Biochemistry Labs

Question 1

6.Paper and Thin Layer Chromatography

Answer 1

Theory;

• Method to measure purity of a compound.
• components are separated between stationary and mobile phase
• Mobile phase: Water adsorbed to a cellulose sheet.
• Stationary phase: Is a developing solution move up the stationary phase.
• Components separate depending on: their ability to absorb to stationary or mobile phase.
• Acetic Acid is used to set the pH.
• Hydrophobic components: - move along the organic front
• Hydrophilic components: - remain near the stationary phase.

Aim: Determine the unknown amino acid in a solution by comparing it’s RF value to known RF Values.

Procedure:

1. cut 10x50 cm paper
2. mark a line with pencil 6cm from the bottom.
3. use a pencil to mark points on the line separated by 1.5cm, and apply solutions with a microcapillary.
4. place into chamber, where line is 1cm above the solvent.
5. Allow to develop for 2hours then spray with ninhydrine solution.
6. Measure RF value.

Thin Layer:

• Similiar to Paper however it takes about 40mins to develop and gives better seperation.
• Involves use of a polyscilinate layer on 0.2mm thickness.
• Water serves as stationary + mobile phase.

Conclusion:

• Proline = yellow colour
• all the others = purple.
• using rf value find out which amino acid it is.

Question 2

1. Reversible Precipitation of Proteins

Answer 2

a)”Salting out” using Ammonium Sulphate.:

Background:

• Globular proteins are soluble in water due to their hydrate envelope.
• When this envelope is disturbed then they precipitate.
• Ammonium Sulphate does not affect it’s secondary or tertiary structure thus this is reversible.

Aim:
To salt out proteins from the Blood Serum with Ammonium Sulphate.

Procedure:

• Add 5ml of Blood serum to a test tube followed by 5ml of Ammonium Sulphate.
• Allow to stand for 10mins (until half saturation point).
• Pour through filter paper - will see Globins on the paper.
• ADD SOLID Ammonium sulphate until saturation, then allow to stand for a few mins.
• Then filter and will see Albumins removed on the paper.

Conclusion:
-Salting out of different proteins occurs at different stages

b)With Organic Solvents

Aim: Show that ethanol causes the precipitation of proteins and that it is reversible.

Theory:

• Ethanol decreases protein solubility.
• It’s polar hydroxyl group interacts with the protein’s groups.
• It’s apolar Hydrocarbon chain is insoluble causing precipitation of proteins.

Procedure:

• Add 5ml of ethanol to 2ml of Blood Serum.
• See precipitation occur.
• Carefully pipette more ethanol on the boundary and see even more precipitation.
• Adding water causes redissolving.

Question 3

2.Irreversible precipitation of Proteins

Answer 3

a) By Heavy Metal Salts

Aim: -To show effect of Heavy metal salts on protein
-effect of EDTA on the complex.
Background:
-Ions form covalent co-ordinate bonds with O and N in protein.
-Complex forming ability causes denaturation (irreversibility) and precipitation.

Procedure:

1) put 3ml of Blood serum in 2 test tubes..
2) In test tubes place 1.5ml of lead acetate.
3) in first test tube place 1.5ml EDTA-NA2 and other put 1.5ml of water.

Conclusion:

• Lead acetate causes precipitation, additional drops causes redissolving.
• EDTA prevents complex forming so no precipitation.

b) By Inorganic Acid

Aim: Show effect of inorganic acid on protein solution.

Theory: Inorganic Acids drastically lower the pH which affects the secondary and tertiary structure.

Procedure:

1) Get 3 test tubes.
2) Place 3ml of Blood serum then add drops of HCl/H2SO4/HNO3
3) See that precipitate forms then even more drops causes redissolving.

c)by Heating

Aim: Show precipitation due to heating and influence of various acid/base.

Theory: Heating breaks H-bonds in secondary and tertiary structure.

Procedure:
1) Prepare 5 test tubes.
2) Place Blood serum 2ml in each.
3) 1. Heat only.
    2. few drops of 1% acetic acid.
    3. 0.5ml of 10% acetic Acid
    4 0.5ml of 10% acetic Acid + nacl
    5. 0.5ml of 10% NaOH

Conclusion:

1: precipitate form
2. Precipitate forms because pH close to Isoelectric pH>
3: No precipitate as pH too low
4. Precipitate forms due to treatment with salts.
5: pH too high so no precipitate forms.

Question 4

5. Gel Filteration

Answer 4

Aim: To seperate proteins based on size differences.

