1.10 Protein desnaturalitation

Introduction

Desnaturalitation es a process in wich proteins or nucleicacids lose the quaternary, terciary and secondary structure that is present in their native State. Denaturatiom is the result of the application ofsome external stress (heat and pH change) or compounds such as a strong acid or base, a concentrated inorganic salt or organic solved.

Materials
-2x250mL beaker
-4 test tube
-Test tube rack
-10mL pipet
-Knife
-Glass marking pen
-Potato
-Distilled water
-Hydrogen perioxide
-NaCl
-HCl

Objectives

1-Study the relation between the structure and the function of proteins.                                                     
2-Understand how temperature, pH and salinity affect to the protein structure.

Catalasa activity

Caalasa is a common enzyme found in nearly all-living organisms exposed to oxygen. It catalyzes the decomposition of hidrogen perioxide (H2O2) to water an oxygen. It is a very important enzyme in protecting the cell from oxidative damage and preventing the accumulation of hidrogen perioxide.

2 H2O2 ----> 2H2O + O2

Procedure

In this experiment we are going to test the catalase activity in diferent environment situations. We  are going to measure the rate of enzyme activity under various conditions, such as different pH values and temperatures. We will measure catalase activity by observin the oxygen gas bubbles when H2O2 is destroyed. If lots of bubbles are producted, it means the reaction is happening quickly and the catalase enzyme is very active.

1-Prepare 30mL of H2O2 10% in a beaker (use a pipet).

2-Prepare 30mL of HCl 10% in a beaker.      à Solutions

3- Prepare 30mL of NaCl 50% in a beaker.

4-Peel a fresh potato tuber and cut the tissure in five cubesof 1cm3. Weigh them and equal the mass.

5-Label 5 test tubes (1, 2, 3, 4, 5)

6- Immerse 10 minutes your piece of potato inside the HCL beaker 

7- Immerse 10 minutes another piece of potato inside NaCL beaker.

8- Boil another piece of potato.

9- With a mortar, mash up the third piece of potato.

10- Prepare 5 test tubes as indicated below:

TUBE	TREATMENT
1	Raw potato
2	Boiled potato
3	Potato with HCl treatment
4	Potato with Nacl tratment
5	Mashed up potato

11- Add  5ml H2O2 in each test tube

12- With a glass-marking pen mark the heigh of the bubbles. Measure it with a ruler.

13- Compare the results of the 5 test tubes.

Table with the important parts of this experiment:

Parts:	In this experiment this was...
Independent variable	Treatment
Dependent variable	Bubble’s height
Experimental group(s)	2, 3, 4, 5 (test tube)
Control groups	1 (test tube)
Constant	Concentration of distilled water, the weight of the potato and oxigeneted water

Results

Observations

-We had to put more oxygenated water since almost no reaction occurs.

-The Mashed potato produced more activity.
-The boiled potato and tehe potato with HCl treatment produced less activity.

Questions

1.How did the temperature of the potato effect the activity of catalase? Temperature

desnaturated the catalase.

2.How did the change of the pH of the potato effects the activity of catalase? The change of the pH denaturate the catalase.

3.In wich potato treatment was catalase the most active? Why do you think this was? Mashed potato, because we had broken the cells and the catalase was more quikly.

4.An experiment was performed to test the effect of temperature and pH on the activity of Enzyme X. The following data was collected during the experiment:
a) What is the optimun pH of enzyme X? 8 (maximum activity).
b) What is the optimun temperature of enzyme X? 20 (maximum activity). c) Why do you tink enzyme X has low activity at a pH of 10? Because has been denatured.
d) Enzyme X performs critical life functions. Use the data above to explain why a fever of 40 degrees may be dangerous. Bacause is more lower than the maximum activity.

1.9 Protein identification

Introduction

Biuret's test is a chemical test used for detecting the presence of peptide bonds. Polypeptides as proteins, are chains of amino acids link together by peptide bonds.

A peptide bond can be broken by hydrolysis (the adding water). In organisms, protein molecules called enzymes facilitate the process.

Materials
-7x250mL beaker
-6 test tube
-Test tube rack
-6x10mL pipet

-Mortar
-Glass marking pen
-Gloves
-Goggles
-Milk
-Rice milk
-Egg
-Potato
-Distilleted water
-NaOH 20%
-10 drops of CuSO4 

Objectives

1. Identify peptide bonds.

2.Compare protein concentration in different foods.

