PROTEIN ANALYSIS Abdul Rohman Laboratorium Kimia Analisis, Bagian Kimia Farmasi, Fakultas Farmasi UGM

Protein

Protein Content of Some Foods

PROTEIN ANALYSIS Protein analysis is required 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

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

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.

(A Guide to Kjeldahl Nitrogen Determination Methods and Apparatus) 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)

Kjeldahl method 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).

Total organic nitrogen - Kjeldahl method Calculation %Protein = %N  conversion factor Conversion factor: generally 6.25 most protein: 16% N Conversion factor egg or meat 6.25 milk 6.38 wheat 5.33 soybean 5.52 rice 5.17

Kjeldahl Apparatus

Kjeldahl method measuring total N not just protein N time consuming 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 Reaksi antara protein dengan formaldehid membentuk dimetilol. Gugus karboksilat bebas selanjutnya dititrasi dengan NaOH

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

UV ABSORPTION (280 nm) 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

Biuret Method Cupric ions react with peptide bonds under alkaline conditions (copper sulfate + K-Na-tartrate + alkali) Measure color in SPECTOPHOTOMETER at 540 nm

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- Disadvantages This 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 concentration Recent evidence suggests that sucrose, lipids, some buffers, monosaccharides and hexosamines react to varying degrees with the reagents in the Lowry test d. 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.

Faster and more convenient that Kjeldahl Disadvantages are 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.

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.

DYE BINDING METHOD 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. 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 Lowry Disadvantages - color not stable with time  precise timing - reducing sugars interfere more than Lowry - color variations between proteins occur absorbance 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).