BIOCHEMISTRY SYLLABUS STATEMENTS B.7 (Part 1)

These syllabus statements are basically the HL part of B.2.

If you're not familiar with the B.2 content, I suggest you go and refresh your memory so this post makes more sense :)

---

Protein Assays

Protein assays commonly use UV-vis spectroscopy and a calibration curve based on known standards.

Determination of the concentration of a protein in solution from a calibration curve using the Beer–Lambert law.

Testing for protein

A qualitative test for proteins that you've probably done before is the Biuret's test. A volume of Biuret's reagent is added to the sample to be analysed.

The Biuret's test can be used to measure the concentration of a solution of proteins using colourimetry because of it's purple colour.

COlourimetry

Colourimetry is the determination of the concentration of a solution based on how much light it absorbs. A colourimeter can be used to measure how much light passes through the solution and can then calculate an absorbance value. This technique works for any solution as long as it's not colourless.

​FINDING THE OPTIMUM WAVELENGTH

In order to find the wavelength that is absorbed most by the solution, an absorption spectrum should be plotted. This is basically an absorbance against frequency/wavelength graph that will show how well each wavelength is absorbed. The wavelength with the highest absorption should be chosen

​HOW IT WORKS:

The complementary colour wheel

Coloured solutions absorb certain wavelengths of light depending on their colour. Like in topic 13.2, if a solution is a certain colour, it means that the complimentary colour of light is being absorbed by the sample. If we know how much light is absorbed by a known concentration value, we can estimate the concentration of unknown samples by comparing the amount of light it absorbs compared to the known sample.

CALIBRATION CURVES

Calibration curves are absorbance-concentration graphs that are used to compare unknown samples to. Absorbance values for samples of known concentration are taken and plotted on a graph, and a line of best fit is drawn. From this graph, any unknown concentrations can be determined by finding their absorbance values on the line of best fit and tracing back to the concentration.

​METHODOLOGY (For finding protein concentration)

1. Use the absorbance values of known concentrations of the protein solution to plot a calibration curve
2. Add Biuret solution to the unknown protein sample and test absorbance in a colourimeter.
3. Use absorbance value and line of best fit to find concentration value.

​THE BEER-LAMBERT LAW

A typical colourimetry setup. An incident beam, a cuvette of solution and a detector.

The light source will shine a beam of light through the cuvette of sample. The intensity of that light beam is measured, and given the symbol I0. The beam then passes through the sample in the cuvette, and a portion of the light is absorbed. The remaining light beam is detected at the detector, and it's intensity is given the symbol I.

​THE MATHS

In the data book, you're given the following formula:

What they don't tell you in this formula, is that both sides equal A, the Absorbance of the solution.

Above, I've added the absorbance to the formula and annotated the formula to show what each variable is.

​THE VARIABLES

Io

• The intensity of the initial beam

I

• The intensity detected after the beam has passed through the solution

A

• The absorbance value for the solution

ε (Epsilon)

• The molar absorptivity constant which is different for every substance 

l

• The path length. This is how much solution the light has to travel through. In a standard colourimetry experiment standard cuvettes are used which are 1cm, so it usually cancels.

c

• The concentration of the solution, which you're usually trying to find.

---

I think I'll have to split this into 3 parts...

There are 3 main parts to the B.7 syllabus statements that require quite lengthy explanations.

1. Beer-Lambert law and colourimetry
2. Enzyme inhibition (competitive and non-competitive)
3. Buffer calcs for amino acids