Procedure

Firs of all we are going to dilute the protein

1. Add 100ml of distilled water to each 250ml beaker. Lebel them with M(milk), EW (egg white), EY (yolk), P (potato) and RM (rice milk)

2. Separate the egg white and the yolk in another beaker.

3. Smash the potato in a mortar and add some amount of the smashed potato to the P beaker.

Prepare the samples

4. Add 10ml of a dispersion of each food (M, RM, EY, EW and P) to the indicate beaker. Calculate the final concentration. All the groups will use the same dispersion from the beakers.

5. Prepare 6 test tubes (cleand and dry) and lebel (M, RM, EY, EW and P). Add 2ml of the every food dilution of each beaker.

6. Add 2ml of 20% NaOH dissolution. 

7. Shake gently and add 5 drops of CuSO4 in each tube. Allow the mixture 5 minutes. 

8. Note any colout change. Remember that proteins will turn solution pink or purple. 

9. Compare the test tubes. 

1.8 Saponification

Introduction

Lipids are a heterogeneous group of compounds synthesized by organisms that are present in all biological tissues.
One of the properties is saponification of fatty acids. If one is reacted acilglicerid (oil or grease) with a strong base, such as sodium hydroxide or potassium hydroxide forms soap (sodium or potassium salt of a fatty acid) and glycerin released and is dissolved in water. The soap forms a solid layer of racipient superfice wing.


Material
-Test tube 
-Test  tube rack
-Bunsen burner
-Wire gauze
-Beaker
-NaOH
-Oil


Objectives 
-Carry out the saponification reaction.


Procedure
1.Take a test tube rack.
2.Take one test tube and put 2mL of NaOH.
3-Put 2mL of olive oil.
4-You need to mix with a stirring rod.
5-Put the test tube in a beaker with water in the bunsen burner, ten minutes.
6-You will see the soap (solid part and yellow).


Observations

Reactions:
H3C-(CH2)7-CH=(CH2)7-COOH ---------> Oleic acid


H3C-(CH2)7-CH=(CH2)7-COO-CH2
                                                /
H3C-(CH2)7-CH=(CH2)7-COO-CH       --------------------> Triolein   
                                                /
H3C-(CH2)7-CH=(CH2)7-COO-CH2


              + NaOH

  (3x)  H3C-(CH2)7-CH=(CH2)7-COONa    ---------------------> Sodium oleic

    
-one glycerine and three molecules of sodium oleic.



Second part


Objectives
1.To know if there arepresence of lipids


Procedure

-Put 2 mL of milk in a test tube .
-Put 10ml of water and 5 drops of Sudan III and stir .
Observes that everything is dyed.

Observation

We can see when we put the Sudan II the colour of the milk change to red, that means that there are a presence of lipids.

1.8 Lipids properties

Introduction
Lipids are a heterogeneous group of compounds synthesized by organisms that are present in all biological tissues.
These compounds are characterized as natural substances that do not mix with water but dissolve in organic solvents.
There are several classes of lipids, including: fatty acids, waxes, triacylglycerols, phospholipis. terpens and steroids.


Material
-Test tube rack
-250mL beaker
-water
-Test tube rack
-Cellulose paper
-Dropper
-Scissors
-Glass rod
-Olive oil
-Soap (detergent)
-Milk with different fat content: full-cream, semiskimmed, and skimed milk.
-Petroleum ether
-Ethanol
-Sudan III


Objectives 
1. Test the solubility of lipids.
2. Identify lipids in liquids compounds.
3. Understan what are an emulsion ant the effect of detergents.

Procedure
Solubility of some lipids:
1.Clean  and dry three test tube. Label as w (water), E (ethanol), PE (ethet).
2.Add 3 dropps of oleic acid to 3 small test tube.
3.Add 1mL of water in the first test tube (W).
4.Ads 1mL of ethanol in the second test tube.
5.Add 1mL ot petroleum ether in the third test tube (PE).
6.Shake carefully each test tube and record solubility and observations in your worksheet.

Observations:
-The water inthe first test tube create a monolayer and the ethanol and ethet dissolved.


Lipids identification:
Translucent mark:
7.Cut two pieces (10x10cm) of cellulose paper.
8.Put 1 drop of water in the first squared pices. You will see a transluced spot. Wait for a while and observed what is happening.
9.Put 1 drop of olive oil in thesecond squared pieces ofcellulose paper. You will see a translucent spot. Wait for a while andpbserve what is happening. Has the spot disappeared? Why?


