Protein digestion, enzymatic

FIGURE 2.16 pH versus enzymatic activity. The activity of enzymes is very sensitive to pH. The pH optimum of an enzyme is one of its most important characteristics. Pepsin is a protein-digesting enzyme active in the gastric fluid. Trypsin is also a proteolytic enzyme, but it acts in the more alkaline milieu of the small intestine. Lysozyme digests the cell walls of bacteria it is found in tears. 

If pronase digestion of the biotinylated protein is to be done, heat 100 pi of the sample at 56°C for 10 minutes, then add 10 pi of the pronase solution. Allow the sample to digest enzymatically at room temperature overnight. If no pronase digestion is desired, simply use the biotinylated protein solution prepared in step 3 without further treatment. 

A3. Albanese, A. A., Davis, V. I., Smetak, E. M., Lein, M., and Fisher, M., Amino acid analysis of the nondiffusible fraction of enzymatic protein digests and human urine. Arch. Biochem. Biophys. 20, 47-54 (1949). 

The scent mark of the saddle-back tamarin, Saguinusfuscicollis, contains water-soluble proteins. The major protein (66kDa) of the scent mark occurs in the urine, while another major protein (ISkDa) derives from gland secretions. If the proteins are removed by enzymatic degradation, tamarins still discriminate scent marks from different donor types. However, they are able to discriminate scents with intact proteins from those with the proteins digested. Consequently, the proteins are most likely an integral part of the scent image (Belcher et ah, 1990). 

Pedersen, B. and Eggum, B.O. 1983. Prediction of protein digestibility by an in vitro enzymatic pH-stat procedure. Z. Tierphysio. Tierernachr. Fultermittelkd. 49 265-277. 

While the determination of total lysine is rather straightforward, the determination of free or available lysine is more problematic. In this situation, the term free is meant to imply that the e-amine of the lysine side chain has not reacted with various components of the sample matrix (most commonly carbohydrates via Maillard browning). This is important because reaction of the e-amine can render lysine nutritionally unavailable and the nutritive value for that protein is then diminished if lysine is the limiting amino acid (which is often the situation with soy proteins). While enzymatic digestion in the human gut may not release the modified lysine in a nutritionally available state, often these lysine adducts are labile to the standard acid hydrolysis in 6N HC1 at 110°C. This results in total lysine values that overestimate the amount of nutritionally available lysine. 

Genetic variants and protein characterization Enzymatic digest a-S 1 -casein, /3-lactoglobulin, thionins, gliadins, and glutenins... 

It is commonly assumed that proteins undergo enzymatic digestion in the gastrointestinal tract and thus do not enter the bloodstream as physiologically... 

Enzyme-Hydrolyzed (Source) Protein Partially Hydrolyzed (Source) Protein (Source) Peptone Enzyme-Modified (Source) Protein Partial Enzymatic Digest of (Source) Protein Partial Acid Digest of (Source) Protein... 

Electrophoretic microfluidic chips feature a number of microreactor characteristics and have been used for conducting chemical and biochemical reactions in channels and microfabricated chambers, mixing reagents, microextraction and microdialysis, post- and preseparation derivatizations, etc. The most recent achievements are reviewed in Ref. 63 and other similar publications. These integrated microdevices perform PCR amplification, cell sorting, enzymatic assays, protein digestion, affinity-based assays, etc. In this section we describe such integrated microsystems and the most recent advances in this field. 

Fig. 24. Catalytic effect of an emulsion of benzaldehyde on the enzymatic condensation of a protein digest (Wasteneys and Borsook, 68).

For identification purposes, two basic approaches have been used. In the first, a purified protein is enzymatically digested, the product peptide mixture is analyzed and a mass spectral pattern is obtained. This pattern, called the MS fingerprint , is used to search in internet-available protein or DNA databases.17 Information about protein origin and an estimate of its MW are required in order to improve the chances of a correct match. The search algorithm then theoretically digests all appropriate proteins in the database with the specified enzyme, and matches the... 

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. 

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