lecture 8 for student

SPECTROSCOPY PROTEIN DETERMINATION PHAM VAN HUNG, PhD Introduction cells, and almost all except storage proteins are important for biological functions and cell structure.. SPECTROSCOPY

SPECTROSCOPY

PROTEIN DETERMINATION

PHAM VAN HUNG, PhD

Introduction

cells, and almost all except storage proteins are important for biological functions and cell structure.

‰Component?

‰Amino acids?

‰Classification?

‰Structure?

‰Functional properties?

Quaternary structure

„ A protein has size and shape as well as unique arrangement of its polypeptide chains (Aggregation of several peptide chains to form a definite molecule by ionic bond, hydrogen bond, and/or hydrophobic bond).

SPECTROSCOPY

Importance of protein analyses

‰Nutrition labeling

‰Pricing

‰Functional property investigation

‰Biological activity determination

Importance of protein analyses

know:

1 Total protein content

2 Content of a particular protein in a mixture

3 Protein content during isolation and purification

of a protein

4 Nonprotein nitrogen

5 Amino acid composition

6 Nutritive value of a protein

Protein Determination Methods

1 Kjeldahl Method

2 Dye Binding Method

3 Biuret Method

4 Lowry Method

5 Ultraviolet Method

Kjeldahl Method - Nitrogen Determination

(1) Digestion to conver nitrogen into an ammonium ion (NH4).

+ conc H2SO4 + a catalyst (Copper sulfate)

(2) Neutralize NH4with NaOH to get NH3 (3) Steam distillation of NH3and trap in boric acid.

(4) Titrate with hydrochloric acid.

Calculation:

Gram nitrogen/ gram of sample =

*(ml of sample - ml of blank) × N (normality) of standard acid × 0.014g/meq

weight of sample

* ml of hydrochloric acid required to titrate sample solution.

Procedure and reactions

[2]

[5]

Conversion Factors from Nitrogen to Protein for Foods

Corns Milk Whole wheat Wheat flour Nuts

6.25 6.38 5.83 5.70 5.30

Beans Millet

SPECTROSCOPY

Advantages:

1 Applicable to all types of foods

2 Inexpensive (if not using an automated system)

3 Accurate; an official method for crude protein content

4 Has been modified (micro Kjeldahl method) to measure microgram quantities of proteins

Disadvantages:

1 Measures total organic nitrogen, not just protein nitrogen

2 Time consuming (at least 2 h to complete)

3 Poorer precision than the biuret method

4 Corrosive reagent

Dye Binding Method

Principle:

- At low pH, basic groups of protein are (+) charged These will

quantitatively bind a (-) charged dye

- Proteins bind the dye to form an insoluble complex The

unbound soluble dye is measured after equilibration of the

reaction and the removal of insoluble complex by centrifugation

or filtration

NH 3

CH

2

CH 2

CH 2

CH 2

N N

H

CH 2

C NH +

CH CH

N HC H

C N

CH 2

CH 2

CH 2

N

NH 2

NH 2

Lysine

Arginine

Histidine

Acid Orange 12:

N = N HO

SO3

-Procedure:

1 Mix protein, dye, buffer pH = 2

2 Filter or centrifuge

3 Measure absorbance of filtrate

Dye Binding Method

SPECTROSCOPY

Absorbance of dye bound by protein = A initial (free) die concentration - A

filtrate die concentration

Dye Binding Method

% Protein (Kjeldahl)

Skim milk

1

1

2

2

x

x

x x

Factors Influencing Dye Binding determination:

1 Temperature

2 Non-proteins

3 Buffers systems

4 Protein quality

Dye Binding Method

Biuret Method

Principles: Cu++in alkaline solution form complexity with

peptide bonds - give pinkish-purple color

Measure the intensity of color at 540 nm Standard is bovine

serum albumin (BSA).

% Protein (Kjeldalh)

Lowry Method

The Lowry method combines the biuret reaction with the reduction

of the Folin–Ciocalteau phenol reagent (phosphomolybdic-phosphotungstic acid) by tyrosine and tryptophan residues in

the proteins

• Cu++in alkaline solution to form complexity with protein

• Cu++ catalyses oxidation of phenol group of tyrosine with phosphomolybdic-phosphotungstic acid

Procedure

1 Proteins to be analyzed are diluted to an appropriate

range (20–100 μg)

2 K Na Tartrate-Na2CO3 solution is added after cooling

and incubated at room temperature for 10 min

3 CuSO4-K Na Tartrate-NaOH solution is added after

cooling and incubated at room temperature for 10min

4 Freshly prepared Folin reagent is added and then the

reaction mixture is mixed and incubated at 50◦C for 10

min

5 Absorbance is read at 650 nm

6 A standard curve of BSA is carefully constructed for

estimating protein concentration of the unknown

Application

Advantages:

1 Very sensitive

„ (a) 50–100 times more sensitive than biuret method

„ (b) 10–20 times more sensitive than 280-nm UV absorption method

2 Less affected by turbidity of the sample.

3 More specific than most other methods.

4 Relatively simple; can be done in 1–1.5 h.

Disadvantages:

1 Color varies with different proteins to a greater extent than the biuret method.

2 Color is not strictly proportional to protein concentration.

3 The reaction is interfered with to varying degrees by sucrose, lipids, phosphate buffers, monosaccharides, and hexoamines.

4 High concentrations of reducing sugars, ammonium sulfate, and sulfhydryl compounds interfere with the reaction.

SPECTROSCOPY

Ultra-violet Absorption (UV) at 280 nm

1 Proteins show strong absorption in the region at ultraviolet

(UV) 280nm, primarily due to tryptophan and tyrosine

residues in the proteins.Chromophoric side chains of

aromatic amino acids (Tyrosine, Tryptophan)

2 Because the content of tryptophan and tyrosine in proteins

from each food source is fairly constant, the absorbance at

280nm could be used to estimate the concentration of

proteins, using Beer’s law.

Procedure

1 Proteins are solubilized in buffer or alkali

2 Absorbance of protein solution is read at 280nm against a reagent blank

3 Protein concentration is calculated according to the equation

A = εlc

where:

A = absorbance

ε = absorptivity

l = cell or cuvette path length

c = concentration

Determination for Amino Acid Compositions of Proteins

A Hydrolysis

1 Overnight in 6 M HCl at 100 C

2 Enzymes

B Separation by ion exchange chromatography

Mechanism of Ion-Exchange Chromatography of

Amino Acids

Na +

Na +

H + OH - = H 2 O

= H2O

OH

H +

Na +

Na +

COO

-H 3 N +

Na +

OH

COO

-N +

H 3

H 3 N +

COOH

OH

COOH COOH

H 3 N +

So 3

-SO 3

-SO3

-So 3

-So 3

-SO3

-H3N +

Exchange Resin

pH 2

pH3.5

pH4.5

LYS

HIS

ASP GLU

ALA VAL

LEU

Moles/Liter

Chromatogram of Amino Acids

The end!