Sudan III dye:
Sudan III is a red fat-soluble dye that is utilized in the identification of presence of lipids, triglycerides and lipoproteins in liquids.
10.Take the W test tube of the first experiment and add 2 drops of Sudan III.
11.Prepared four test tubes: 3 with milk with differents fat content (M1, M2, M3) an soda (S). Add two drops of Sudan III and observe the results.

Observations:
-We can see that the colous of the oil change bacause it hasfatty acids. The water doesn't change the colour because doesn't have fats.


Permanent emulsion:
12.Take a 250mL beaker and put 100mL of water.
13.Add 1mL of olive oil. With a glass rod stir the mixture vigorously and let it stand for a few minutes.
14.Make note of what is happening.
15.Add 2 dropps of soap and stir the mixtue again. Let it stand for a few minutes and notice the differents between both mixture.

Observations:
Water whith olive oil. We can see that whenwe stir the water with oil, the oil never dissolved the micelles come together.
Water with oil and soap. The micelles are not together because there are soap and create a membrane that don't f¡let the micelles come together. 

1.7 Starch

Introduction

Materials
-Bunsen burner
-tripod stand
-wire gauze
-250 cm3 beaker
-boiling tube
-forceps
-test tube holder
-leaf to be tested
-90% ethanol
-iodine/potassium iodine solution


Objectives 
1. Identify the presence of the starch in leaves.
2.Relate the presence of starch with photosyntesis.
3.Identify the presence of starch in some food.


Procedure
1.Remove a green leaf from a plant that has been exposed tosunlight for a few hours.
2.Half-fill a 250cm3 beaker with water. Heat the water until it boils. Keep the water at boiling point.
3.Use the forceps to place the leaf in the boiling water. Boil for 2 minutes.
4.Turn offthe Bunsen Burner.
5.Place the boiling leaf in a boiling tube containing 90% ethanol.
5.Place the boiling tube in hot water and boil for10 minutes or until the leaf decolourizes. (It may benecessary to replace the ethanol)
6.Gently remove the leaf and wash with a fine trickle of cold tap water.
7.Spread the leaf evenly on a white tile.
Add a few drops of iodine/potassium iodiene solution to the leaf and note any observations.


Observations
we can see thet in the

Questions
1.Which is the origin of the starch that you can see in the leaf? From the glucose that was synthesized the starch, photosynthesis.
2.Complete this reaction: And write the name of this process. 
CO2+H2O------>C6H12O6+O2
3.Explain the significance of boiling the leaf in water. Lose the turgent conditions
4.Explain the significance of boiling the leaf in ethanol. To lose the color (chlorophyll dissapears)

5.Explain the significance of rinsing the leaf in water. Because the lugol isn't soluble with ethanol

1.6 Fehling's test: reducing sugars

Introduction

Fehling's solution is a chemical test used to differentiate between reducing and non-reducing sugars. This test is based on the reaction of a functional group of a sugar molecules with fehling's reagent.

Fehling's reagent has two separate solutions: Fehling's A is a blue aqueous soluion of copper (II) sulphate; and Fehling's B clear and colourless solution of potassium sodium tartrate and sodium hydroxide.

Materials

-Test tube rack

-10 mL pipet

-Distilled water

-5 test tubes

-5 spatula

-Lactose
-Maltose
-Glucose
-Sucrose
-Starch
-Fehling's A and B solutions
-HCL

Objectives

1.Identify reducing sugars.
2.Comprehend redox reactions.
3.Understend the relation between structure and reducing ability of some sugars.

Procedure

In this experiment you will first determine with sugars give a positive test with Fehling's reagents and then, by testing the reaction of some organic molecules containing only a single functional group, you should de able to deduce with functional group of sugars is reacting with Fehling's reagent.

          1.Take 5 test tubes and label: G, M, S, L, ST.
          2.Put 2mL of distilled water inside each tube. 
          3.With different spatula put a small amound of each sugar. Dissolve the sugar.
          4.Add 2mL of Fehling's A solution and then Fehling's B.

          5.Place each test tube in a boiling water dath (250mL beaker on a hotplate stirrer).
          6.Observe what is happening.

