Bicarbonate protein digestion

Most pancreatic secretion takes place during the intestinal phase. The intestinal hormone secretin stimulates release of a large volume of pancreatic juice with a high concentration of bicarbonate ion. Secretin is released in response to acidic chyme in the duodenum (maximal release at pH < 3.0). The intestinal hormone cholecystokinin is released in response to the presence of the products of protein and lipid digestion. Cholecystokinin then stimulates the release of digestive enzymes from the pancreas. 

A study of by Palmer-Toy et al.,12 summarized in Table 19.1, provides further empirical evidence of the utility of techniques coupling heating with efficient protein extraction for the proteomic analysis of FFPE tissue. A specimen from a patient with chronic stenosing external otitis was divided in half and preserved as fresh-frozen tissue or FFPE. Ten micromolar sections of the FFPE tissue were vortexed in heptane to deparaffinize the tissue and were then co-extracted with methanol. The methanol layer was evaporated, and the protein residue was resuspended in 2% SDS/lOOmM ammonium bicarbon-ate/20mM dithiothreitol (DTT), pH 8.5 and heated at 70°C for lh. After tryptic digestion, 123 total confident proteins were identified in the FFPE tissue, compared to 94 proteins identified from the fresh-frozen tissue. Hwang et al. also reported up to a fivefold increase in protein extraction efficiency for samples extracted in a Tris-HCl/2% SDS/1% Triton X-100/1% deoxycholate solution at 94°C for 30 min versus samples extracted in 100 mM ammonium bicarbonate/30% acetonitrile at the same temperature.14... 

As the acidic stomach contents pass into the small intestine, the low pH triggers secretion of the hormone secretin into the blood. Secretin stimulates the pancreas to secrete bicarbonate into the small intestine to neutralize the gastric HC1, abruptly increasing the pH to about 7. (All pancreatic secretions pass into the small intestine through the pancreatic duct.) The digestion of proteins now continues in the small intestine. Arrival of amino acids in the upper part of the intestine (duodenum) causes release into the blood of the hormone... 

Alkylammonium acetate buffer systems have also attracted limited attention for the separation of tryptic digests of proteins on octadecyl silica 60). The ammonium formate or trifluoroacetate buffers have also been used in the reversed-phase HPLC separation of the tryptic, chymotryptic, and carboxypeptidase A digests 136) of a-melanotropin and its N,0-diacetylserine analog as well as for enzymatic digests of the adrenocorticotropic family 137). Ammonium bicarbonate eluent systems have proved useful in mapping studies due to the eluent s volatility and differ-... 

A brilliant success of modern biochemistry has been to show that all or almost all proteins of bovine pancreatic juice are enzymes (1). It may be said in a cursory way that this juice is a solution of enzymes in bicarbonate and that its composition is strictly utilitarian. Bicarbonate is present in order to neutralize hydrochloric acid coming from the stomach and to keep enzymes in solution. Enzymes are present for digesting alimentary products in the intestine. On the other hand, pancreatic juice seems to have been created for the protein chemist s delight, since its proteins are biologically active, relatively simple, and endowed with unusual properties. 

Ammonium bicarbonate stock (100 mMl was made and stored at 4° C. Stock solutions of all proteins were prepared in 0.1% TFA, 50% acetonitrile solutions and stored at -20°C until use. Aliquots of the stocks, containing 10 to 100 pg of protein, were dried in a centrifugal dryer prior to digestion. To 5 mg of N,N-diethylaminopropyl-bis-(3-hydroxypropyl) phosphine, 1 ml of 50% isopropyl alcohol/50 mM ammonium bicarbonate was added. The samples were then resuspended in 20 pi reagent solution per 10 pg of protein, heated at 80 C for 2 hours, and dried to remove volatile components. 

A stock solution of rabbit Cytochrome c was made up in water at a concentration of 4.3 mg/ml. The concentration of the stock solution was determined by quantitative amino acid analysis of a 5pJ aliquot that was hydrolyzed and analyzed as previously described (4). The stock solution was diluted 5-fold with 0. IM ammonium bicarbonate or 0. IM sodium borate (both at pH 8.2) and TPCK-Trypsin in lOmM HCl was added to give a 1 20 enzyme protein solution (w/w). Digestion was carried out at 37°C for 24 hours after which the enzymatic activity was terminated by heating at 100°C for 5 min. 

The enzymatic digestion of the purified proteins was carried out as described (13). The enzymes TPCK-treated pancreatic trypsin (EC 3.4.21.4 Type XIII) and TLCK-treated pancreatic chymotrypsin (EC 3.4.21 Type VII) were from Sigma Chemical Company. The amount of proteinase used was 2% of the concentration of the purified protein. The proteins were dissolved in 200 oL of 0.1 N ammonium bicarbonate buffer (pH=7.0), followed by addition of the enzyme dissolved in deionized water. The enzymatic digestion was carried out by incubating the samples for 18 hours at 37° and the reaction was stopped by addition of 10% trifluoroacetic acid solution. The proteins were lyophilized. 

The chemical phase of digestion also involves the hormone cholecystokiiun (CCK). Dietary fats and proteins elicit the release of CCK from cells of the intes tines. The versatility of this hormone in digestion is revealed, later in this chapter, via studies involving dogs, rats, and humans. These studies address the influence of CCK on the release of pancreatic enzymes, bile salts, and pancreatic bicarbonate into the lumen of the small intestines-... 

A common duct from the pancreas and gall bladder enters the duodenum. Duodenal pH is 6 to 6.5 due to the presence of bicarbonate that neutralizes the acidic ch5une emptied from the stomach. The pH is optimum for enzymatic digestion of protein and peptide food. Pancreatic juice containing enzymes is secreted into the duodenum from the bile duct. Trypsin, ch5unotrypsin, and carboxypeptidase are involved in the hydrolysis of proteins into amino acids. Amylase is involved in the digestion of carbohydrates. Pancreatic lipase secretion hydrolyzes fats into fatty acids... 

The digestion of proteins involves the salivary glands, the gastric mucosa, and the pancreas, all which introduce digestive enzymes into the gastrointestinal tract (Chaney, in Devlin, 1986, p. 11350). These actions are assisted by hydrochloric acid secreted in the gastrointestinal tract, and by the presence of sodium chloride and sodium bicarbonate. 

Hydrochloric acid (HCl), also called gastric acid, is secreted by parietal cells of the stomach into the stomach lumen, where the strong acidity denatures ingested proteins so they can be degraded by digestive enzymes. When the stomach contents are released into the lumen of the small intestine, gastric acid is neutralized by bicarbonate secreted from pancreatic cells and by cells in the intestinal lining. 

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