PROTEIN ANALYSIS

What is protein:

polymer of 20 a– amino acids

N is most distinguished element: among the composing elements of C,H, N, O, S, for some proteins: P, Cu, Fe, I.

N content in different proteins ringing from 13.4% -19.1%, and averagely 16%.

PROTEIN ANALYSIS

Ó Proteins are important components of food, providing biological and structural function.

Ó There importance for analysis is primarily related to their unique biological activities or functional properties, and for nutritional benefits.

Protein analysis: You need to know..

1. total protein

2. amino acid composition

3. amount of a particular protein in a mixture

4. protein content during isolation and purification

5. Nonprotein nitrogen

6. Nutritional value of a protein

Protein Methods

Soluble & Insoluble Protein – Kjeldahl & Dumas (combustion) – measures total nitrogen (N)

Soluble protein – Biuret, Lowry, UV, Dye binding, Bradford, Ninhydrin

KJELDAHL NITROGEN

Based on conversion of protein nitrogen to ammonia (NH3). At the same time, carbon and hydrogen are oxidized to carbon dioxide and water.

In the presence of sulfuric acid (H2SO4), the ammonia picks up a hydrogen forming ammonium sulfate (NH4)2SO4.

Then concentrated sodium hydroxide (NaOH) is added to the solution of ammonium sulfate + sulfuric acid. This raises the pH transforming ammonium (NH4+) into ammonia.

When the sample containing ammonia, sodium hydroxide and sodium sulfate is heated, the ammonia is driven off as a gas.

We can condense the ammonia gas and introduce it into a fluid that contains boric acid

Ammonia reacts with boric acid to form ammonium borate. This is a base.

Titrate ammonium borate to an endpoint with hydrochloric acid (we must know the normality of the hydrochloric acid used).

3 Stage Process:

1. Digestion with acid + catalysis

2. Distillation with steam and alkali

3. Titration with acid and indicators.

KJELDAHL Reactions

l Protein + H2SO4 à (NH4)2SO4 (Digestion)

l NH4+ + NaOH àsteam and heatà NH3 (g) + H2O (Distillation)

l NH3 (l) + H3BO3 à NH4+ + H2BO3- (Trapped)

l H2BO3- + HCl à H3BO3 (Titration)

KJELDAHL Advantages are

1. applicable to most food samples

2. simple

3. inexpensive

4. accepted as Official method

5. can measure mg levels of proteins

Disadvantages are

1.Measures total N not protein
2.Time consuming – at least 2 hrs
3. Poor precision when compared to other methods
4. Corrosive (dangerous) method

BIURET METHOD

Applications: Suitable for cereals, meat, soybean, and isolated proteins

Advantages:

Cheaper and faster than Kjeldahl

– Less problem with color deviations than other protein methods

– Few substances interfere

– Does not measure non-protein nitrogen (NPN)

Disadvantages:

not sensitive, only to 2-4 mg level

– bile pigments interfere

– ammonium salts interfere

– color depends on type of protein

– lipids and carbos can affect clarity of solution

– PROTEIN MUST BE SOLUBLE

Going to test soluble protein against a standard of BSA.

The reaction of peptide bonds in protein with copper ions in alkaline conditions will produce a purple color.

The intensity of the color is directly proportional to the amount of protein.

Quantify using a spectrophotometer (540 nm)

The lowry’s Method (Folin-phenol method)

Principle:

Folin reagent phosphomolybdic and phosphotungstic acid is reduced to a blue molybdenum complex, mainly by the phenolic groups of tryptophan and tyrosine.

Lowry greatly increased the sensitivity of the determination by preceding the reaction by pretreatment with a copper reagent in a basic medium.

DYE BINDING METHOD

l Proteins will bind to certain types of dye. When this binding occurs, the protein-dye complex will precipitate.

l The unbound dye is then easily determined with a spectrophotometer using a standard curve with varying dye concentrations.

l Using the amount of dye initially added to the protein solution, the amount of protein can be calculated.

Bradford assay-Coomassie Brilliant Blue

Coomassie Brilliant Blue solution will directly bind to specific amino acids and protein tertiary structures

The dye’s color changes from reddish-brown to blue

Absorbance at 595 nm read.

Pros:

Rapid assay

Useful when accuracy is not crucial

Applications: Finds use as rapid protein method in food and biomedical industry.

Ninhydrin

Primary amino groups on the end of proteins, peptides, and free amino acids will react with ninhydrin.

This reaction forms a strongly colored purple solution referred to as Ruheman’s purple. Read at 570 nm.

Sample can be tested for the amount of primary amino acids currently present, or sample can be alkaline hydrolyzed to increase the amount of these amino acids.

Ninhydrin

Advantages are

Faster and more convenient that Kjeldahl

Disadvantages are

Large dilutions are necessary for spec. reading.

Proteins differ in the dye binding capacity

Make standard curve based on predominant primary amino acid present in the food.

NPN, calcium, or phosphorous constituents will bind to the dye or to protein, causing interference.

Addition of a metal chelator (i.e.. oxalic acid) may help reduce binding.

1 Response so far »

  1. 1

    rara87 said,

    wahh,infonya bisa bwt ngerjain tugas, tq yup(^0^)v


Comment RSS · TrackBack URI

Tinggalkan Balasan

Isikan data di bawah atau klik salah satu ikon untuk log in:

Logo WordPress.com

You are commenting using your WordPress.com account. Logout / Ubah )

Gambar Twitter

You are commenting using your Twitter account. Logout / Ubah )

Foto Facebook

You are commenting using your Facebook account. Logout / Ubah )

Foto Google+

You are commenting using your Google+ account. Logout / Ubah )

Connecting to %s

%d blogger menyukai ini: