DETERMINATION OF PROTEIN

Dr. Hasnah HaronNutrition Programme, PPSJK, FSK, UKMKL

PENGENALAN

n Protein mkn adalah kompleksn Berat molekular yg. berbeza : 5,000 hingga > 1 juta Daltonshingga > 1 juta Daltons

n H,C,N,O,Sn 20 ∝-asid amino bentuk blok proteinn Kandungan N dlm mkn : 13.4 - 19.1%

INTRODUCTIONn Proteins are polymers of amino acids. n Twenty different types of amino acids occur naturally in proteins.

n Proteins differ from each other according to the type, number and sequence of amino the type, number and sequence of amino acids that make up the polypeptide backbone.

n Thus, protein have different molecular structures, nutritional attributes and physiochemical properties.

INTRODUCTIONn Proteins are important constituents of foods for a number of different reasons.

n They are a major source of energy, as well as n 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

n Classification of protein based on composition, structure, biologikal function, solubility.Eg : Simple protein – amino acid.Conjugate protein – non amino acid component.

INTRODUCTION

Conjugate protein – non amino acid component.

n Denaturation of protein - heat, acid , alkali, organic solvent and detergent. Function and solubility of protein change as well.

INTRODUCTION

n Protein : komponen terbanyak dalam sel jalankan fungsi biologikal.

n Fungsi utama protein:1. Sumbang tenaga dan nutrien penting 2. Beri ciri fizikokimia – kualiti dan atribut sensori3. Fungsi biologikal in vivo

– Enzim– Hormon– antibodi

INTRODUCTIONn 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, ureapeptide, nucleic acid, fosfolipid, uric acid, urea

n Lipid and carbohydrate can interfere with protein analysis.

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

Importance of protein analysisq Determination of biological activity

Proteins are also the major structural components of manynatural foods, often determining their overall textureqProteolitic enzyme to soften the meatqPectine for ripeness of fruit.

q Research on the functional characterization.Isolated proteins are often used in foods as ingredientsbecause of their unique functional properties. Their abilityto provide desirable appearance, texture or stability.qGlutenin in flour - breadqCasein – cheese

qGelling agents, emulsifiers, foaming agents andthickeners.

Keperluan lain analisis protein

q Kandungan total proteinq Komposisi asid aminoq Kandungan protein tertentu dlm

campurancampuranq Nitrogen bukan proteinq Nilai pemakanan protein

(Penghadaman, Protein Efficiency Ratio)

Kandungan protein dlm mkn

n Sumber penting protein: mkn bersumber haiwan dan kekacang

n Eg : Daging lembu (stik) 29%Daging ayam (dada) 27%Ikan kod (di masak) 29% Telur 13%Kekacang 22%

Determination of protein concentration

1. Determination of nitrogen total2. Determination of colorimetric and

determination of protein total determination of protein total quantitative.

3. Determination protein concentration using spectrophotometry

Determination of total nitrogen

1. Kaedah Kjehdahl

Johan Gustav Christoffer Thorsager Kjeldahl

n Johan Gustav Christoffer Thorsager Kjeldahl (1849–1900),

n Danish chemist who n 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 Kjeldahln Kjeldahl worked in Copenhagen at theCarlsberg Laboratory, associated withCarlsberg Brewery, where he was head ofthe Chemistry department from 1876 tothe Chemistry department from 1876 to1900.

n He was given the job to determine the amount of protein in the grain used in the malt industry.

Kjeldahl method

n 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 n 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.

n Kjeldahl equipment is used extensively all over the world.

Kjeldahl

Kjehdahl method

n A food is digested with a strong - releases nitrogen which can be determined by a suitable titration technique.

n The amount of protein present is then calculated from n 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

n It is usually considered to be the standard method of determining protein concentration.

Kjehdahl method

n 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. protein concentration.

n A conversion factor of 6.25 (equivalent to 0.16 g nitrogen per gram of protein) is used for many applications.

n 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

HeatKjehdahl digestion flask

1. Digestionn Digestion converts anynitrogen (N) in the food andothers in the form of nitratesor nitrites) into ammonia.

n Ammonia is in the form of theammonium ion (NH4

+) whichbinds to the sulfate ion (SO4

2).

