PROTEIN DIGESTION

Presented by: GROUP 1 – DAACoojacinto

De AsisGo

Saunar

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

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

Protein Digestion is the degradation of proteins by cellular

enzymes enzymes in a process called hydrolysis.

Protein digestion takes place in two different phases: In the stomach In the small intestine

Both of these phases of digestion are based on several types of enzymes that are called proteinases and proteases.

Proteases- endo- & exo- peptidases; enzymes that degrade proteins by hydrolysis of peptide bonds

Proteinases- endo-peptidases; proteases that show specificity for intact proteins

Peptide bond hydrolasePeptidase(=Protease)

Endo-acting peptide bond hydrolase

Endopeptidase(=Proteinase)

Exo-acting peptide bond hydrolaseExopeptidase

Mouth and Salivary Glands

Chewing and crushing of protein rich foods and mix them with saliva to be swallowed

In the Stomach : start of protein Digestion

Gastrin-stimulates Parietal cells to secrete HCL; Chief cells of the gastric glands to secrete pepsinogen

Hydrochloric acid-Denatures protein structure-Activates pepsinogen(zymogen) to pepsin

Pepsin -hydrolyzes proteins to smaller polypeptides and some free amino acids.

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).

In the Small Intestine : enzymes

Secretin- stimulates the pancreas to secrete bicarbonate into the small intestine to neutralize the gastric HCl

Cholecystokinin-stimulates secretion of several pancreatic enzyme with activity optima pH 7 to 8.

In the Small IntestineCont....Trypsin

- activates chymotrypsinogen chymotrypsin procarboxypeptidasescarboxypeptidases

proelastaseelastase-further hydrolyze the peptides that were produced by pepsin in the stomach specifically the peptide bonds next to lysine and arginine

Chymotrypsin-cleaves peptide bonds next to phe, tyr, trp,met, asp, and his

Cont....Carboxypeptidase A & B

-cleave amino acids from the acid (carboxyl) ends of polypeptides

Elastase and collagenase-cleave polypeptides into smaller polypeptides and tripeptides

Cont....Intestinal tripeptidases

-Cleave tripeptides to dipeptides and amino acids

Intestinal dipeptidases-cleave dipeptides to amino acids

Intestinal aminopeptidases -cleave amino acids from the amino ends of small polypeptides(oligopeptides)

Amino acids absorbed

Free amino acid small intestine(villi)Liverblood circulation

Materials: Commercial pepsin Trypsin Concentrated HCl NaOH CuSO4 solution 0.5% Na2CO3 Hard Boiled Egg (Protein) albumin

Biuret Test a chemical test used to detect the presence

of peptide bonds Reagent: Potassium hydroxide (KOH) and

hydrated copper(II) sulfate, sodium tartarate

Result:o (+) test = purple (presence of proteins)o (−) test = blue blue to pink when combined with short-

chain polypeptides (it will not cleave on all peptide chains)

Procedure

A. Action of the Gastric Protease (Pepsin) on Proteins

B. Action of Pancreatic Protease (Trypsin) on Proteins

A. Action of the Gastric Protease (Pepsin) on Proteins

TEST TUBE BIURET TESTOUR RESULT IDEAL RESULT

A: warm H20, protein(hard boiled egg), pepsin, 2 drops of HCl

clear Pinkish lavender

B: warm H20, protein, pepsin

blue Purple

C: warm H20, protein

blue Purple

B. Action of Pancreatic Protease (Trypsin) on Proteins

TEST TUBE BIURET TESTOUR RESULT IDEAL RESULT

A: warm H20, protein, 0.5g trypsin, 2ml 0.5% Na2CO3

Pinkish lavender or Blue

B: warm H20, protein, 0.5g trypsin

Purple

C: warm H20, protein,2 mL Na2CO3

Purple

B. THE ACTION OF PANCREATIC PROTEASE (TRYPSIN) ON PROTEINS

1. Label 3 tubes, each half full of warm H2O2. To the first tube add a small amount of grated white of hard boiled

egg.3. To the first tube add a 0.5g of trypsin and 2ml of 0.5% NaCO3.4. To the second tube add 0.5g trypsin.5. To the third tube add 2ml of Na2CO3 solution.6. Keep the tube in a warm H2O bath kept at body temperature for an

hour.7. Filter the contents of each tube and the Biuret test on each of the

filtrates.8. Observe and record the results.