Starch Hydrolysis:
Hydrolysis is the reaction of a compound with water. As you know, starch is a polymer, consisting of many units of -D-glucose covalent linked together.
          7.Place 2mL of 1% starch in a test tube and add 0,5mL of 3M HCL. Mix and place this           mixture in a boiling water bath for 10 minutes.
          8.After 10 minutes, remove the tube fromthe water bath and let it cool. Neutralize this           solution with 1 M NaOH and mix well.
          9.Transfer 8-10 drops of this solution to a small test tube.
          10.Add 1mL of Fehling's Ac solution and 1mL of Fehling's B.
          11.Heat for a few minutes in a boiling water bath.
          12.Record your observations. Compare the results of this test with your results for                 unhydrolyzed starch in the steo 1 of this experiment.
          13. You can test the absence of starch with iodine solution too!

Observations

We can see that the differents test tube have an orange colour.

Questions
1.From your observation and the structures of the sugars given above, indicate whith functional group in the sugar molecules reactys with Fehling's reagent.
2.Compare the results you obtained for the Fehling's test of starch and Fehling's test of hydrolyzed starch. Explain your results.
3.Would have you obtained a Fehling's positive test if you had hydrolyzed the sucrose (as you have done with starch)? why?
4.What does "reducing sugars" term means?

1.5 Saccharides properties

Introduction 
Saccharides are organic molecules consisting of C, H and O atoms; and the empirical formula is Cn H2n On.

Are divided into thte goups:
-Monosaccharides: formed by a linear carbon chain, the oligo and polysaccharides are formed by these.
They have a one funcional group: cetone (C=O) or aldehyde (COH).
Pentoses and hexoses in dissolution can cyclic forming shapes called furanoses and pyranosres.
-Oligosaccharides: small polymer containing between 2 and 10 monosaccharides.
-Polysaccharides: big polymers with more than 10 monosaccharides.

These molecules perform numerous roles in living organisms like storage if energy, structural components .

Saccharides yield 4,2 Kcal/g. They are a common source of eneregy in living organisms.


Materials
-Test tube rack
-10 mL pipet
-Distilled water
-5 test tube
-1 dropper
-5 spatula
-Lactose
-Maltose
-Glucose
-Sucrose
-Starch
-Lugol's iodine


Objectives
1.Identify differents sugars from it's properties.
2.Differentiate monoand disaccharides.
3.Understand the relation bettween structure and some properties.


Procedure
1.In the firspartof the experiment we are going to test some physcal properties of the saccharides you have in the lab: flavour, crysta structure and colour. How?
          a. Flavour:Put a small amount of each saccharides in your hand and test it! Is it sweet            or not sweet?
          b. Crystals: Observe a small amount of each saccharide on a clock glass under                      magnification.
          c. Colour: White, transparent or creamy.

The propierties of 5 saccharides:



To test solubility:
2. Clean and dry 5 test tubes and label them "G, M, L, SU, S" 
3. Put 5 mL of water in each test tube.
4. With the aid of a spatula, put a small amount of each saccharide inside the labelles test tube and test if they are soluble or insoluble.
5. Observe if each saccharide forms a mixture called dissolution or a colloidal suspension.

Lugol's iodine Test:
6. Finally, add 2 drops of Lugol's iodine to each test tube and test if the reaction is positive or negative. Lugol's is a solution of elemental iodine (I) and potassium iodine (KI) in water that is use to test a saccharide. The reaction is positive when iodine reacts by turning from yellow to a purple, dark-blue/dark colour.

Finally we are going to test if some foods contain starch. Try with potato!
1. Add 2 or 3 drops of Lugol's solution to one piece of potato observe what is happening.


Observations
We can see that different test tube we put lugol's iodine, and one is in the different colour, is ti the strach (positive).
Questions
1.Write the empirical formula of each saccharide thet you have use. Show structures ofthe five saccharides.Classify each one in one group: mono, oligo, or polysaccharide.
-
2.Which of the monosaccharide are aldoses and wich are cetoses?
-
3.Which bond links monosaccharide?
-
4.Which saccharide/s is/are sweet? Is this property related to the structure of the molecules?
-
5.Whichsaccharide/s is/are insoluble? Is this property related to the structure of the molecules?
-
6.Which saccharide has reacted whith Lugol's iodine solution?
-
7.Which kind of food contains starch?
-
8.Calculate the energy from the nutrition facts label from a cereals:
   a.Calculate the energy that comes from the saccharides.
   b.Which % of the total energy comes from the saccharides?