PROTEIN ANALYSIS
Endrika WIDYASTUTI
FOOD ANALYSIS AND BIOCHEMISTRY PRACTICE Food Science and Technology Department 2012
THE OUTLINE INTRODUCTION OF PROTEIN TYPES PROTEIN ANALYSIS NINHIDRIN TEST KJEDAHL METHOD FORMOL TITRATION
PROTEIN Proteins are made up of amino acids Food analysts are interested in knowing the total concentration, type, molecular structure and functional properties of proteins in foods Amino acids are the building blocks of protein
Total organic nitrogen = protein + non-protein nitrogen
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PROTEIN 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.
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PROTEIN Protein is analyzed for: 1. determination of biological activity 2. investigation of functional properties 3. nutritional labeling Protein analysis is required for you 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
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TYPES OF PROTEIN
ANALYSIS • NINHIDRIN • ELECTROFORESIS • CHROMATOGRAPHY
QUALITATIVE METHOD
• Kjeldahl – measures the amount of nitrogen in a sample • Lowry- measures the tyrosine/tryptophan residues of proteins
QUANTITATIVE METHOD
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PROTEIN ANALYSIS Primary amino groups on the end of proteins, peptides, and free amino acids will react with ninhydrin.
NINHIDRIN TEST
This reaction forms a strongly colored purple solution referred to as Ruheman's purple
Sample can be tested for the amount of primary amino acids currently present, or sample can be alkaline hydrolyzed to increase the amount of these amino acids.
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PROTEIN ANALYSIS NINHIDRIN TEST Ninhidrin mengalami deaminasi oksidatif dan asam amino dekarboksilasi menjadi CO2, NH3 dan aldehid
Ninhidrin yang tereduksi akan bereaksi dengan amonia dan dengan molekul ninhidrin lain sehingga terbentuk senyawa kompleks berwarna ungu ( ungu Ruhemann)
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PROTEIN ANALYSIS ADVANTAGES
NINHIDRIN TEST
Faster and more convenient that Kjeldahl
DISADVANTAGES •
• • • •
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 NPN, calcium, or phosphorous constituents will bind to the dye or to protein, causing interference Addition of a metal chelator (i.e.. oxalic acid) may help reduce binding
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PROTEIN ANALYSIS NINHIDRIN TEST BAHAN larutan susu skim (10%) gelatin (5%) putih telur enzim keju, pemanis sintetik diasweet MSG (5%) akuades sebagai (kontrol)
- Pelabelan tabung reaksi - Pengambilan sampel 2 ml + 2 ml larutan ninhidrin - Pemasukan tabung reaksi pada air mendidih (1520 detik) Pengamatan warna larutan : (+ ) jika hasil test positif (berwarna ungu, berarti sampel mengandung gugus amina bebas) (– ) jika hasil test negatif. Jika terbentuk warna lain seperti (kuning, orange dan merah) maka uji negatif prolin, hydroxyproline, dan 2-, 3-, and 4-asam aminobenzoat
PROTEIN ANALYSIS CRUDE PROTEIN-KJEDAHL • Crude protein content • Johan Kjeldahl (1883) developed the basic process • PRINCIPLE: total organic N released from sample and absorbed by acid – Digestion: sulfuric acid + catalyst – Neutralization and distillation; Sodium hydroxide – Titration; Hydrochloric acid
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PROTEIN ANALYSIS CRUDE PROTEIN-KJEDAHL DIGESTION
Protein
SULFURIC ACID HEAT, CATALYST
(NH4)2SO4
(ammonium sulfate)
Protein N NH4+ + H2SO4 (NH4)2SO4 melepaskan unsur N dari protein yang diubah menjadi amonium sulfat
NEUTRALIZATION AND DISTILLATION
(NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O -
NH3 + H3BO3 NH4+ : H2BO3 + H3BO3 (boric acid) (ammonium-borate complex) amonium sulfat diubah menjadi amoniak yang excess ditangkap oleh larutan asam standar berlebih
Color change
PROTEIN ANALYSIS CRUDE PROTEIN-KJEDAHL TITRATION Titration (direct titration) H2BO3 + H+ H3BO3 (HCl) – Calculation moles HCl = moles NH3 = moles N in the sample Sisa asam yang tidak bereaksi dengan amoniak dititrasi, sehingga dapat diketahui jumlah amoniak dari N protein sampel
%N = (ml NaOH blanko – ml NaOH contoh) x N HCl x 100 x 14.008 g sampel x 1000 % protein = %N x faktor konversi (tergantung jenis sampel)
PROTEIN ANALYSIS CRUDE PROTEIN-KJEDAHL • Calculation %Protein = %N conversion factor Conversion factor: generally 6.25 – most protein: 16% N
egg or meat milk wheat soybean rice
Conversion factor 6.25 6.38 5.33 5.52 5.17
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PROTEIN ANALYSIS CRUDE PROTEIN-KJEDAHL • ADVANTAGES