Ideal Results:TEST TUBE Content BIURET

TESTA Small amount of

grated white of hard boiled egg ; Trypsin; Na2Co3

Pinkish-Lavender or Blue

B TrypsinPurple

C Na2Co3Purple

POST LAB QUESTIONS

POST LAB QUESTIONS1. What are the enzymes used in

Protein Digestion? Gastric Protease (Pepsin) Pancreatic Protease (Trypsin) Small intestine enzymes (peptidases)

POST LAB QUESTIONS2. What are the similarities and

differences of these enzymes? These enzyme hydrolyze proteins

and their main difference is the location where they are found.

POST LAB QUESTIONS3. What are the factors that would

bring about Protein digestion? the acidity of the food and of the

stomach Temperature the presence of any digestion

inhibitors, such as antacids

POST LAB QUESTIONS4. Trace the pathway of Protein Digestion

Stomach Gastrin stimulates:

Parietal cells secrete HCl Chief cells secrete pepsinogen then HCL convert it to

pepsin but acts only on certain amino acids Small Intestine

released in the lumen Cholecystokinin stimulates pancreatic enzymes such

as:a) Trypsin Trypsinogen (active form)

specific for Lys and Arg (carboxyl side)b) Chymotrypsin Chymotrypsinogen (active form)

specific for Tyr, Trp, Phe, Leu and Met (carboxyl side)

Protein Digestion Pathway

( digestion doesn’t happen yet)

Mouth and Salivary Glands

Stomach( Gastric Phase)

Small Intestine and Pancreas

(Pancreatic Phase)

Blood

POST LAB QUESTIONS5. What tests are used to detect the

completeness of protein digestion?

MILLON’S TEST NITROPRUSSIDE TEST HOPKIN’S COLE TEST XANTHOPROTEIC TEST SAKAGUCHI TEST PAULY TEST

5. MILLON’S TEST

Test for tyrosine (tyr) Used to detect the presence of soluble proteins Given by phenols or phenolic substances such

as Salicylic acidPrinciple Mercuric sulphate forms a colored

compound with hydroxyl group of Tyr. Result: (+) red ppt

5. HOPKIN’S COLE TEST

specific for tryptophan --- indole group.

Principle The indole ring reacts with glyoxylic acid

in the presence of a strong acid: H2SO4 Reagent: glyoxylic acid Result: violet cyclic product

5. PAULY’S TEST

For tyr, trp, and his

Principle Diazotised sulphanilic acid couples with

amino phenol and immidazole to form a colored azo comp’d in cold condition.

Result: (+) Deep red color dye

5. SAKAGUCHI TEST

Test for Arg -- guanido group

Principle Alkaline solution Sakaguchi Reagent:α-naphthol and

sodium hypochlorite Result: (+) reddish wine color

5. XANTHOPROTEIC TEST

For aromatic groups: tyr, trp, phe(unactivated)

Principle Nitration of a Benzene ring with

Nitric Acid Reagent: Nitric acid (HNO3) and NaOH Result: (+) yellow (tyr); orange (trp)

--alkali

5. SODIUM NITROPRUSSIDE TEST

Bollin’s test specific for cys – free thiol group (S—H)Principle Cystine which contains disulphide linkage (S

—S) may be reduced to cysteine reducing agent/s: sodium cyanide, sodium

brohydride or sodium bisulphate Result: (+) red complex

6. What are the factors affecting protein digestion?

pH: acidity and alkalinity of environment Regulating activity of inhibitors