N(food) (NH4)2SO4

2. NeutralizationProduct of digestion + H20 +

addition of NaOH)

NH to NHNH4 to NH3

(NH4)2SO4 + 2 NaOH

2NH3 + 2H2O + Na2SO4

3. Distillation

nThe ammonia gas goes into thereceiving flask - which contains anexcess of boric acid.

nThe low pH of the solutionnThe low pH of the solutionconverts ammonia gas into theammonium ion and converts theboric 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 HClor 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:

Wherevs and vb - titration volumes of the sample and blank14g - molecular weight of nitrogen N.

Calculation

Once the nitrogen content has beendetermined it is converted to a proteincontent using the appropriate conversionfactor:factor:

%Protein = %N X Protein factor

Faktor protein

n Campuran protein secara am mengandungi 16%

n Penentuan dari kandungan nitrogen perlu X faktor protein iaitu 6.25(100/16).

Faktor ProteinSumber protein Faktor protein

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

Kebaikan dan keburukan kaedah Kjehdahl

Kebaikan Keburukan

Diguna – semua jenis makanan

Ukur nitrogen organik secara totaljenis makanan organik secara total

Ringkas Masa agak panjang

Murah Reagen menghakis

Tepat Kurang persis

Kaedah Kjehdahl

n Prosedur Kjehdahl yg asal AOAC 955.04.

n Jenis automasi, semiautomasi telah n Jenis automasi, semiautomasi telah dikembangkan dlm kaedah AOAC 976.06, 976.05 dan 960.52

Prosedur Kjeldahl guna semi auto machine

qPenyediaan sampelqSampel dihomogen.

qPenghadamanqSampel + H SO + CuSO + K SOqSampel + H2SO4 + CuSO4 + K2SO4dipanaskan

qHasil penghadaman menjadi ‘clear’qAmmonium sulfat (NH4)2SO4 terbentuk

Penghadam ‘kjeltec’

qPeneutralan dan penyulinganqHasil penghadaman dicairkan dengan air suling.

qSodium thiosulfat ditambah untuk

Prosedur Kjehdahl guna semi auto Kjeltec

qSodium thiosulfat ditambah untuk neutralkan asid sulfurik

(NH4)2SO4 + 2NaOH

2NH3 + Na2SO4 + 2H2O

Prosedur Kjehdahl guna semi auto Kjeltec

nNH3 terbentuk disuling ke dalam asid borik

H BO (Boric acid) + NHH3BO3 (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

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

H BO- (Anion Borat) + H+ H BOH2BO-3 (Anion Borat) + H+ H3BO3

qAnion borat terhasil ∝ amaun N

qmol HCl = mol NH3 = mol N dlm sampel

Pengiraann % N =Isipadu HCl (sampel-blank) × N HCl × 14 × 100

Berat sampel × 1000

n % Protein = %N × Faktor protein

Fully automated unit

Advantage and disadvantages

ØThe Kjeldahl method's universality,precision and reproducibility have made it theinternationally-recognized method forestimating the protein content in foods and itis the standard method against which allis the standard method against which allother methods are judged.

ØIt does not give a measure of true proteincontent, as it measures non-proteinnitrogen in addition to the nitrogen inproteins.

Pro and Consn 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.

n Different correction factors are needed for different n Different correction factors are needed for different proteins to account for different amino acid sequences.

n 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

n Pembakaran sampel pada suhu tinggi (about 900 °C) dalam ‘chamber’ mengandungi oksigen.

n Pembebasan karbon dioksida, air dan nitrogen -kolum serap karbon dioksida dan air.

n Kolum – pengesan konduktiviti terma - asing dan ukur nitrogen.

n Instrumen dikalibrasi dgn bahan rujukan yang diketahui kandungan nitrogen

n Kepekatan nitrogen ditukar kepada protein kasar menggunakan faktor penukaran protein

SISTEM PEMBAKARAN DUMATHERM

Kebaikan dan kelemahan kaedah Dumas

n Kebaikan– Mudah diguna. – Lebi cepat berbanding kaedah Kjeldahl -beberapa minit.beberapa minit.

– Tidak guna katalis atau bahan kimia toksik

n 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

INTRODUCTIONn A number of methods have been devised to measure

protein concentration, which are based on UV-visible spectroscopy. Kaedah paling sesuai untuk protein tulen.

n The basic principle behind each of these tests is n 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

n Penyerapan cahaya UV oleh asid amino triptofan, tirosina, sistina dalam larutan proteinprotein

n Kepekatan protein 20 – 3000 µg/mln Lengkuk piawai guna 3mg/ml larutan stok protein piawai (BSA).

