitative analysis by paper or thin-layer chromatography or by elec-... 

Fig. 7.6. Amino acid analysis of ninhydrin-amino acid complexes of a standard protein hydrolysate. 10 nmol of each amino acid was applied, except where stated otherwise.

HPLC has been used particularly in the analysis of amino acids and a large number of amino acid analysers are available commercially. For example, the use of a high-pressure, single-column amino acid analyser that can give a complete analysis of a protein hydrolysate in 42 min was described by Benson [254]. 

The analysis of cell culture media and supernatants, as well as non-standard protein hydrolysates such as collagens and glycoproteins, has created the demand for techniques that accurately quantify additional amino acids not normally found in hydrolyzed samples, Methods of amino acid analysis (AAA) based on precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) have previously been shown to quantify hydrolyzed samples with a high degree of accuracy (1,2). The AQC-based method has also been shown to derivatize effectively in the presence of salts and lipids (3). Taking into account the above strengths, the excellent stability of the derivatives, and the unique fluorescence properties that allow for direct injection of the reaction mixture without cleanup, the AQC methodology represents an ideal choice for the analysis of complex samples. 

Additional development work will make the economic picture even more favorable. It should be remembered that a few short years ago it took over a day to perform a semiautomated amino acid analysis on a protein hydrolysate, whereas it can now be performed in a highly automated way in less than 2 hours (Bl). We may even reach the point where it will be less expensive, faster, and more accurate to make a high-resolution analysis of a body fluid sample even when we are interested only in a few of the constituents. This has certainly been true for the somewhat analogous case of trace metal analysis by the newer spectrographic methods instead of the more specific, but now less acceptable, wet chemistry methods. 

H7. Heathcote, J. G., and Haworth, C., An improved technique for the analysis of amino acids and related compounds on thin layers of cellulose. II. The quantitative determination of amino acids in protein hydrolysates. J. Chromatogr. 43, 84-92 (1969). 

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... 

The process of separation and quantitation of amino acids has been automated. In one automated method, a single cation exchange resin column separates all the amino acids in the protein hydrolysate. The analyzer is capable of detecting as little as 1-2 nmol of an amino acid and a complete analysis can be obtained in about 4 hours. In newer procedures, the complete analysis can be performed in about Ihour and permit detection of as little as 1-2 nmol of an amino acid. Picomole amounts of amino acids can be determined when the separated amino acids are coupled to fluorescent reagents such as o-phthalaldehyde. Amino acid separation and quantitation can also be accomplished by reverse-phase high-pressure liquid chromatography of amino acid derivatives—a rapid and sensitive procedure. 

The rapid development of biotechnologies, as in biochemistry, requires the analysis of amino acids (proteins hydrolysates) photometric detectors can be used with the condition that prior to passage in the measuring cell a post-column reaction with ninhydrin is carried out (cf. Chapter 8). 

Of course, derivatization methods can also be used for the identification of organic acids. For example, volatile fatty acids in urine and plant protein hydrolysates were esterified with phenyldiazomethane and the resulting benzyl esters were separated by glass capillary GC [254]. Janos et al. [255] described a method for the analysis of dimedone derivatives of formaldehyde and other aliphatic aldehydes on capillary columns. Phenolic amines, 3-methoxycatecholamines, indoleamines and related amines can be determined as their N,0-ethyloxycarbonyl derivatives [256]. The reaction of dithiols and certain monothiols with phenylarsine oxide was used for derivatization prior to GC [257]. Destructive GC methods for the identification of microorganisms were described in refs. 258-261. 

An interesting fact, first noticed by Wright (1937, 1939) is that the infrared spectrum of the DL-form of an amino acid is usually markedly different from the spectrum of either the d- or the L-form of the same acid when each is examined in the solid state. Wright attributes this to compound formation between the d- and L-forms. Darmon et al. (1948) have confirmed this observation, which is extremely important if infrared methods are to be used for the analysis of mixtures of amino acids, e.g., the estimation of leucine iso-leucine ratios in protein hydrolysates. In this connection, Gore and Petersen (1949) have reported differences between the spectra of L-threonine and D-threonine when examined in the solid state. They point out that this might arise from a polarization effect in the spectrometer. 

The history of the discovery of amino acids is closely related to advances in analytical methods. Initially, quantitative and quaHtative analysis depended exclusively upon crystallization from protein hydrolysates. The quantitative precipitation of several basic amino acids including phosphotungstates, the separation of amino acid esters by vacuum distillation, and precipitation by sulfonic acid derivatives were developed successively during the last century. 

A second approach to Isomer separation by HPLC Is to use a non-optlcally active stationary phase and an optically active solvent. If the amino acids can Interact with both the stationary and mobile phases, but one of the Isomers Interacts more strongly with the mobile, optically active phase, separation of the Isomers Is possible (49). In 1979, several laboratories reported methods Involving the use of chiral mobile-phases containing zlnc(II) or copper (II) complexed to an L-amlno acid (51-53). A distinct advantage of these methods Is that they do not require derlvatlza-tlon of the sample prior to analysis. However, separation of a complete mixture of amino acids (such as that obtained from a protein hydrolysate) has not been reported. 

Peptide hydrolysis, complete hydrolysis of peptides and proteins for amino acid analysis or the production of individual amino acids from the peptide or protein hydrolysate. For this purpose, numerous chemical and enzymatic protocols are known, but none of these procedures alone is fully satisfactory. Besides hydrolysis with 6 M hydrochloric acid at 120 °C for 12 h, or with dilute alkali (2-4 M NaOH) at 100 °C for 4-8 h, mixtures of peptidases can also be used for complete peptide hydrolysis. Restricted or limited peptide hydrolysis ( peptide cleavage) is important for - sequence analysis and peptide mapping. 

Amino acids arising from protein hydrolysate This analysis is used often in food industry as well in basic biochemistry. It is important in determination of the structure of protein and in the assessment of the nutritional value of different proteins. Here the analysis of about 20 amino acids is sufficient. This type of analysis is relatively more difficult than that for a single amino acid. 

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