DETERMINATION OF PROTEIN

Dr. Hasnah Haron Nutrition Programme, PPSJK, FSK, UKMKL

PENGENALAN
Protein mkn adalah kompleks Berat molekular yg. berbeza : 5,000 hingga > 1 juta Daltons H,C,N,O,S 20 -asid amino bentuk blok protein Kandungan N dlm mkn : 13.4 - 19.1%

INTRODUCTION
Proteins are polymers of amino acids. Twenty different types of amino acids occur naturally in proteins. Proteins differ from each other according to the type, number and sequence of amino acids that make up the polypeptide backbone. Thus, protein have different molecular structures, nutritional attributes and physiochemical properties.

INTRODUCTION
Proteins are important constituents of foods for a number of different reasons. They are a major source of energy, as well as containing essential amino-acids, such as lysine, tryptophan, methionine, leucine, isoleucine and valine, which are essential to human health, but which the body cannot synthesize.

Asid amino secara umum

Ikatan peptida

INTRODUCTION
Classification of protein based on composition, structure, biologikal function, solubility. Eg : Simple protein amino acid. Conjugate protein non amino acid component. Denaturation of protein - heat, acid , alkali, organic solvent and detergent. Function and solubility of protein change as well.

INTRODUCTION
Protein : komponen terbanyak dalam sel jalankan fungsi biologikal. Fungsi utama protein:
1. Sumbang tenaga dan nutrien penting 2. Beri ciri fizikokimia kualiti dan atribut sensori 3. Fungsi biologikal in vivo
Enzim Hormon antibodi

INTRODUCTION
Analysis protein is complex food component that shows same physicochemicals as protein.
Eg : Nitrogen non protein free amino acid, small peptide, nucleic acid, fosfolipid, uric acid, urea

Lipid and carbohydrate can interfere with protein analysis.

INTRODUCTION
Total nitrogen in food consisted of mainly protein & non protein containing nitrogen.

Importance of protein analysis

Determination of biological activity Proteins are also the major structural components of many natural foods, often determining their overall texture Proteolitic enzyme to soften the meat Pectine for ripeness of fruit. Research on the functional characterization. Isolated proteins are often used in foods as ingredients because of their unique functional properties. Their ability to provide desirable appearance, texture or stability. Glutenin in flour - bread Casein cheese

Gelling agents, emulsifiers, foaming agents and thickeners.

Keperluan lain analisis protein

Kandungan total protein Komposisi asid amino Kandungan protein tertentu dlm campuran Nitrogen bukan protein Nilai pemakanan protein (Penghadaman, Protein Efficiency Ratio)

Kandungan protein dlm mkn

Sumber penting protein: mkn bersumber haiwan dan kekacang Eg : Daging lembu (stik) 29% Daging ayam (dada) 27% Ikan kod (di masak) 29% Telur 13% Kekacang 22%

Determination of protein concentration

1. 2.

3.

Determination of nitrogen total Determination of colorimetric and determination of protein total quantitative. Determination protein concentration using spectrophotometry

Determination of total nitrogen

1. Kaedah Kjehdahl

Johan Gustav Christoffer Thorsager Kjeldahl

Johan Gustav Christoffer Thorsager Kjeldahl (1849 1900), Danish chemist who developed a method for determining the amount of nitrogen in certain organic compounds using a laboratory technique which was named the Kjeldahl method after him.

Johan Gustav Christoffer Thorsager Kjeldahl

Kjeldahl worked in Copenhagen at the Carlsberg Laboratory, associated with Carlsberg Brewery, where he was head of the Chemistry department from 1876 to 1900. He was given the job to determine the amount of protein in the grain used in the malt industry.

Kjeldahl method
His laboratory technique for nitrogen and protein analysis is still the universally accepted method for this analysis. Other methods claim to be faster and more efficient, but cannot cope with the variety of sizes or conditions of samples than Johan Kjeldahl's original method. Kjeldahl equipment is used extensively all over the world.

