Protein digestion enzymatic methods

A method of sample preparation proposed for MS/MS analysis of proteins in wine is by enzymatic hydrolysis of gel pieces from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using trypsin (Kwon, 2004). Hydrolysis occurs at the carboxyl side of lysine and arginine residues. Protocols of protein precipitation, enzymatic digestion, and sample preparation are reported in Table 10.5. 

Gravimetric methods are the methods of choice for the determination of dietary fibers (cf. 15.2.4.2). In the defatted sample, the digestible components (1,4-a-glucans, proteins) are enzymatically hydrolyzed (heat-stable a- amylase, glucoamylase, proteinase). After centrifugation. 

Most enzymatic techniques follow a similar protocol feed samples are incubated at optimal temperature and pH with an enzyme or combination of enzymes. After the incubation period, protein digestion is measured as the amoimt of amino acids and small peptides released from the protein divided by the amount of protein in the original sample. Determination of protein digestion requires that the method used wiU estimate amounts of these amino acids and peptides that are released. Various techniques differ in the enzyme or combination of enzymes used and the method selected for deterrnining the digested protein. 

Urea Enzymatic Dialysis Method. This method (16) uses 8 M urea [57-13-6] to gelatinize and facUitate removal of starch and promote extraction of the soluble fiber at mild (50°C) temperatures. EoUowing digestion with heat-stable a-amylase and protease, IDE is isolated by filtration or I DE is obtained after ethanol precipitation. Values for I DE are comparable to those obtained by the methods described eadier, and this method is less time-consuming than are the two AO AC-approved methods. Corrections for protein are required as in the AO AC methods. 

The theory and application of this fluorescence method have been discussed in detail by LePecq and others (3,8). The assay requires that there is sufficient ionic strength to minimize ionic binding (e.g., O.IM sodium chloride), that the pH is 4-10, that no heavy metals are present, that the fluorescence is not enhanced on binding to other excipients (e.g., proteins) and that at least portions of the nucleic acids are not complexed. These requirements can usually he met when dealing with recombinant products in some cases the samples must he manipulated to create the appropriate conditions. In the intercalative method of dye binding, proteins rarely interfere with the assay, and procedures have been developed to remove the few interferences they may cause (e.g., the use of heparin or enzymatic digestion of the protein 9). 

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