Practical Session 4

Question 1

What is the title and aims of practical 4?

Answer 1

Colorimetric Assay of Serum Protein

1. Make a standard curve for protein serum and calculate ε for this compound.
2. Use micropipettes.
3. Understand the prediction error from results calculated from callibration curves.

Question 2

How is the biuret test known?

Answer 2

Piotrowski’s test.

Question 3

What is the biuret test?

Answer 3

A chemical test used for detecting peptide bonds.

Question 4

What does a copper (ll) ion form when peptides are present?

Answer 4

Mauve-coloured coordination complexes in alkaline solution.

Question 5

At which wavelength does the blue adduct absorb?

Answer 5

540 nm = λ.

Question 6

What do biuret and peptide bonds form when they react with cupric ion?

Answer 6

A blue adduct.

Question 7

What does the biuret reagent include?

Answer 7

Sodium potassium tartrate.

Question 8

What does sodium potassium tartrate do?

Answer 8

Stabilize cupric ions that are sensitive to reduce to Cu+.

Question 9

How is the biuret reaction characterised?

Answer 9

The most common technique for measuring total protein using automated chemistry analysers.

Question 10

What do modifications of the biuret method include?

Answer 10

Lowry and Folin-Ciocalteu techniques.

Question 11

Where are the Folin-Ciocalteu techniques useful?

Answer 11

Detecting minute amounts of protein presented after isolation/separation.

Question 12

What does the measurement of total protein determine?

Answer 12

Concentration of all proteins present in serum (except clotting factors).

Question 13

Which are the major proteins?

Answer 13

Albumin.

Immunoglobulins: IgG, IgA, IgM.

Question 14

How is the total protein measurement used clinically?

Answer 14

1. Total protein measured in serum –> gives indication of total immunoglobulin concentration: total protein - albumin = globulins.
2. Total protein included in ‘liver function tests’: chronic liver diseases –> increase immunoglobulin –> increase total protein.
3. Total protein measured in suspected humoral immunodeficiency: diagnosis by immunoglobulins measurements.
4. Total protein measured when a patient is suspected of having paraprotein but not high: serum protein electrophoresis.

Question 15

What is total protein measurement without measurement of albumin?

Answer 15

Of little value.

Question 16

What is the proportion of light absorbed by a solution of a particular compound?

Answer 16

A function of the concentration of the compound.

Question 17

What does spectrophotometry allow?

Answer 17

Quantitative analysis of concentration of a substance from Beer-Lambert relationship.

Question 18

What does a spectrophotometer do?

Answer 18

Direct light of a specific wavelength on solution.

Calculate absorbance.

Question 19

What is the light from the spectrophotometer?

Answer 19

The incident light.

Question 20

What is the light that passes through the solution?

Answer 20

The transmitted light.

Question 21

What is the absorbance of the solution?

Answer 21

The log of the ration of the incident light divided by transmitted light.

A (Absorbance) = log10 (Intensity of incident light (Io) / Intensity of transmitted light (I))

Question 22

How can we find the concentration of the solution?

Answer 22

Know absorbance.

Beer-Lambert equation.

Question 23

What is the Beer-Lambert equation?

Answer 23

A = ε.l.c

Question 24

What does the Beer-Lambert equation mean?

A = ε.l.c

Answer 24

ε = Absorbance of 1M solution of substance measured through 1cm light path, constant.

c = concentration, moles/litres.

l = length of light path through solution, cm = 1 cm.

Question 25

So, since l = 1 how is the equation of Beer-Lambert law then?

Answer 25

C = A / ε

Question 26

What do we need to know to apply the Beer-Lambert equation?

Answer 26

The Molar Absorption Value = ε for substance.

Wavelength.

Question 27

How can we know the ε?

Answer 27

Absorbance of 1M solution.

Question 28

How can we measure absorbance in substances with strong absorbance?

Answer 28

With a standard curve.

Question 29

How is the standard curve produced?

Answer 29

Measuring absorbance of dilute solutions of known concentrations.

Question 30

What is the slope when absorbance is plotted against concentration?

Answer 30

Relationship between concentration and Absorbance.

ε = Α/ C 
Slope = ε.

Question 31

What do we measure with A/C?

Answer 31

ε.

Question 32

What can we find once we measure ε?

Answer 32

Absorbance at same wavelength.

Question 33

What can we find once we know ε and Absorbance?

Answer 33

Concentration of any solution.

Question 34

What are the units of concentration?

Answer 34

mg / mL.

Question 35

What are the units of ε?

Answer 35

mL -1 x mg.cm

Question 36

What is the first part of Serum Protein assay consist?

Answer 36

Making a standard curve with known quantities of pure protein.

Question 37

What will we use as the protein in the experiment?

Answer 37

Bovine Serum Albumin (BSA).

Question 38

With what will the BSA react?

Answer 38

The biuret agent.

Question 39

What will the standard curve of BSA + biuret give us?

Answer 39

ε value which can be applied to any protein solution that reacts with biuret.

Question 40

What does the second part of the serum protein assay consist?

Answer 40

Measure quantities of protein + biuret reactions.

Measuring Absorbance of each reaction mixture.

Question 41

What are the methods of the experiment?

Answer 41

1. Using the 10 mg/ml Bovine Serum Albumin (BSA) stock prepare 1 ml of the following concentrations of BSA: 0 mg/ml (Blank), 1 mg/ml, 2 mg/ml, 4 mg/ml, 6 mg/ml, 8 mg/ml, 10 mg/ml in test tubes. You can dilute with the saline solution (0.9% NaCl).
2. Mix thoroughly
3. Take 1 ml of the solution you have been assigned (A, B or C)
4. Dilute the assigned sample 1 in 10 to give a final volume of 10 ml
5. Pipette 1 ml of the solution into 3 tubes
6. To all of the 10 tubes (1 blank, 6 calibration and 3 unknown) add 4 ml of biuret reagent.
7. Wait 30 minutes
8. Measure the absorbance of each tube at 550 nm
9. Plot the Absorbance readings of the BSA samples (on the Y-axis) as a function of BSA (i.e. protein) concentration in each reaction mixture (on the X-axis).
10. Calculate the gradient of the graph to determine .
11. Calculate the concentration of the unknown (don’t forget the dilution!)
12. You will need to enter your data on the spreadsheet at the front of the class.
13. Determine the prediction error associated with your result (this will be covered in the statistics sessions)