Kjeldahl

Kjehdahl method
A food is digested with a strong - releases nitrogen which can be determined by a suitable titration technique. The amount of protein present is then calculated from the nitrogen concentration of the food. The same basic approach is still used today, although a number of improvements have been made to speed up the process and to obtain more accurate measurements It is usually considered to be the standard method of determining protein concentration.

Kjehdahl method
Kjeldahl method does not measure the protein content directly a conversion factor (F) is needed to convert the measured nitrogen concentration to a protein concentration. A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of protein) is used for many applications. The Kjeldahl method can conveniently be divided into three steps: digestion, neutralization and titration.

Principals of the Kjehdahl method

1. Digestion
Sample + sulfuric acid + catalyst (anhydrous sodium sulfate ) + copper Heat

Kjehdahl digestion flask

1. Digestion
Digestion converts any nitrogen (N) in the food and others in the form of nitrates or nitrites) into ammonia. Ammonia is in the form of the ammonium ion (NH4+) which binds to the sulfate ion (SO42). N(food) (NH4)2SO4

2. Neutralization
Product of digestion + H20 + addition of NaOH) NH4 to NH3
(NH4)2SO4 + 2 NaOH 2NH3 + 2H2O + Na2SO4

3. Distillation
The ammonia gas goes into the receiving flask - which contains an excess of boric acid. The low pH of the solution converts ammonia gas into the ammonium ion and converts the boric acid to the borate ion.

NH3 + H3BO3 (boric acid)

NH4+ + H2BO3- (borate ion)

4. Titration
The nitrogen content is then estimated by titration of the ammonium borate by H2SO4 or HCl

H2BO3- + H+

H3BO3

5. Calculation
Protein kasar dikira berdasarkan kandungan nitrogen (dari amaun ion ammonia) X faktor protein

Calculation
The following equation can be used to determine the nitrogen concentration of a sample that weighs m grams using a xM HCl acid solution for the titration:

Where vs and vb - titration volumes of the sample and blank 14g - molecular weight of nitrogen N.

Calculation
Once the nitrogen content has been determined it is converted to a protein content using the appropriate conversion factor:

%Protein = %N

X Protein factor

Faktor protein
Campuran protein secara am mengandungi 16% Penentuan dari kandungan nitrogen perlu X faktor protein iaitu 6.25(100/16).

Faktor Protein
Sumber protein Sumber haiwan Susu Nasi Gandum Kacang soya Kacang Tepung Bijirin lain Faktor protein 6.25 6.38 5.95 5.83 5.70 4.6 5.70 6.25

Kebaikan dan keburukan kaedah Kjehdahl

Kebaikan Diguna semua jenis makanan Ringkas Murah Tepat Keburukan Ukur nitrogen organik secara total Masa agak panjang Reagen menghakis Kurang persis

Kaedah Kjehdahl
Prosedur Kjehdahl yg asal AOAC 955.04. Jenis automasi, semiautomasi telah dikembangkan dlm kaedah AOAC 976.06, 976.05 dan 960.52

Prosedur Kjeldahl guna semi auto machine

Penyediaan sampel Sampel dihomogen. Penghadaman Sampel + H2SO4 + CuSO4 + K2SO4 dipanaskan Hasil penghadaman menjadi clear Ammonium sulfat (NH4)2SO4 terbentuk

Penghadam kjeltec

Prosedur Kjehdahl guna semi auto Kjeltec Peneutralan dan penyulingan Hasil penghadaman dicairkan dengan air suling. Sodium thiosulfat ditambah untuk neutralkan asid sulfurik (NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O

Prosedur Kjehdahl guna semi auto Kjeltec

NH3 terbentuk disuling ke dalam asid borik

H3BO3 (Boric acid) + NH3 NH4 + H2BO-3 (Anion Borate)

Semi auto kjeltec in our lab

Latest unit of semi auto kjeltec

Prosedur Kjehdahl guna semi auto Kjeltec Pentitratan

Hasil sulingan di dlm asid borik iaitu anion borat dititrat dgn asid HCl

H2BO-3 (Anion Borat) + H+

Anion borat terhasil amaun N

H3BO3

mol HCl = mol NH3 = mol N dlm sampel

Pengiraan
%N=
Isipadu HCl (sampel-blank) N HCl 14 100 Berat sampel 1000

% Protein = %N Faktor protein

Fully automated unit

Advantage and disadvantages

The Kjeldahl method's universality, precision and reproducibility have made it the internationally-recognized method for estimating the protein content in foods and it is the standard method against which all other methods are judged. It does not give a measure of true protein content, as it measures non-protein nitrogen in addition to the nitrogen in proteins.

