exercise 6 - warszawski uniwersytet medyczny€¦ · the aim of this investigation is to...

Biochemistry Exercise 6

Department of Biochemistry

Second Faculty of Medicine with the English Division and the Physiotherapy Division 1

Student’s name: _____________________________

Index numer: _________________________

Date: __________________

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EXERCISE 6

DIGESTIVE SYSTEM.

Digestive enzymes are substances produced by our bodies that help us to digest the foods we eat. These

enzymes are secreted by the various parts of our digestive system and they help to break down food

components such as proteins, carbohydrates, and fats. This breakdown allows the nutrients from foods to be

absorbed into our bloodstreams so that they can support the functioning of all of the cells in our bodies.

The release of digestive enzymes

Digestive enzymes are released in both anticipation of food and in response to food. This means that just

thinking about or looking at food is enough to get your juices flowing. As we smell and eventually taste our

food, the amount of enzymes that are being secreted increases.

Enzymes are secreted from our salivary glands, and then from the cells lining our stomach, pancreas, and large

and small intestines. Different types of enzymes are secreted depending on the types of foods that we eat.

Types of digestive enzymes

Our bodies produce many different types of digestive enzymes to help our bodies to take advantage of the

various nutrients found within the foods we consume. Here are examples of some of the more prevalent types

of enzymes:

Amylase is a digestive enzyme essential for our digestion of carbohydrates, as amylase breaks down

starches into sugars.

Amylase is secreted by both our salivary glands and from our pancreas. The measurement of amylase

levels in the blood is sometimes used as an aid in diagnosing various pancreas or other digestive tract

diseases.

Lactase is a type of enzyme that breaks down the sugar, lactose, found in dairy products.

Supplemental lactase may be used to assist people who are lactose intolerant to digest dairy products.

Lipase is the enzyme responsible for the breakdown of fats that we consume. Specifically, lipase breaks

fats into fatty acids and glycerol. Within your body, lipase is produced in small amounts by your mouth

and stomach, and in larger amounts by your pancreas.




Maltase is secreted by the small intestine and is responsible for breaking down maltose (malt sugar).

Proteases are digestive enzymes that break down proteins into amino acids. Here are the major types of

proteases found within the human digestive tract:

Carboxypeptidase

Aminopeptidase

Chymotrypsin

Pepsin

Trypsin

Sucrase is secreted by the small intestine where it breaks down sucrose into the simpler sugars of fructose

and glucose.

Digestive enzyme supplements

There are a wide variety of health conditions that interfere with the secretion of sufficient amounts of digestive

enzymes for full digestion of foods. In such a case a person might benefit from taking a digestive enzyme

supplement. One of the areas in which digestive enzyme supplements are most helpful is for people who have

chronic pancreatitis. For these individuals, digestive enzyme supplements not only help with digestion, but also

can help to ease the pain associated with the disease.

Summary of human digestive enzymes:




Experiment 1

Amylase

Saliva contains hydrolitic enzyme -amylase (EC 3.2.1.1). It belongs to the hydrolase class of enzymes (EC 3).

Amylase optimal pH is 6.6-6.8 but it also needs Ca2+

which keeps proper conformation of the protein and Cl-

(enzyme’s activator).

Salivary amylase acts in mouth

(buccal cavity) and it produces:

dextrins (fragmented starch molecules,

most commonly containing one

-1,6-glycosidic bond)

maltose (isomaltose) molecules

maltotriose molecules

Aim:

The aim of this investigation is to detect the digestive properties of saliva.

Method used:

Iodine is the indicator for starch because it forms an intense blue

complex. Starch is not a redox indicator; it responds specifically

to the presence of I2, not to a change in redox potential. The

active fraction of starch is amylose, a polymer of the sugar α-D-

glucose. In the presence of starch, iodine forms I6 chains inside

the amylose helix and the color turns dark blue.

Procedure:

Take a test tube (large) and add:

1 ml of phosphate buffer (pH 6.6)

2.5 ml of 0.9% NaCl

2.5 ml of 1% starch solution

Mix the content and place the test tube into water bath (37°C) and leave it until the end of investigation.




Prepare 6 test tubes (labelled 1-6) and add to each of them 1 drop of iodine solution and 1 drop of 2M HCl.

The determination of activity of amylase is performed in 5x diluted saliva. Take test tube and make 5x diluted

saliva solution (take your own saliva, split into test tube and add approx. 4 volumes of water). Mix it well to

obtain homogeneous solution.

1. Take 1 ml of diluted saliva and add to the incubation mixture in water bath

2. Immediately mix the content, take 2 drops using a pipette and transfer it to the first test tube containing the

iodine indicator (test tube 1 = time 0)

3. Take another samples of incubation mixture with saliva every 1 min and transfer them to labelled test tubes

4. If the starch is still present in the last test tube you must extend the experiment and take more test tubes

with iodine solution.

Observations and conclusions:




Experiment 2

Pepsin

Pepsin is an endopeptidase found in gastric juice. It catalyzes the hydrolysis of peptide bonds within

polypeptide chain: X┼Tyr; X┼Phe; X┼Trp; X┼Leu; X┼Glu; X┼Gln.

