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. Protein DigestionPresented by: GROUP 1 DAACoojacintoDe AsisGoSaunar 2.

2. Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues 3. Protein digestion The process of digestion is defined as the process by which macromolecules in food are broken down into their component small-molecule subunits.The macromolecules are the proteins or polypeptides themselves, and the subunits are the amino acids. The bonds holding the subunits together are peptide bonds 4. Protein Digestionis the degradation of proteins by cellular enzymes enzymes in a process called hydrolysis.Protein digestion takes place in two different phases:In the stomachIn the small intestineBoth of these phases of digestion are based on several types of enzymes that are called proteinases and proteases. 5. Proteases- endo- & exo- peptidases; enzymes that degrade proteins by hydrolysis of peptide bondsProteinases- endo-peptidases; proteases that show specificity for intact proteins 6. Peptide bond hydrolasePeptidase(=Protease)Endo-acting peptide bond hydrolaseEndopeptidase(=Proteinase) Exo-acting peptide bond hydrolaseExopeptidase 7. Mouth and Salivary Glands Chewing and crushing of protein rich foods and mix them with saliva to be swallowed 10. In the Stomach : start of protein DigestionGastrin -stimulates Parietal cells to secrete HCL; Chief cells of the gastric glands to secrete pepsinogenHydrochloric acid -Denatures protein structure -Activates pepsinogen(zymogen) to pepsinPepsin -hydrolyzes proteins to smaller polypeptides and some free amino acids. 11. In the intestine. The remainder of protein digestion occurs in the small intestine as the result of the action of enzymes such as trypsin (secreted by the pancreas) and peptidases (located in the cells that line the small intestine). 12. In the Small Intestine : enzymesSecretin - stimulates the pancreas to secrete bicarbonate into the small intestine to neutralize the gastric HClCholecystokinin-stimulates secretion of several pancreatic enzyme with activity optima pH 7 to 8. 13. In the Small IntestineCont....Trypsin - activates chymotrypsinogenchymotrypsinprocarboxypeptidasescarboxypeptidasesproelastaseelastase further hydrolyze the peptides that were produced by pepsin in the stomach specifically the peptide bonds next to lysine and arginineChymotrypsin -cleaves peptide bonds next to phe, tyr, trp,met, asp, and his 14. Cont....Carboxypeptidase A & B -cleave amino acids from the acid (carboxyl) ends of polypeptidesElastase and collagenase -cleave polypeptides into smaller polypeptides and tripeptides 15. Cont....Intestinal tripeptidases -Cleave tripeptides to dipeptides and amino acidsIntestinal dipeptidases -cleave dipeptides to amino acidsIntestinal aminopeptidases -cleave amino acids from the amino ends of small polypeptides(oligopeptides) 16. Amino acids absorbedFree amino acid small intestine(villi)Liverblood circulation 17. Materials:Commercial pepsinTrypsinConcentrated HClNaOHCuSO4 solution0.5% Na2CO3Hard Boiled Egg (Protein) albumin 18. Biuret Testa chemical test used to detect the presence of peptide bondsReagent: Potassium hydroxide (KOH) and hydrated copper(II) sulfate, sodium tartarateResult:(+) test = purple

(presence of proteins)() test = blueblue to pink when combined with short-chain polypeptides (it will not cleave on all peptide chains) 19. ProcedureAction of the Gastric Protease (Pepsin) on ProteinsAction of Pancreatic Protease (Trypsin) on Proteins 20. Action of the Gastric Protease (Pepsin) on Proteins 21. Action of Pancreatic Protease (Trypsin) on Proteins 22. B. THE ACTION OF PANCREATIC PROTEASE (TRYPSIN) ON PROTEINSLabel 3 tubes, each half full of warm H2OTo the first tube add a small amount of grated white of hard boiled egg.To the first tube add a 0.5g of trypsin and 2ml of 0.5% NaCO3.To the second tube add 0.5g trypsin.To the third tube add 2ml of Na2CO3 solution.Keep the tube in a warm H2O bath kept at body temperature for an hour.Filter the contents of each tube and the Biuret test on each of the filtrates.Observe and record the results. 23. Ideal Results: 24. POST LAB QUESTIONS 25. POST LAB QUESTIONSWhat are the enzymes used in Protein Digestion?Gastric Protease (Pepsin)Pancreatic Protease (Trypsin)Small intestine enzymes (peptidases) 26. POST LAB QUESTIONSWhat are the similarities and differences of these enzymes? These enzyme hydrolyze proteins and their main difference is the location where they are found. 27. POST LAB QUESTIONSWhat are the factors that would bring about Protein digestion?the acidity of the food and of the stomachTemperaturethe presence of any digestion inhibitors, such as antacids 28. POST LAB QUESTIONSTrace the pathway of Protein DigestionStomachGastrin stimulates:Parietal cells secrete HClChief cells secrete pepsinogen then HCL convert it to pepsin but acts only on certain amino acidsSmall Intestinereleased in the lumenCholecystokinin stimulates pancreatic enzymes such as:TrypsinTrypsinogen (active form)specific for Lys and Arg (carboxyl side)ChymotrypsinChymotrypsinogen (active form)specific for Tyr, Trp, Phe, Leu and Met (carboxyl side) 29. Protein Digestion Pathway ( digestion doesnt happen yet) Mouth and Salivary GlandsStomach( Gastric Phase)Small Intestine and Pancreas (Pancreatic Phase) Blood 30. POST LAB QUESTIONSWhat tests are used to detect the completeness of protein digestion?MILLONS TESTNITROPRUSSIDE TESTHOPKINS COLE TESTXANTHOPROTEIC TESTSAKAGUCHI TESTPAULY TEST 31. 5.MILLONS TESTTest for tyrosine (tyr)Used to detect the presence of soluble proteinsGiven by phenols or phenolic substances such as Salicylic acidPrincipleMercuric sulphate forms a colored compound with hydroxyl group of Tyr. Result: (+) red ppt 32. 5.HOPKINS COLE TESTspecific for tryptophan --- indole group. PrincipleThe indole ring reacts with glyoxylic acid in the presence of a strong acid: H2SO4Reagent: glyoxylic acidResult: violet cyclic product 33. 5.PAULYS TESTFor tyr, trp, and his PrincipleDiazotisedsulphanilic acid couples with amino phenol and immidazole to form a colored azocompd in cold condition.Result: (+) Deep red color dye 34. 5.SAKAGUCHI TESTTest for Arg -- guanido groupPrincipleAlkaline solutionSakaguchiReagent:-naphthol and sodium hypochloriteResult: (+) reddish wine color

35. 5.XANTHOPROTEIC TESTFor aromatic groups: tyr, trp, phe(unactivated)PrincipleNitration of a Benzene ring with Nitric AcidReagent: Nitric acid (HNO3) and NaOHResult: (+) yellow (tyr); orange (trp) --alkali 36. 5.SODIUM NITROPRUSSIDE TESTBollins testspecific for cys free thiol group (S H)PrincipleCystine whichcontains disulphide linkage (SS) may be reduced to cysteinereducing agent/s: sodium cyanide, sodium brohydride or sodium bisulphateResult: (+) red complex 37. What are the factors affecting protein digestion?pH: acidity and alkalinity of environment Regulating activity of inhibitors