Pro and Cons

This can be mirrored from 2008 Chinese milk powder scandal when melamine, a nitrogen-rich chemical, was added to raw material to fake high protein contents. Different correction factors are needed for different proteins to account for different amino acid sequences. The need to use concentrated sulfuric acid at high temperature and the relatively long testing time (an hour or more), compare unfavorably with the Dumas method for measuring crude protein content

Penentuan nitrogen total

2. Kaedah Pembakaran Dumas

Apparatus for the estimation of nitrogen by Dumas' method

Prinsip kaedah Dumas

Pembakaran sampel pada suhu tinggi (about 900 C) dalam chamber mengandungi oksigen. Pembebasan karbon dioksida, air dan nitrogen kolum serap karbon dioksida dan air. Kolum pengesan konduktiviti terma - asing dan ukur nitrogen. Instrumen dikalibrasi dgn bahan rujukan yang diketahui kandungan nitrogen Kepekatan nitrogen ditukar kepada protein kasar menggunakan faktor penukaran protein

SISTEM PEMBAKARAN DUMATHERM

Kebaikan dan kelemahan kaedah Dumas

Kebaikan
Mudah diguna. Lebi cepat berbanding kaedah Kjeldahl beberapa minit. Tidak guna katalis atau bahan kimia toksik

Kelemahan
Kos tinggi Bukan pengukuran nilai protein sebenar Faktor penukaran berbeza diperlukan utk protein yang berbeza

Kaedah Spektrofotometri
Penentuan kepekatan protein

Spektrofotometer versi lama

Spektrofotometer versi baru

INTRODUCTION
A number of methods have been devised to measure protein concentration, which are based on UV-visible spectroscopy. Kaedah paling sesuai untuk protein tulen. The basic principle behind each of these tests is similar.
A calibration curve of absorbance (or turbidity) versus protein concentration is prepared using a series of protein solutions of known concentration. The absorbance (or turbidity) of the solution being analyzed is then measured at the same wavelength, and its protein concentration determined from the calibration curve

Penentuan kepekatan protein pada penyerapan A280 nm

Penyerapan cahaya UV oleh asid amino triptofan, tirosina, sistina dalam larutan protein Kepekatan protein 20 3000 g/ml Lengkuk piawai guna 3mg/ml larutan stok protein piawai (BSA).

Penentuan kepekatan protein pada penyerapan A280 nm

20, 50, 100, 250, 500, 1000, 2000 g/ml Patuhi hukum Beers Kepekatan protein piawai 3mg/ml pada A280 nm = 1.98 Jika abs sampel > 2.0, sampel perlu dicairkan.

Penentuan kepekatan protein pada penyerapan A280 nm

Triptofan dan tirosina - konstan Prosedur:
Larutkan sampel protein dalam penimbal atau alkali Dibaca berbanding pengosong (Blank) Kepekatan protein dikira A=aXbXc

Penentuan kepekatan protein pada penyerapan A280 nm

The advantages of this method are that
procedure is simple to carry out non destructive no special reagents are required.

Suitable for
Natural protein system (milk and meat products) Protein is extract in alkali.

Penentuan kepekatan protein pada penyerapan A280 nm

The major disadvantage is that nucleic acids also absorb strongly at 280 nm and could therefore interfere with the measurement of the protein if they are present in sufficient concentrations.