Penentuan kepekatan protein pada penyerapan A280 nm

n 20, 50, 100, 250, 500, 1000, 2000 µg/mln Patuhi hukum Beer’sn Kepekatan protein piawai 3mg/ml pada n Kepekatan protein piawai 3mg/ml pada A280 nm = 1.98

n Jika abs sampel > 2.0, sampel perlu dicairkan.

Penentuan kepekatan protein pada penyerapan A280 nm

n Triptofan dan tirosina - konstann Prosedur:

– Larutkan sampel protein dalam penimbal atau alkaliatau alkali

– Dibaca berbanding pengosong (Blank)– Kepekatan protein dikira

A = a X b X c

Penentuan kepekatan protein pada penyerapan A280 nm

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

n Suitable for – Natural protein system (milk and meat products)

– Protein is extract in alkali.

Penentuan kepekatan protein pada penyerapan A280 nm

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

Penentuan kepekatan protein pada penyerapan A205 nm

n Penyerapan pada A205 nm - kepekatan peptida.

n Penyerapan protein pada A205 nm n Penyerapan protein pada A205 nm adalah 7 X > tinggi berbanding A280 nm

n Kelemahan : Penimbal dan bahan lain beri penyerapan pada A205 nm

Other methods for Other methods for determination of protein

n Principal : A violet-purplish color is produced when cupric ions (Cu2+) interact with peptide bonds under alkaline conditions.

n The biuret reagent, which contains all the chemicals required to carry out the analysis,

Kaedah Biuret (540-560 nm)

chemicals required to carry out the analysis, can be purchased commercially

n Left for15 min before reading at 540 nm

n Kalibrasi: Lengkuk piawai berdasarkan penentuan protein tulen (BSA)

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

n Advantages– no interference from materials that adsorb at lower wavelengths

– less sensitive to protein type because it utilizes absorption involving peptide bonds

Kaedah Biuret

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

n Disadvantages1. Tidak berapa sensitif berbanding

kaedah Lowrykaedah Lowry2. Keamatan warna berbeza3. Pelbagai warna muncul jika

kandungan lemak dan karbohidrat tinggi

Kaedah Lowry

Kaedah Lowry

n The Lowry method combines the biuret reagent with another reagent (the Folin-Ciocalteau phenol reagent) which reacts with tyrosine and tryptophan residues in proteins. .

n This gives a bluish color which can be read n This gives a bluish color which can be read somewhere between 500 - 750 nm depending on the sensitivity required.

n 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

n This method is more sensitive to low concentrations of proteins than the biuret method. biuret method.

n Less affected by the turbidity of the samples.

n More specific than most other method

Coloured binding method

Penentuan protein

ANIONIC COLOURED BONDING

n 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).

n 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

n The anionic dye binds to cationic groups of the basic amino acid residues (histidine, arganine and lysine) and to free amino terminal groups.

n The amount of unbound dye remaining in solution n 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.

n The amount of protein present in the original solution is proportional to the amount of dye that bound to it: dyebound = dyeinitial - dyefree.

Kaedah Bradfordn Pewarna *Coomassie Brilliant Blue G-250 (reagen bradford) + PROTEIN

Kemerahan Kemerahan

kepada

Kebiruan

465 nm

kepada

595 nm

Kaedah Bradford

n Prosedur– Pewarna* dilarutkan dlm ethanol dan asid fosforikfosforik

– 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

n Kelebihan– Pantas (2 min)– Hasil yang sama berulangkali– 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

n Kekurangan– Kompleks pewarna-protein terikat kpd kuvet kuarza.kuvet kuarza.

– Guna gelas atau plastik– Warna berubah ikut jenis protein

PERBANDINGAN KAEDAH

Dari segi penyediaan sampel

n Kjehdahl : requires little preparation (20 mesh or smaller is required).

n Other method: Fine particles –extraction of proteins from the complex food system.

Dari segi prinsip

n Kjehdahl : Measure directly the total amount of organic nitrogen elements in the food/the food/

n Other methods : Measure the various physicochemical properties of proteins.Biuret method = peptide bondsLowry method = peptide and amino acids

Dari segi sensitiviti

n Kjehdahl, Biuret, Anionic colouredbinding LESS SENSITIVE compared toUV, Lowry, BradfordUV, Lowry, Bradford

Dari segi Speed

n Methods involving spectrophotometric must separate proteins from the interfering insoluble materials before interfering insoluble materials before mixing with the colour reagent.

n BUT it is faster than Kjehdahl method.