Bio
1510: Amino Acids (Moti Nissani’s Lab)
Glycine is the simplest amino
acid. The N2H is the amine
group. The COOH is the carboxyl group. The C at the center, which is attached to
the C of the carboxyl group, is called the alpha C (α–carbon). This C atom is attached to one H and to an R
group. Each amino acid has its own R
group (in glycine, it’s H; in alanine, it’s CH3—see p. 21).
When two amino acids combine into a single molecule, they do so by releasing water (dehydration synthesis), and by joining the C of the carboxyl group of one amino acid with the N of the amino group of the other. This C-N bond is called a peptide bond. When the chain contains only a few amino acids, the resultant midsize molecule is called a peptide (just 2 amino acids=dipeptide; 3=tripeptide . . . ). When the molecule is large, containing many amino acids, it’s called a polypeptide. A protein, in turn, is made up of one or more polypeptide chains.
Substances used today:
· Seven amino acids
·
Urea 
Highly soluble in water. Found in urine. Irritating to skin and
eyes.
· Biuret (“bi-urea”) is made up when two molecules of urea combine (by eliminating ammonia, NH3, see p. 23). At room temperature, it’s a white solid, soluble in hot water.
· Casein (from Latin caseus "cheese") is a protein common in milk and cheese. It has a lot of prolines, and is poorly soluble in water.
· Hydrolyzed casein—the protein had been broken down to smaller peptides, or to its constituent amino acids.
· Beef broth should also contain amino acids.
· Cow Milk: Casein is prominent, but there are dozens of other proteins (also, the disaccharide lactose)
How do you detect the presence of amino acids? Today we
shall combine two approaches in one test.
The first, ninhydrin test, involves a chemical reaction that occurs at high temperatures. Ninhydrin (triketohydrindene hydrate) is a chemical used to detect ammonia and other amine group containing substances such as amino acids. This test can, on its own, uniquely identify the amino acid proline (it’s the only amino acid that turns bright yellow in this test). But the colors produced by the other amino acids overlap, hence requiring an additional test. To overcome this problem, the ninhydrin is added to a thin layer chromatography solvent. Now, in addition go gaining color (by heating), and each amino acid travels to its own spot. So, at the end, we get colors (caused by a chemical reaction to ninhydrin) and a unique retardation factor (Rf), for each acid.
This in turn provides us the standard for the 7 amino acids—in this lab we find out the color and location of each on our chromatogram. Now we can ask ourselves: Which of these 7 amino acids are present in the other 6 natural substances in our sample? (the 6 are: urea, biuret, casein, hydrolyzed casein, beef broth, milk).
The Biuret reagent (=Benedict Reagent) contains copper in alkaline solution (it does not contain the chemical biuret). Rather, it is used in the biuret test (for 3 or more peptide bonds) to test for the presence of the substance biuret (each biuret molecule has 4 C-N bonds, so here we only expect a slight change in color), and also for the presence of at least 3 C-N bonds per molecule. When 3 or more peptide bonds (C-N) are present in a molecule, the copper is reduced and the solution turns from blue to varying shades of purple (the exact shade depending on the number of C-N bonds in each molecule, and the quantity of the substances involved).
ŕA positive reaction in the biuret tests tells us that we have peptide bonds, NOT proteins. If we want to establish the presence of proteins, we can, e.g., hydrolyze the mixture, then carry out the ninhydrin test.
Here is a reminder of chromatography and Rf values (courtesy of Wikipedia):
|
|
Retardation
factor, RF, is a ratio of the distance traveled by the
center of the spot to the distance simultaneously traveled by the mobile
phase: RF = b/a The RF value is characteristic for any given compound on the same stationary phase using the same mobile phase for development of the plates. Hence, known RF values can be compared to those of unknown substances to aid in their identifications. |
As far as I can tell (??), today we expect something like the following:
|
|
1-glu |
2-pro |
3-tyr |
4-cys (has S) |
5-ala |
6-arg |
7-gly |
8-urea |
9-biuret |
10-casein |
11-hyd casein |
12-beef |
13-milk |
|
N-TLC (for aa) |
Purple? |
yellow |
Purple? |
Purple? |
Purple? |
Purple? |
Purple? |
? |
? |
No? |
Light Yellow? |
yes |
No? |
|
Biuret (for >3 C-N) |
no |
no |
no |
Yellow /green/ black? |
no |
no |
no |
no |
Weak pink? |
Purple? |
No? |
No? |
purple? |
How-To Comments:
· Make the chromatography spot as small as possible (allow drying between applications)
· Handle TLC sheet gently, and do not touch them with your fingers
· Draw lines gently with dull-pointed pencil