Procedure note: talk procedure before theory in this one:

1) In this experiment we used a Sephadex 25 Column.
2) We placed 0.3ml of the Bovine Serum/Potassium iodide mix on top of the gel.
3) We then add water as the elution buffer.
4) collect twenty 2ml samples in tubes.
5) after collection add: 1ml SULFOSALACILIC which causes protein precipitation
6) then add 0.5ml of NaOCl (turns iodide brown thus allowing detection).

Theory + Results:

• Gel filteration seperates proteins based on the size differences.
• The gel has pores with a certain diameter.
• In our experiment the iodide is able to enter the Pores thus distributed between pores and amongst the beads.
• Whereas proteins too big to enter to pass between the beads thus move through the column much quicker so are seen in first samples.

Important Formulas:

1.
(ClO-) + (2H+) + 2I-) —-> I2 + Cl- + H20

2.
I2 + I- I3- yellow colour

3.
n-I3 + starch –> Dark blue colour.

Question 5

10.Determination of Urease Enzyme Kinetics.

Answer 5

Aim:
-To determine the Vmax and Km of Urease.

Theory:
-Urease is an enzyme which catalyses the hydrolysis of Urea into Ammonia and CO2.

EXTRACTION
-We extract it from crushed soybean with a 3:1 Water to Acetone mixture. Also EDTA + 2-Mercaptoethanol is in the micture.

BERTHELOT

• We follow the Urease Activity by using the Berthelot Reaction.
• The Berthelot Reagent is an Alkaline solution consisting of Phenol and Hypochlorite.
• it reacts with Ammonia to form a BLUE-coloured product

There is a direct proportionality with between product formed and time of incubation at all concentrations of substrate/enzyme.

We find that [S} (x-axis) and V (y-axis) forms a hyperbolic curve.

Procedure:

1. Prepare 9 Test-tubes.
2. 9th Test tube = blank, has 1ml 1% EDTA.
3. Remaining test tubes also have 1ml 1% EDTA.

1,2: 50ul of 5mM Uurea
3,4: 50uL of 10mM urea
5,6: 50uL of 20mM urea
7,8: 50ul of 30mM Urea

4. Measure Absorbance vs the blank tube.
5. Calculate velocity;=Ab/Min
6. Plot Line-weaver Burk Plot.

FIND KM AND Vmax

Question 6

11. Reversible and Irreversible Inhibition of Urease Activity.

Answer 6

Aim:
To examine the properties of Competitive and Non-Competitive Inhibitors

Theory:
A ….
->Irreversible Inhibitor: dissociates slowly from enzye as it is tightly bound.
->Reversible inhibitor: dissociates rapidly from the enzyme as it is loosely bound.
->Competitive Inhibitor:
These bind to the active site of an enzyme, thus preventing substrate from binding. They affect the Km.
Their degree of inhibition depends on:
- Binding constant
- Substrate/Enzyme Concentration.

->Non Competitive Inhibitor:
These bind to region apart from the active site. They affect Vmax. They prevent conversion of substrate into product.

In our Experiment:

• Guanidine = Competitive inhibitor
• PCMB = Non-Competitve Inhibitor

Procedure:
Test Tubes are prepared,
1,2: Blanks, 10mM urea + UREASE

Competitive:
3,4: 10mM Urea + Urease + Guanidine
5,6: 20mM Urea + Urease + Guanidine

Non-Competitive:
7,8: 10mM Urea + Urease + PCMB
9,10: 20mM Urea + Urease + PCMB
11,12: 10mM Urea + Urease + PCMB + 2-Mercaptoethanol.

Question 7

7. Principle of Denaturing Polyacrimide Gel Electrophoresis of Proteins.

Answer 7

Aim:
-To seperate Proteins based on the Molecular Mass of the Proteins.

Theory:

• In Gel Electrophoresis proteins are seperated by their size.
• We prepare the Sample by boiling the protein mixture with Bromophenol, Glycerol and SDS.
• The FUNCTION OF SDS:
  - >It keeps the insoluble denaturised Proteins soluble by binding to the surface.
  - > It equalises the negative charge of the polypeptide chain thus separation is only determined by Mr.

-Smaller molecules experience less resistance thus travel greater distances in the gel.

• THE GEL in our experiment consists of two parts:
  - > Stacking Gel (pH 6.8)
  - > Lower Gel (pH 8.8)

Procedure:

1) Pour Running Gel in the Outer and Inner Chamber
2) Remove the Comb and apply the protein sample with a syringe.
3) Then apply Bromophenol into the well (this is a negative charged dye which allows us to visibly follow the process.
4) Connect the apparatus to a Power Supply and allow separation until blue line reaches the end of the Gel.
5) Apply Commaise Blue to make Protein Bands visible.