1. applicable to most food samples 2. simple 3. inexpensive 4. accepted as Official method 5. can measure mg levels of proteins
• DISADVANTAGES
1. Measures total N not protein 2. Time consuming – at least 2 hrs 3. Poor precision when compared to other methods 4. Corrosive (dangerous) method
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PROTEIN ANALYSIS CRUDE PROTEIN-KJEDAHL Asam sulfat pekat, berat jenis 1.84 Air raksa oksida Kalium sulfat Larutan natrium hidroksida-natrium tiosulfat Larutan asam borat jenuh Larutan asam klorida 0.02 N NO 1 2 3 4 5
KELAS A Susu cair segar Susu bubuk Yoghurt Keju Yakult
KELAS D Kedelai mentah Tempe kedelai Kecap Keripik tempe Tahu
KELAS E Ikan Kecap ikan Bakso ikan Kacang tanah mentah Tempe kacang
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PROTEIN ANALYSIS CRUDE PROTEIN-KJEDAHL Pemasukan bahan kedalam labu kjeldahl. Penambahan 7.5 g K2S2O4 dan 0.35 g HgO (Awas: zat ini beracun) ,tambahkan 15 ml H2SO4 pekat. Pemanasan semua bahan dalam labu kjeldahl dalam almari asam sampai berhenti berasap dan cairan menjadi jernih. Penambahan aquades dalam labu kjeldahl yang didinginkan dalam air es dan beberapa lempeng Zn, juga tambahkan 15 ml larutan K2SO4% (dalam air) dan akhirnya tambahkan perlahan-lahan larutan NaOH 50% sebanyak 50 ml yang sudah didinginkan dalam lemari es. Pasanglah labu kjeldahl dengan segera pada alat distilasi. Panaskan labu kjeldahl perlahan-lahan sampai dua lapisan cairan tercampur kemudian panaskan dengan cepat sampai mendidih. Distilat ini ditampung dalam erlenmeyer yang telah diisi dengan 50 ml larutan standar HCl (0.1 N) dan 5 tetes indikator metil merah. Lakukan distilasi sampai distilat yang tertampung sebanyak 75 ml. Titrasilah distilat yang diperoleh dengan standar NaOH (0.1 N) (lampiran) sampai warna kuning. 17
PROTEIN ANALYSIS BIURET
Cupric ions react with peptide bonds under alkaline conditions (copper sulfate + K-Na-tartrate + alkali) Measure color in SPEC at 520 nm
• • •
O
O
O H H
N R
H
2+
Cu
H
N R
2+
N H
H R
Cu O H-
O H
N
O
O H
N
N
H
P u rp le b iu re t c o m p le x
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PROTEIN ANALYSIS • ADVANTAGES
BIURET
– most sensitive (20-200g) - Cheaper and faster than Kjeldahl - Less problem with color deviations - Few substances interfere - Does not measure non protein nitrogen (NPN)
• DISADVANTAGES – color development not proportional to protein concentration – color varying with different proteins – interference (sugars, lipids, phosphate buffers, etc)
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PROTEIN ANALYSIS BIURET BAHAN Pereaksi biuret Larutkan 3 gram CuCO4.5H2O dan 9 gram Na-K-Tartrat dalam 500 ml NaOH 0.2 N. Tambahkan 5 gram KI kemudian encerkan sampai 1000 ml dengan menggunakan NaOH 0.2 N. Larutan protein standar Larutan bovine serum albumin atau kasein 5 mg/ml
PEMBUATAN KURVA STANDAR PERSIAPAN SAMPEL Penimbangan Sampel Penghancuran Sampel Penyaringan dan Pensentrifuga asian Supernatan didekantasi Jika supernatan keruh (atau bahan mengganggu), ada perlakuan tambahan
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PROTEIN ANALYSIS Prinsip: larutan protein dinetralkan dengan basa (NaOH), kemudian penambahan formalin akan membentuk dimethilol
FORMOL TITRATION (N-AMINO)
Pembentukan dimethilol ini menunjukkan gugus amino sudah terikat dan tidak akan mempengaruhi reaksi antara asam (gugus karboksil asam amino) dengan basa NaOH perubahan warna menjadi merah muda yang tidak hilang selama 30 detik. Titrasi formol hanya tepat untuk menunjukkan proses hidrolisis protein dan kurang tepat untuk menentukan kadar protein
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PROTEIN ANALYSIS FORMOL TITRATION (N-AMINO) BAHAN
K-oksalat Fenolftalein 1% NaOH Rosanilin klorida Formaldehid 40 % Akuades Sampel seperti analisis protein metode Kjeldahl
titrasi formol % N = ____________ x N NaOH x 14.008 g bahan x 10
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PROTEIN ANALYSIS FORMOL TITRATION (N-AMINO) Pindahkan larutan protein ke dalam erlenmeyer 125 ml dan tambahkan 20 ml aquades dan 0.4 ml larutan Kalium oksalat jenuh (kalium oksalat : air = 1:3) dan 1 ml phenolphtalein 1%. Diamkan selama 2 menit. Titrasilah larutan contoh dengan 0.1 N NaOH sampai mencapai warna seperti warna standar di bawah ini atau sampai warna merah jambu. Warna standar : 10 ml susu + 10 ml aquades + 0.4 ml K-oksalat jenuh + 1 tetes 0.01% indikator rosanilin-chlorida. Setelah warna tercapai, tambahkan 2 ml larutan formaldehid 40% dan titrasilah kembali dengan larutan NaOH sampai warna seperti warna standar tercapai lagi. 23
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