Lab 5 - Reactions of amino acids

Question 1

Methods to determinate a.a.

Answer 1

R. of reactive groups:

a) General: amino- and carboxyl groups
b) Specific: other groups

a) R. of amino groups
- Acylation
- Arylation
- Oxidation
- Ninhydrin R.
- Formol titrations
- Deamination of a.a. w. FDNB
- Danzyl reaction

b) Detection of other reactive groups
- Identification of sulphydryl groups
- Million´s R for detection of tyrosine

Question 2

Ninhydrin R.

• What does ninhydrin do?
• Observation?
• Materials?

Answer 2

• Deamines alpha-a.a. by condensation
• Purple, with proline yellow. Collidine-ninhydrin gives various colours w. diff. a.a.
• Chromotograph, ninhydrin sol., hairdryer, vaporizer

Question 3

Formol titration

• What does the formaldehyde do?
• How can the a.a. conc. be calculated?
• Materials?

Answer 3

• R. w. amino groups and form methylol compounds
• H+ obtained in the R. can be titrated.
• A.a. sol, formaldehyde, NaOH, Phenolphtalein indicator

Question 4

Deamination of a.a. w. FDNB

• FDNB stands for?
• What does FDNB do?
• Criterias?
• Obs.?
• How can we determine the Rf?
• Hydrolysis?

Answer 4

• 1-fluoro-2,4-dinitrobenzene
• R. w. N-term. of a.a. and form DNP-a.a.
• Alkaline milieu, room temp.
• Yellow colour
• With chromotography
• Resistant to acidic hydrolysis

Question 5

All labtests?

Answer 5

• Ninhydrin R.
• Formol titration
• Identification of sulphydryl groups

Question 6

Danzyl reaction

• What does the danzyl-reagent do?
• Criterias?
• How can the prod. be visualized?
• Hydrolysis?

Answer 6

• R. w. N-term. of a.a. and form danzyl-a.a.
• Alkaline milieu
• With flueroscence
• Resistant to acidic hydrolysis

Question 7

Identification of sulphydryl groups

• Which a.a.?
• Criterias?
• How?
• Obs.?
• Materials

Answer 7

• Cysteine
• Alkaline milieu
• Two cysteine bound together form cystine, two H+ released reduced Fe3+ to Fe2+.
• Purple (free -SH with Fe3+) -> colourless (Fe2+)
• Cysteine sol., Na2CO3, Fe2(SO4)3

Question 8

Million´s R for detection of tyrosine

• Principle?
• Materials?

Answer 8

• Comp. cont. p-hydroxyphenyl groups R. with mercuric salts in nitric acid forming red complex
• Diluted blood serum, tyrosine, glycine, Million´s reagent

Question 9

Hopkins-Cole´s R. for detection of tryptophan

• Principle?
• Materials?

Answer 9

• Tryptophan forms a violet condens. prod. w. aldehydes in alkaline milieu
• Milk(tryptophan), acetic acid, sulphuric acid

Question 10

Sep. of a.a. from their mixture

Answer 10

1. Thin layer chomotography

2. Ion exchange chomotography

Question 11

Thin layer chromatography

• Support media?
• Principle?
• Which flow fastest?
• How is the a.a. faciliated in the sep.?
• How is the components visualized?
• How is the retention factor measured?
• Materials?

Answer 11

• Cellulose, silicagel, alumina oxide layer on glass/plastic
• Princ.: Solubility of diff. a.a. depends on solvent
• Fastest: Apolar solvents->furthest away from start line
• Faciliated by the fact they have diff. affinity to the medium. Apolar a.a. binds with lower strength.
• Visualized by staining R.-ninhydrin R.
• Rf=dist. moved by solvent/dist. moved by a.a.(Rf

Question 12

Ion exchange chromatography

• Support media?
• Principle?
• What does the strength of the bond depend on?
• How can the a.a. be eluted?

Answer 12

• Diff. synthetic resins cont. diff. ionized groups
• A medium w. neg.charged group binds to a.a.-cations. (DEAE-cellulose, w. SO3^-).
• Depends on pH and dissociation properties of a.a.
• Eluted by: gradually increasing pH.