Proteins, analysis hydrolysis

Additional factors that may affect the reliability of the chemical scoring methods lie with the inherent difficulties of amino acid analysis. The analytical procedure for amino acid analysis can affect both the recovery and reliable quantitation of amino acids. Proteins must first be hydrolyzed to amino acids before analysis. Hydrolysis methods affect the amino acid recovery. Cystine, methionine, tryptophan, threonine, serine, and tyrosinecan bedestroyed during hydrolysis. Valine and isoleucine are released slowly and may not be completely... 

Fluorimetric methods for the determination of amino acids are generally more sensitive than colorimetric methods. Fluorescamine (4-phenyl-spiro[furan-2(3H),l -phthalan]-3,3 -dione) and o-phthaldialdehyde (OPA) substances are used for protein analysis. Fluorescamine reacts with amino groups to form fluorophores that excite at 390 nm and emit at 475 nm (Weigele et al., 1972). Applications of fluorescamine include monitoring the hydrolysis of K-casein (Beeby, 1980 Pearce, 1979) and quantification of proteins, peptides, amino acids in extracts (Creamer et al., 1985). OPA produces fluorescence on reaction with 2-mercaptoethanol and primary amines, with strong absorption at 340 nm. Lemieux et al. (1990) claimed that this method was more accurate, convenient, and simple for estimating free amino acids than the TNBS, ninhydrin, or fluorescamine methods. 

Scheme 2.4 The products of hydrolysis of e-N-deoxyfructosyllysine under protein-analysis conditions...

Succinimidyl esters are an excellent first choice to activate amine-reactive probes, but their low solubility has led to the alternative use of sulphonyl chlorides (Figure 4.17). The resultant sulphonamide link is extremely stable, even more stable than an amide link, and will survive even complete protein hydrolysis - a property that can be exploited in protein analysis. The disadvantage of sulphonyl chlorides is that they are unstable in aqueous buffers under mildly alkaline conditions (typically the pH required for the reaction with aliphatic amine ). Hence extreme care must be taken to perform bioconjugations with sulphonyl chlorides at low temperatures (approx 4 °C). Alternatively, amine-reactive probes may be equipped with isothiocyanate traps , from which thiourea links are formed post-reaction with amine functional groups, or with aldehydes, from which Schiff sbase links can be formed with amine functional groups (Figure 4.17). 

In addition to being detected in routine amino acid analysis, several specific methods have been developed for determination of LAL in food or feed proteins. Since LAL is most frequently bound in the peptide, all analytical procedures start with an acid hydrolysis of the protein. The hydrolysis conditions most frequently used are 6N hydrochloric acid at llO C for 24 hrs. From the hydrolysis step on, a variety of chromatographic procedures have been attempted. 

The objective of cleaving a protein at specific sites is to generate peptides of suitable size for mass spectrometry analysis. Two methods are commonly used to cleave proteins (1) hydrolysis with a suitable chemical reagent and (2) digestion... 

In the past, one of the main problems of mass spectrometric analysis of proteins or other macromolecules was that their mass was outside the mass range of most mass spectrometers. For the analysis of larger molecules, such as proteins, a hydrolysis and the analysis of the resulting peptide mixture had to be carried out. With ESI, it is now possible to ionize large biomolecules without prior hydrolysis and analyze them by using MS. 

The method seems promising and useful also for protein analysis, as checked with ot-chymotrypsin. Hydrolysis was carried out with 2.7 N Ba(OH)2 at 120° C, which had been carefully boiled to remove dissolved air. An aliquot of the hydrolyzate was applied to the Sephadex column, which was connected to the automatic analyzer. The tryptophan peak was completely resolved from those of other amino acids. 

Methods have been developed for analysis or deterrnination of free amino acids in blood, food, and feedstocks (116). In proteins, the first step is hydrolysis, then separation if necessary, and finally, analysis of the amino acid mixture. 

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. 

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