Penentuan kepekatan protein pada penyerapan A205 nm

Penyerapan pada A205 nm - kepekatan peptida. Penyerapan protein pada A205 nm adalah 7 X > tinggi berbanding A280 nm Kelemahan : Penimbal dan bahan lain beri penyerapan pada A205 nm

Other methods for determination of protein

Kaedah Biuret

(540-560 nm)

Principal : A violet-purplish color is produced when cupric ions (Cu2+) interact with peptide bonds under alkaline conditions. The biuret reagent, which contains all the chemicals required to carry out the analysis, can be purchased commercially Left for15 min before reading at 540 nm Kalibrasi: Lengkuk piawai berdasarkan penentuan protein tulen (BSA)

Kaedah Biuret
Digunakan untuk menganalisa protein dalam bijirin, daging, protein kacang soya, makanan ternakan

Kaedah Biuret
Advantages
no interference from materials that adsorb at lower wavelengths less sensitive to protein type because it utilizes absorption involving peptide bonds that are common to all proteins, rather than specific side groups. Not sensitive to component non protein cheap

Kaedah Biuret
Disadvantages Tidak berapa sensitif berbanding kaedah Lowry Keamatan warna berbeza Pelbagai warna muncul jika kandungan lemak dan karbohidrat tinggi

1. 2. 3.

Kaedah Lowry

Kaedah Lowry
The Lowry method combines the biuret reagent with another reagent (the Folin-Ciocalteau phenol reagent) which reacts with tyrosine and tryptophan residues in proteins. . This gives a bluish color which can be read somewhere between 500 - 750 nm depending on the sensitivity required. There is a small peak around 500 nm that can be used to determine high protein concentrations and a large peak around 750 nm that can be used to determine low protein concentrations.

Kaedah Lowry
This method is more sensitive to low concentrations of proteins than the biuret method. Less affected by the turbidity of the samples. More specific than most other method

Coloured binding method

Penentuan protein

ANIONIC COLOURED BONDING

A known excess of a negatively charged (anionic) dye is added to a protein solution whose pH is adjusted so that the proteins are positively charged (i.e. < the isoelectric point). The proteins form an insoluble complex with the dye because of the electrostatic attraction between the molecules, but the unbound dye remains soluble.

ANIONIC COLOURED BONDING

The anionic dye binds to cationic groups of the basic amino acid residues (histidine, arganine and lysine) and to free amino terminal groups. The amount of unbound dye remaining in solution after the insoluble protein-dye complex has been removed (e.g., by centrifugation) is determined by measuring its absorbance. The amount of protein present in the original solution is proportional to the amount of dye that bound to it: dyebound = dyeinitial - dyefree.

Kaedah Bradford
Pewarna *Coomassie Brilliant Blue G250 (reagen bradford) + PROTEIN
Kemerahan kepada Kebiruan 465 nm kepada 595 nm

Kaedah Bradford
Prosedur
Pewarna* dilarutkan dlm ethanol dan asid fosforik Pewarna* ditambah kepada piawai BSA dan sampel Dibaca pd 595 nm berbanding blank Kepekatan protein dlm sampel ditentukan dari lengkuk piawai BSA

LENGKUK PIAWAI

Kaedah Bradford
Kelebihan
Pantas (2 min) Hasil yang sama berulangkali 7 X > sensitif dari kaedah Lowry Tiada gangguan dari kation spt K+ Na+ Mg2+ X gangguan dari polifenol dan karbohidrat

Kaedah Bradford
Kekurangan
Kompleks pewarna-protein terikat kpd kuvet kuarza. Guna gelas atau plastik Warna berubah ikut jenis protein

PERBANDINGAN KAEDAH

Dari segi penyediaan sampel

Kjehdahl : requires little preparation (20 mesh or smaller is required). Other method: Fine particles extraction of proteins from the complex food system.

Dari segi prinsip

Kjehdahl : Measure directly the total amount of organic nitrogen elements in the food/ Other methods : Measure the various physicochemical properties of proteins.
Biuret method = peptide bonds Lowry method = peptide and amino acids

Dari segi sensitiviti

Kjehdahl, Biuret, Anionic coloured binding LESS SENSITIVE compared to UV, Lowry, Bradford

Dari segi Speed

Methods involving spectrophotometric must separate proteins from the interfering insoluble materials before mixing with the colour reagent. BUT it is faster than Kjehdahl method.