Amino acids, Proteins and DNA

general structure of amino acid

optical activity of amino acids

all amino acids, except glycine, display optical isomerism bc they are chiral

• they can rotate plane polarised light

zwitterions

• molecules with both positive and negative ions

  • only exist at the amino acids isoelectric point (the pH at which the overall charge is zero)

• the ionic interaction between zwitterions explains the relatively high melting points of amino acids

  • as opposed to the weaker hydrogen bonding that would occur in the no charge form

acidity & basicity of amino acids

• the amine group is basic and the carboxylic acid group is acidic

• so amino acids are amphoteric

• when in an alkaline solution with high pH

  • at high pH, the NH3+ is likely to lose a H+ ion, forming NH2

• when in an acidic solution with low pH

  • at low pH, the COO- is likely to accept a H+ ion, forming COOH

• amino acids act as weak buffers

  • will only gradually change the pH if small amounts of acid or alkali are added

dipeptides

combination of two amino acids with one amide (peptide) link

• must have CONH link !!

esterification reaction of amino acids with alcohol

• shows the reaction of carboxylic acid with an alcohol (e.g. methanol) in the presence of a strong acid catalyst (H+ e.g. sulfuric acid)

• forms an ester and water (byproduct)

esterification reaction of amino acids with acyl chloride

• shows the reaction of carboxylic acid with an acyl chloride

• basically the Cl gets removed alongside one H, and the NH then bonds with COCH3

what happens when an amino acid reacts with an excess of a haloalkane

excess haloalkane (e.g. bromomethane) + amine (NH2) in amino acid results in a successive nucleophilic substitution reaction

• (rmbr that NH2 is a nucleophile!)

• results in the formation of a quaternary ammonium ion

draw the structure of the species formed when glycine reacts with an excess of bromoethane

hydrolysis of dipeptides/proteins

• if proteins are heated with concentrated hydrochloric acid or concentrated strong alkalis they can be hydrolysed and split back into their constituent amino acids

• the composition of the protein molecule may then be deduced using TLC chromatography

TLC chromatography steps

1. Wearing gloves, draw a pencil line 1 cm above the bottom of a TLC plate

2. Use a capillary tube to add a tiny drop of each solution to a different spot on TLC plate

3. allow the plate to air dry

4. Add solvent to a chamber with a lid

   • ensure solvent is below pencil line

5. When the level of the solvent reaches about 1 cm from the top of the plate, remove the plate and mark the solvent level with a pencil

6. Allow the plate to dry in the fume cupboard.

7. Spray paper with ninhydrin

   • Draw around them lightly in pencil.

8. Calculate the Rf values of the observed spot

importance of wearing gloves

to prevent contamination form the hands to the plate

importance of pencil line

so it will not dissolve in the solvent

importance of tiny drop

too big of a drop will cause different spots to merge

importance of the depth of the solvent

if the solvent is too deep it will dissolve the sample spots/mixture from the plate

importance of the lid

to prevent evaporation of toxic solvent

importance of drying in a fume cupboard

important as the solvent is toxic

is allowing the solvent to rise to the top of the plate essential?

not essential

• will get more accurate results if the solvent is allowed to rise to near the top of the plate

• but the Rf value can be calculated if the solvent front does not reach the top of the plate

why is ninhydrin sprayed on the amino acids?

bc amino acids are transparent and cannot be seen

• can also shine UV light to see the position of the spots

outline the steps needed to locate the positions of the amino acids on the TLC plate and to determine their Rf values

• spray with developing agent or use UV

• measure distance from initial pencil line to the spots (x)

• measure distance from the initial pencil line to the solvent front line (y)

• Rf value = x/y

how to identify the amino acid using the Rf value

1. measure how far each spot travels relative to the solvent front and calculate the Rf value

2. each amino acid has its own Rf value

3. compare Rf values to those for known substances

explain why different amino acids has different Rf values

• amino acids have different polarities

• therefore they have different retention on the stationary phase and different solubility in the developing agent

suggest why lysine leaves the column after alanine

• lysine has a more positive charge

• so it has a greater affinity to the stationary phase

  • so it adheres better to the stationary phase

proteins definition

polymers made from combinations of amino acids formed by condensation reactions

• linked by peptide bonds

primary structure

the sequence of amino acids joined together via condensation reaction, forming peptide bonds

secondary structure

alpha-helix & beta-pleated sheets

• held in place by H-bonds between the H in N-H group and the O in C=O group

tertiary structure

folding of secondary structure to form 3D shapes

• held in place by interactions between R groups (e.g. H-bonds, ionic bonds and disulfide bridges)

stereospecific active site

If the substrate is chiral then it’s likely that only one enantiomer will have the correct stereochemistry to fit in the active site of the enzyme

• so only one isomer will be catalysed.

Drugs as Enzyme Inhibitors

• Many drugs act as an enzyme inhibitor by blocking the active site

• The inhibitor will often bind to the active site strongly so stopping the substrate attaching to the enzyme

• Some inhibitors can also attach elsewhere on the enzyme but in doing so can change the shape of the active site, which also stops its effectiveness

how sugar-phosphate chain is formed

explain how cytosine forms a base pair with guanine

• the top N-H on cytosine forms a H-bond to the l.p. of electrons on the oxygen on guanine

• the l.p. of electrons on nitrogen in cytosine H-bonds to the H-N on guanine

• a l.p. of electrons on the oxygen on cytosine bonds to the lower H-N on guanine

why can no other base pairs in DNA beside A-T and C-G form?

no other base pairing can be formed because:

• the partially charged atoms would be too close to each other and repel

• the bases would not get close enough to each other for H-bond to form

complementary base pairing

cisplatin

• Cisplatin prevents DNA replication in cancer cells by a ligand replacement reaction with DNA

  • coordinate bond is formed between platinum and a nitrogen atom on guanine

• Cisplatin can also prevent the replication of healthy cells by attaching to healthy DNA in normal cells

why does cisplatin work as an anticancer drug but transplatin doesn’t?

The cisplatin version only works as two chloride ions are displaced and the molecule joins on to the DNA

• In doing this it stops the replication of cancerous cells

side effects of cisplatin and how it can be minimised

use of cisplatin may lead to unwanted side effects like hair loss.

• unwanted side effects can be minimised by giving cisplatin in small doses/amounts and targeting the application to the tumour