PROTEIN ANALYSIS

Abdul RohmanLaboratorium Kimia Analisis, Bagian Kimia Farmasi, Fakultas Farmasi

UGM

Protein

H2N CH C

R

OH

O

H2N CH C

R

OH

O

+ .........

H2N CH C

R

O

HN CH C

R

O

HN CH C

R

OH

O

n

+

asam amino asam amino

ikatan peptidaikatan peptida

Protein Content of Some Foods

Milk (dry) 22-25 Rice 7.5-9

Milk (skim) 3.2 Wheat flour 9.8-13.5

Egg (dry) 35 Walnuts 15-21

Beef (dry) 81-90 Potato 10-13

PROTEIN ANALYSIS Protein analysis is required to know:

1. total protein2. amino acid composition3. amount of a particular protein in a mixture4. protein content during isolation and purification5. Nonprotein nitrogen6. Nutritional value of a protein

Methods for Protein Analysis• Kjeldahl (Volumetri)• Formol (Volumetri)• Spektroskopi (Biuret dan Lowry)• Chromatography

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Kjeldahl method

• Crude protein content• Johan Kjeldahl (1883) developed the basic process• Principle: total organic N released from sample and absorbed

by acid

Protein analysis in foods has been mostly done by determining Nitrogen Content

• Nitrogen is: largely unique to protein – only MAJOR constituent of foods containing N.

• Other nitrogenous compounds (NPN): Chlorophyll, nucleic acids, some vitamins, lecithins, urea, amino sugars, alkaloids, ammonium ions, etc.

• On the average, food proteins contain 16% nitrogen. 100% divided by 16% = 6.25. Therefore multiply N content by 6.25 to get protein content.

Protein analysis in foods has been mostly done by: Determining Nitrogen content

• The most common and well accepted method for determining nitrogen in food is the Kjeldahl method….(Kel-Dall)

• Labconco is one of the industry leaders in Kjeldahl equipment:

• http://www.labconco.com/pdf/kjeldahl/index.shtml• Click on “Kjeldahl” and download the brochures.

(A Guide to Kjeldahl Nitrogen Determination Methods and Apparatus)

3 Stage Process:

• 1. Digestion with acid + catalysis

• 2. Distillation with steam and alkali

• 3. Titration with acid and indicators.

Kjeldahl method

• 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.

Kjeldahl method

• 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 and pH indicators.

• 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).

Kjeldahl method

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Total organic nitrogen - Kjeldahl method

• Calculation%Protein = %N conversion factorConversion factor: generally 6.25 • most protein: 16% N

Conversion factoregg or meat 6.25milk 6.38wheat 5.33soybean 5.52rice 5.17

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Kjeldahl Apparatus

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Kjeldahl method

• Advantages:• applicable to any foods• simple, inexpensive• accurate, official method for crude protein content

• Disadvantages:

• measuring total N not just protein N• time consuming• corrosive reagents

ALTERNATES TO DISTILLATION AND TITRATION

1. NESSLERIZATION = reaction of ammonia with mercuric iodide to produce ammonium dimercuric iodide which can be read in a SPEC (visible @ 440nm)

2. Reaction with phenol and hypochlorite to form indophenol which can be read in a SPEC (visible @ 630nm)

Titrasi Formol• Pada titrasi formol digunakan formaldehid untuk menutup

gugus amin dan membentuk metilol. • Metode ini digunakan untuk penetapan kadar protein dalam

susu secara cepat. • Oleh karena protein mempunyai gugus karboksilat dan gugus

amina, maka protein bersifat netral. Bila gugus –NH2 dinonaktifkan oleh formaldehid menjadi bentuk dimetilol, maka gugus karboksilat akan bersifat asam yang selanjutnya dapat dititrasi secara alkalimetri dengan larutan baku NaOH

Reaksi antara protein dengan formaldehid membentuk dimetilol. Gugus karboksilat bebas selanjutnya dititrasi dengan NaOH

N-dimetilol

H2N CH C

R

O

HN CH C

R

O

HN CH C

R

OH

O

n

N CH C

R

O

HN CH C

R

O

HN CH C

R

OH

O

n

HOH2C

HOH2C

NaOH

2 HCOH

Spectroscopic-based techniques

• UV spectroscopy• Visible spectroscopy

• BIURET• FOLIN• LOWRY• NINHYDRIN• DYE BINDING METHOD• BICINCHONIC ACID (BCA) METHOD

UV Absorption (280 nm)

• Most proteins exhibit strong UV light absorption at 280 nm because they contain “chromophoric” side chains such as tyrosine, tryptophan, and phenylalanine.