Aim:

The aim of this investigation is to determine the influence of pH on the pepsin activity.

Procedure:

Prepare 4 test tubes and label them (1-4).

Reagents (mL) 1 2 3 4 (control)

0.2M HCl 0.5 - - -

Buffer (pH 6) - 0.5 - -

0.2M NaOH - - 0.5 -

Buffer (pH 7) - - - 0.5

Pepsin 0.5 0.5 0.5 0.5

a piece of chicken egg + + + +

Note observations after 5, 15, 30, 60 minutes.





Experiment 3

Trypsin

The pancreatic juice after mixing with a food content from the stomach neutralizes its pH in duodenum. The

same effect has a bile produced in the liver and released from gallbladder. In such conditions different enzymes

which act in the intestines can be active: trypsin, chymotrypsin, elastase, carboxypeptidases, aminopeptidases,

pancreatic amylase, nucleases, maltase, lactase or pancreatic lipase. After digestion all products can be

absorbed.

Trypsin is an endopeptidase which works the best in pH 7-9. It breaks down peptide bonds between: Lys┼X;

Arg┼X.

Aim:

The aim of this investigation is to detect the activity of trypsin.

Method used:

In protein hydrolysate formalin blocks the amino groups of short peptide chains. Free carboxylic groups are

then titrated with the sodium alkaline in the presence of formaldehyde (Søerensen method1). The amount of

base is used to determine the level of trypsin activity.

Procedure:

Prepare 4 Erlenmeyer flasks and label them (1-4).

1. In flask 1 make an incubation solution: add 10 ml of gelatin solution and place it into water bath (37°C)

2. Add 3 ml of formalin with phenolphthalein to flasks 2, 3 and 4 and leave them at room temperature.

3. After 5 min add 3 ml of trypsin buffer solution (pH 8) to the flask 1, mix the content quickly (without

taking the flask out of the water). Take 2 ml of mixture and transfer it into flask 2 (time 0).

4. Titrate the content of flask 2 with the 0.02M NaOH solution until the pinkish colour appears.

You’ve got 5 minutes to do the titration (as the next sample of mixture must be taken from flask 1 after 5 minutes).

5. After 5 minutes take another sample of mixture (2 ml) and transfer it into flask 3. Do the titration.

6. After another 5 minutes take another sample of mixture (2 ml) and transfer it into flask 4. Do the titration.

Calculate the amount of free carboxylic groups formed during protein digestion. Remember, that the amount of

NaOH used correlates with the amount of carboxylic groups due to the proportion:

1 ml 0.02 M NaOH ↔ 20 µmol of carboxylic groups

1 details not needed.




Calculations :

Draw a line graph showing the correlation between the time and the amount of hydrolysis product.




Experiment 4

Vitamin C

Ascorbic acid (vitamin C) is a derivative of carbohydrates.

It is a coenzyme of oxidoreductases and acts as a donor of protons, e.g. during the synthesis of collagen,

adrenalin or bile acids). It is also needed for Fe3+

ions absorption in the intestines.




Aim:

The aim of this investigation is to quantitative determination of vitamin C content in a fruit homogenate.

Method used:

DCPIP (2,6-Dichlorophenolindophenol) is a common indicator for vitamin C.

The amount of ascorbic acid is determined using the titration method.




Procedure:

Take 3 test tubes and label them “C” for control test, “S” for standardization test and “E” for experimental test.

Reagents (mL) “C” ”S” “E”

Phosphoric(V) acid in acetic acid 1 - -

Standard solution of vitamin C - 1 -

Fruit homogenate - - 1

Titrate all three solutions with a DCPIP solution until decolourisation (until pinkish colour persists).

Calculate the amount of vitamin C (in g/100g of fruit).

1 ml of vitamin C standard solution contains 0.12 mg of ascorbic acid.

1 ml of homogenate contains 0.2 g of fruit.

Calculations:




Experiment 5

Lactase

Lactose consists of galactose and glucose molecules connected with -1,4-glycosidic bond.

Lactose intolerance results due to decreased activity of lactase and may be caused by:

genetics

damage of cells of intestine lining as a result of different drugs taken by the patient or due to the protein

deficiency

The signs and symptoms of lactose intolerance usually begin 30 minutes to two hours after eating or drinking

foods that contain lactose. Common signs and symptoms include: diarrhoea (because of water retention in the

intestines), nausea (sometimes vomiting), abdominal cramps, bloating, gases production (H2, CO2, CH4).

lactose intolerance




Aim:

The aim of this investigation is to confirm the hydrolytic properties of lactase.

Procedure:

Take 2 test tubes and label them (1-2).

Reagents (mL) 1(control) 2

4% glucose solution 1 -

2% lactose solution - 1

Lactase in buffer (pH 7) 1 1

Mix the content and incubate (37°C) for 15 minutes. Use the glucose-detecting strips to test for the presence of

products of lactose digestion.


exercise 6 - warszawski uniwersytet medyczny€¦ · the aim of this investigation is to...

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