Chemistry 3251 Laboratory Report

Experiment #5 – Protein Analysis

Ryan Moore: 960514735

Purpose: This experiment was designed to understand more of the chemistry involved with proteins and their reactions. It was broken into 7 parts:

1) Biuret test – to test complexes with Copper ions with the protein and measure the absorbance of standard protein concentrations and determine the concentration of an unknown sample. We will be using unknown sample I.

2) Strong Acids – to note the reactions of proteins with strong acids (HCL, HNO₃ and H₂SO₄).

3) Heavy Metal Cations – to note the reactions (and precipitations) that proteins have with heavy metal ions (namely Copper, Lead, and Silver).

4) Alkaloidal Reagents – to note reactions that proteins have with TCA (trichloroacetic acid), PWA (phosphotungstic acid), and TA (tannic acid).

5) Salting – to study the precipitation reactions with proteins.

6) Organic Solvents – to study the affects of an organic solvent on proteins.

7) Sevag Test – to study about the Sevag test (physical shaking of a molecule).

Method: follow the procedures for this experiment as laid out in the Chemistry 3251 Lab Manual.

Theory:

Biuret Test

This test is a little bit of a misnomer due to the fact that there is no biuret being used. What is being used is copper (II) ions in solution. A famous solution is a complex that biuret makes with the copper (II) ion, where 4 biuret molecules complex to the metal to form a highly coloured (violet/purple) solution.

Where the lone pair of electrons attach to the copper. Now it’s the amide type nitrogen that will attach in this way to the copper to give the purple colour. In proteins, since there are many many amide bonds (peptide bonds) we’d find that the biuret molecules would be replaced with individual peptide bonds from a protein. Sometimes even from the same protein. What we will do is prepare a standard protein concentration of varying degrees and measure it’s absorbance at 550nm in a spectrophotometer and make a graph of absorbance vs. concentration. From that we will do the same with an unknown protein concentration and determine it’s concentration from where it lays on the standard graph. A blank solution will be run and subtracted from the rest of the readings to reduce internal errors.

Strong Acids

Taking a neutral pH solution of a protein we add a very strong acid (in this case we will use concentrated HCl, concentrated HNO₃, and concentrated H₂SO₄). We cause the carboxyl end(s) of the protein to remain undissociated and all amino groups to become fully protenated, resulting in an overall high positive charge for the protein. This high positive charge will disrupt the hydrogen bonds in the protein causing the 2^o and 3^o to break down as well as the high positive charge will then make an ionic salt with the strong acid’s conjugate base (Cl^-, NO₃^- and SO₄^2-) and precipitate out.

Heavy Metal Cations

Pretty much the same as the Strong Acids we add Copper, Lead and Silver cations to the solution. At the pH around neutral the protein has an overall negative charge. The protein will make an ionic salt with the available ‘heavy metals’ and precipitate out.

Alkaloidal

Certain acidic reagents react similarily as the Heavy Metals do in that the acid will react with the protein to form an insoluble salt. In this experiment we will use TCA (trichloroacetic acid to form protein trichloroacetate), TA (tannic acid to form protein tannate), and PWA (phosphotungstic acid to form protein phosphotunstinate).

Salting Out

A protein’s solubility is directly dependant upon the ionic strength of the salt that is in solution with it. In this experiment we use ammonium sulphate. With a salt with such a weak ionic character the solubility of the protein is decreased to a point where it will just precipitate out. What we will do is add ammonium sulphate until the protein is precipitated out and use the biuret test on the filtrate and on the remaining solution to determine where the protein is.

Organic Solvents

When a protein is placed into an organic solvent the hydrogen bonding making up the higher order structures of the protein is disrupted and will tend to ‘clump’ together precipitating out.

Sevag Test

In the Sevag test we actually physically ‘jarr’ the molecule into losing it’s higher order structure. Adding chloroform to the solution helps in breaking up the hydrogen bonding when the protein is ‘rearranged.’

Observations:

Biuret Test

(corrected values)

Attached is a graph

From the graph of the standard we find that the equation is:

Absorbance = 0.00109 + 0.63752[Concentration]

So for our unknowns we find a concentration of:

1 - 0.166128121470698958464048186723554 mg/mL

2 - 0.126913665453632827205421006399799 mg/mL

3 - 0.056327644622913790939892081817041 mg/mL

4 - 0.0249560798092608859329903375580374 mg/mL

now we know that in 1 there was 24 mL in the solution 2 of which was the protein. So from calculations there were:

1 - 1.993537457648387501568578240676 mg/mL

2 - 1.903704981804492408081315095985 mg/mL

3 - 1.68982933868741372819676245451 mg/mL

4 - 1.49736478855565315597942025348 mg/mL

since they were all from the same protein sample we find the average to be:

1.77110895 mg/mL

Strong Acid

Heavy Metal

Alkaloidal

Salting Out

1% egg white + 10.03 g of NaSO₄ formed a white precipitate. After filtration the precipitate (being put into a solution and adding biuret reagent) was found to have a violet colour and the solution from which the precipitate was filtered there was no colour change.

Organic Solvent

Sevag Test

Adding a few mL of chloroform to the 1% egg white solution and a vigorous shake a solid white precipitate was seen between layers.

Discussion:

1. The part of the protein structure that produces the coloured complex with Biuret is the amide, or peptide link. The lone pairs on the nitrogen will complex with the copper ion.

   The advantages to the biuret test is that the forming complex is highly coloured and a spectrophotometer would accurately tell the concentration. The disadvantage could be that not only one protein molecule could complex with copper and in the standard there may only have been one molecule per ion and that would interfere with the results.

   The blank was necessary due to some possible internal errors in either the spectrophotometer or due to other molecules in the solution or in the glass which will absorb at 550nm. Doing a test at a 0 concentration we know what to subtract from all the readings.

   For precipitation in the Heavy Metal test the carboxylate anions (or acidic side chains) and in the Alkaloidal the amino (or basic side chains) cations.

   The protein is least soluble at neutral.

   The denaturation of the egg white solution is due to the high concentration of the ammonium sulphate because of the low ionic strength between the two ions. This denaturation is reversible by adding amounts of either ion into solution. By Lechatelier’s principle, it would cause the equilibrium between the salt and the ions to go back towards the salt and decrease the concentration and the egg white would go back into solution.

   The alcohol and acetone affected the solution of the protein in the fact that the non polar solvent disrupted the affect to which the hydrogen bonds which held the protein in it’s higher order structures to the point where it falls apart. Not all proteins will be affected the same way by the same amount of volume, because some proteins are larger and will need a larger amount of non polar molecules to disrupt the structures.

   The chloroform aids the disruption of the higher order structures in the Sevag Test by weakening the hydrogen bonding. Using Urea and Analine in the alkaloidal test is there to show that that type of chemistry not only happens with just any amine or amino acid (as analine is an amino acid and urea is a di amide) and only with specific nitrogen (amino) compounds like peptides or proteins (poly-peptide).

References:

• http://msip.lce.org/vishwa/biuret1.htm

  Chemistry 3251 Lab Manual. Department of Chemistry, Lakehead University. 1998

  Principles of Biochemistry, 2^nd ed. Hortan, Moran, Ochs, Rawn, Scrimgeour. Prentice-Hall ©1996

  http://sophia.hou.midcounty.com/UreaMfg/urea.html