• The concentration of protein in a non-turbid solution is proportional to the absorbance

Advantages- rapid and sensitive-Nondestructive

-Disadvantages- nucleic and phenolic acids also absorb at 280 nm- amounts of Trp and Tyr vary with protein types- turbidity (cloudiness in solution) is a problem

Applications of method? Not widely accepted for general food analysis – more useful for research purposes by monitoring the extraction or separation of proteins

UV ABSORPTION (280 nm)

Biuret Method

• Cupric ions react with peptide bonds under alkaline conditions

• (copper sulfate + K-Na-tartrate + alkali)• Measure color in SPECTOPHOTOMETER at 540 nm

N

N

OH

RH

OH

Cu2+

OH-

ON

N

HH

H

R

O

N

N

OH

RH

OH

Cu2+

Purple biuret complex

BIURET METHOD

Advantages:- Cheaper and faster than Kjeldahl- Less problem with color deviations- Few substances interfere- Does not measure non protein nitrogen (NPN)

Disadvantages:-not sensitive: to 2-4 mg level-color depends on protein-- PROTEIN MUST BE SOLUBLE

Folin-Ciocalteu

• Used to determine reducing compounds• Reduction of phosphomolybdic-phosphotungstic acid by

reducing groups in a sample.• Once reduction has occurred, the solution is made alkaline and

a blue color is formed.• Can be used directly for proteins, pure sugar solutions, or

phenolic compounds.

LOWRY METHOD• The Lowry method is a colorimetric method

based on the formation of a blue color formed

• Tyrosine and/or tryptophan in a protein reduces a phosphomolybdic-phosphotungstic reagent (Folin-Ciocalteu reagent) in the presence of K-Na-tartrate in alkali (Biuret reagent)

• Absorbance values are determined on a spectrophotometer at 750 nm.

• As little as 0.2 mg protein in a sample can be determined.

LOWRY METHOD- Advantages• This is the most sensitive spectrophotometric

method available for determining total protein. It is 10 to 20 times more sensitive than UV absorption at 280 nm (next topic)

• This method is more specific• The Lowry method is relatively rapid,

requiring 1-2 hours for analysis• Widely used in biomedical field

LOWRY METHOD- DisadvantagesThis method requires careful standardization (making a good standard curve) because a. The amount of color varies with different proteins. b. The color is not strictly proportional to the concentrationc. Recent evidence suggests that sucrose, lipids, some buffers, monosaccharides and hexosamines react to varying degrees with the reagents in the Lowry testd. High concentrations of ammonium sulfate, sulfhydryl compounds, and phosphate can interfere

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.

NinhydrinAdvantages 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.

Metode pengikatan warna (Dye binding method)• Gugus polar dalam protein dapat mengikat zat warna yang

bermuatan berlawanan dengan muatan pada protein membentuk kompleks protein-zat warna yang tidak larut.

• Zat warna yang bersifat basa mengikat gugus asidik pada permukaan protein seperti pada asam glutamat dan asam aspartat.

• Zat warna yang memiliki gugus asam seperti COO- dan SO3- akan

mengikat rantai samping asam amino yang bersifat basa seperti lisin, histidin, dan arginin.

• Zat warna yang sering digunakan adalah zat warna asidik seperti Amido Black 10B ( max 615 nm) dan Orange G ( max 485 nm). Amido Black dan orange G bersifat asam karena punya 2 gugus –SO3H.

O2N N N N N

NH2 OH

HO3S SO3HAmino black 10 B

N N

SO3H

HO3S

HO

Orange G

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

The unbound dye is then easily determined with a spectrophotometer using a standard curve

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

DYE BINDING METHOD

DYE BINDINGAnionic dyes and Bradford Main types

Anionic dyes use principle that proteins bind dyeand become insoluble.

Basic groups bind anionic dye (+ charge at neutralpH)

Unbound dye inversely proportional to protein

Ads: quick, can estimate available lysine, nonasty reagents, NPN does not interfere, precise

Disads: not sensitive, calibration curverequired for each protein, many interferences

more proteinless color

More Protein,Less Color

Diagram sebaran yang menunjukkan plot antara kandungan nitrogen protein terhadap metode pengikatan warna untuk 73 sampel serbuk kentang

Sumber: Wu, W.B. and Lakin, A.L. 1993. Estimation of protein in potato tissue by dye binding. Food Chemistry 46: 49-53

BICINCHONIC ACID (BCA) METHOD

Proteins reduce cupric ions to cuprous ions under alkaline conditions. Cuprous ions react with BCA reagent to give a purple color.

The BCA assay primarily relies on two reactions.

• Firstly, the peptide bonds in protein reduce Cu2+ ions from the cupric sulfate to Cu1+ . The amount of Cu2+ reduced is proportional to the amount of protein present in the solution.

• Next, two molecules of bicinchoninic acid chelate with each Cu1+ ion, forming a purple-colored product that strongly absorbs light at a wavelength of 562 nm.

BICINCHONIC ACID (BCA) METHOD

Advantages- as sensitive as Lowry but simpler- reagent more stable than LowryDisadvantages- color not stable with time precise timing- reducing sugars interfere more than Lowry- color variations between proteins occurabsorbance vs concentration not absolutely linear

Dumas Method• Prinsip metode: • sampel dibakar pada suhu yang sangat tinggi (700 – 800

oC). Nitrogen yang dilepaskan dianalisis secara kuantitatif dengan gas kromatografi menggunakan detektor konduktifitas termal. Nitrogen yang ditentukan selanjutnya diubah menjadi kandungan protein dalam sampel.

• Metode pembakaran Dumas ini sesuai untuk semua jenis makanan. AOAC menggunakan metode ini untuk analisis protein dalam daging dan dalam serealia (metode AOAC 992.15 dan metode 992.23).