Hey, this pdf is all about "Applications and Techniques to Biomolecules" e.g. protein and amino acid analysis, peptide sequencing, the reaction of specific amino acid, colorimetric method, fluorimetric method, and microbiological method.

2. APPLICATION OF TECHNIQUES TO
BIOMOLECULES

3. PROTEINS & AMINO ACID ANALYSIS
1. Dansyl chloride.
2. Peptide Sequencing.
3. Reaction of Specific Amino Acids.
4. Colorimetric Methods.
5. Fluorimetric Methods.
6. Microbiological Methods.

4. 1. Dansyl Chloride:
o DimethylAminoNaphthalene-5-Sulphonyl Chloride.
o It is a reagent that reacts with N-terminal or free amino
groups in alkaline solution (pH 9.5-10.5).
o It forms Fluorescent molecules useful in biochemical studies.
o Highly sensitive.
o Best for small amount of proteins (peptides).
o Identified by chromatography and fluorescence detection
after acid hydrolysis.
o It produce stable blue or blue green fluorescent.

6. 2. Peptide Sequencing :
o A technique used to find out the amino acids sequence in protein.
o Treat with phenylisothiocyanate (PITC) or the Edman reagent.
o It removes one amino acid from the N- terminal end of the peptide
without disrupting other peptide bonds.
o Automatic protein sequencer used to analyze data and give results
automatically.
o It plays a vital role in targetting drugs to specific metabolic pathway.

7. 3. Reaction Of Specific Amino Acids:
o Amino acid react with each other in a typical acid-base
neutralization reaction to form a salt.
o It undergoes in reactions like: oxidative deamination,
transamination, decarboxylation, transmethylation, etc.
o Amino acids can also be identified by specific colour reactions, like:
Biuret test identifies two peptide linkages, Ninhydrin reaction is for
α-amino acids that form a purple, blue or pink colour complex &
A.A like Proline and Hydroxyproline give Yellow colour with
Ninhydrin.

9. 4. Colorimetric Method :
o It is the most common analytical technique used in biochemical
estimation in clinical laboratory.
o It involves the quantitative estimation of colour.
o The instrument used for the measurement of coloured substances is
colorimeter or photoelectric colorimeter or absorbtiometer.
o This instrument is operative in the Visible range (400-800 nm) of
the electromagnetic spectrum of light.
o The working of colorimeter is based on the principle of Beer-
Lambert law.

10. Beer-Lambert Law
The amount of energy
absorbed or transmitted by a
solution is proportional to the
solution's molar absorptivity and
the concentration of solute.
In simple terms, a more
concentrated solution absorbs more
light than a more dilute solution
does.

11. Example Of Colorimetric Methods
• Pauly’s Reagent reacts with Histidine &
Tyrosine to give Red coloured products.
• Erhlich’s Reagent gives a Purple-Red product
with Tryptophan & other Indoles. This reagent
gives a Yellow coloured product with aromatic
amines.

12. 4. Fluorimetric Method :
o It is the measurement of fluorescence when the molecules are
excited by radiation at certain wavelength.
o When these molecules return to ground state , emit light .
o It is measured by Fluorimeter or spectrofluorimeter.
o It is more improved method on specificity and sensitivity than
calorimeter.
o The quantitative analysis for the aromatic aminoaacids e.g:
Tyrosine & Phenylalanine.

13. Example(s):
Tyrosine:
 1-Nitroso-2-Naphthol reacts with it in the presence of sodium nitrite
that form unstable red compound which is converted by heating with
nitric acid to form a stable yellow fluorescent product.
 Resulting fluorescence measured at 570 nm with excitation at 460 nm.
Phenylalanine:
 It reacts with Ninhydrin in the presence of dipeptide to form a fluorescent
product.
 It stabilizes by an alkaline copper reagent to adjust pH to 5.8.
 The resulting fluorescence is measured at 515nm after excitation at 365 nm.

15. 6. Microbiological Method:
o Certain microorganisms require amino acid for growth.
o Many strains of such microorganisms have been produced
which show dependence on a particular amino acid.
o The assay is performed on a layer of agar which is incorporated
with a mixture of a suspension of Bacillus Subtilis spores,
growth nutrient and a fixed amount of metabolic antagonist.
o Blood soaked filter paper discs are placed on the surface of the
agar together with the range of phenylalanine standards which
incubated overnight at 37°C.

16. For Example:
Guthrie Test /PKU Test /Heel Prick Test is done to check whether a
newborn baby has the enzyme needed to use phenylalanine in his or
her body. Phenylalanine is an amino acid that is needed for normal
growth and development.