PROTEIN ANALYSIS Abdul Rohman Laboratorium Kimia Analisis, Bagian Kimia Farmasi, FakultasFarmasi UGM
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 (Biuretdan 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.
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 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)
TitrasiFormol • 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
Reaksiantara protein denganformaldehidmembentukdimetilol. GuguskarboksilatbebasselanjutnyadititrasidenganNaOH
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-tartratein 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'spurple. Read at 570 nm.
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.
Metodepengikatanwarna (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 bersifatasamkarenapunya 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 antarakandungan nitrogen protein terhadapmetodepengikatanwarnauntuk 73 sampelserbukkentang • 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 • Prinsipmetode: • sampeldibakarpadasuhu yang sangattinggi (700 – 800 oC). Nitrogen yang dilepaskandianalisissecarakuantitatifdengan gas kromatografimenggunakandetektorkonduktifitastermal. Nitrogen yang ditentukanselanjutnyadiubahmenjadikandungan protein dalamsampel. • Metodepembakaran Dumas inisesuaiuntuksemuajenismakanan. AOAC menggunakanmetodeiniuntukanalisis protein dalamdagingdandalamserealia (metode AOAC 992.15 danmetode 992.23).