Penetapan Kadar Protein dengan Metode Lowry (Protein Concentration Determination by Lowry Method)

Metode Lowry merupakan pengembangan dari metode Biuret. Dalam metode ini terlibat 2 reaksi. Awalnya, kompleks Cu(II)-protein akan terbentuk sebagaimana metode biuret, yang dalam suasana alkalis Cu(II) akan tereduksi menjadi Cu(I).
Ion Cu+ kemudian akan mereduksi reagen Folin-Ciocalteu, kompleks phosphomolibdat-phosphotungstat (phosphomolybdotungstate), menghasilkan heteropolymolybdenum blue akibat reaksi oksidasi gugus aromatik (rantai samping asam amino) terkatalis Cu, yang memberikan warna biru intensif yang dapat dideteksi secara kolorimetri. Kekuatan warna biru terutama bergantung pada kandungan residu tryptophan dan tyrosine-nya. Keuntungan metode Lowry adalah lebih sensitif (100 kali) daripada metode Biuret sehingga memerlukan sampel protein yang lebih sedikit. Batas deteksinya berkisar pada konsentrasi 0.01 mg/mL. Namun metode Lowry lebih banyak interferensinya akibat kesensitifannya.

Reagen:

1. Reagen pembentuk kompleks: Siapkan segera sebelum digunakan campuran dari ketiga larutan-larutan berikut dengan perbandingan 100:1:1
   Larutan A: 2%b/v Na2CO3 dalam akuades
   Larutan B: 1%b/v CuSO4.5H2O dalam akuades
   Larutan C: 2%b/v Kalium Natrium tartrat dalam akuades
2. NaOH 2N
3. Reagen Folin-Ciocalteu 1N
   Ke dalam labu alas bulat 1500 mL masukkan 100 g sodium tungstate, 25 g sodium molibdate, 700 mL akuades, 50 mL asam phosphate, dan 100 mL HCl. Campuran direfluks selama 10 jam, tambahkan 150 mL lithium sulfat, 50 mL akuades dan beberapa tetes bromine (Br2). Didihkan campuran (tanpa pendingin) sekitar 15 menit (hingga kelebihan bromine habis). Dinginkan, encerkan dengan akuades hingga 1 L, saring (filtrat berwarna kehijauan). Sebelum digunakan, encerkan 1 bagian filtrat dengan 5 bagian akuades.

Standard:

Gunakan larutan stok standard protein (misalnya albumin) yang mengandung 4 mg/mL protein dalam akuades, disimpan pada -20oC. Siapkan larutan standard dengan pengenceran larutan stok sbb:

Larutan stok (μL)	0	1.25	2.50	6.25	12.5	25.0	62.5	125	250
Akuades (μL)	500	499	498	494	488	475	438	375	250
Konsentrasi Protein (μg/mL)	0	10	20	50	100	200	500	1000	2000

Prosedur:

1. Ambil 1 mL sampel atau standard, tambahkan 1 mL NaOH 2N. Hidrolisis pada 100oC selama 10 menit pada penangas air.
2. Dinginkan pada suhu ruangan, tambahkan 5 mL reagen pembentuk kompleks. Biarkan larutan selama 10 menit pada suhu kamar.
3. Tambahkan 0.5 mL reagen Folin-Ciocalteu, homogenkan dengan vortex, biarkan selam 30-60 menit (jangan sampai lebih dari 60 menit)
4. Baca absorbansi pada 660 nm jika konsentrasi protein di bawah 500 μg/mL atau 550 nm jika konsentrasi protein antara 100 – 2000 μg/mL.

Catatan:

Jika sampel berbentuk endapan, encerkan dulu dengan NaOH 2N.
Reaksi yang terjadi dalam metode Lowry sangat tergantung pada pH. Oleh karena itu aturlah pH 10 – 10.5

Interferensi:

Beberapa zat yang bisa mengganggu penetapan kadar protein dengan metode Lowry ini diantaranya adalah: buffer, asam nuklet, dan gula/karbohidrat, deterjen, gliserol, Tricine, EDTA, Tris, senyawa-senyawa kalium, sulfhidril, disulfida, fenolat, asam urat, guanin, xanthine, magnesium dan kalsium. Interferensi agen-agen ini dapat diminimalkan dengan menghilangkan interferens tersebut. Sangat dianjurkan untuk menggunakan blanko untuk mengkoreksi absorbansi. Interferensi yang disebabkan oleh deterjen, sukrosa dan EDTA dapat dieliminasi dengan penambahan SDS atau melakukan preparasi sampel dengan pengendapan protein.

Referensi:

Dennison, C., 2002, A Guide to Protein Isolation, Kluwer Academic Publishers, New York
Lowry, O.H., Rosenbrough, N.J., Farr, A.L. & Randall, R.J., 1951, Protein Measurement with the Folin Phenol Reagent, J. Biol. Chem, 193: 265-275

IN ENGLISH

Lowry method is a development of the Biuret method. In this method involved two reactions. Initially, the complex of Cu (II)-proteins will be formed as Biuret method, which in the atmosphere of alkaline Cu (II) will be reduced to Cu (I).
Cu + ions will then reduce the Folin-Ciocalteu reagent, the complex-phosphotungstat phosphomolibdat (phosphomolybdotungstate), produces blue heteropolymolybdenum due to oxidation of aromatic groups (side chains of amino acids) catalyzed Cu, which give an intensive blue color that can be detected by colorimetry. Blue strength mainly depends on the content of tryptophan and tyrosine residues him. The advantage is a more sensitive method of Lowry (100 times) than the Biuret method, thus requiring less protein sample. Limits of detection range at a concentration of 0:01 mg / mL. But Lowry method more interference due to senstively.

Reagents:

1. Complex-forming reagent: Prepare immediately before use a mixture of the three-solution of the following solution with the ratio 100:1:1
Solvent A: 2% w / v Na2CO3 in distilled water
Solution B: 1% w / v in distilled water CuSO4.5H2O
Solution C: 2% w / v Sodium potassium tartrate in distilled water
2. 2N NaOH
3. 1N Folin-Ciocalteu reagent
Into the pumpkin base in 1500 mL round insert 100 g sodium tungstate, 25 g sodium molibdate, 700 mL of distilled water, 50 mL of phosphate acid, and 100 mL of HCl. Mixture refluxed for 10 hours, add 150 mL of lithium sulfate, 50 mL of distilled water, and a few drops bromine (Br2). Boil the mixture (without cooling) of approximately 15 min (until the excess bromine supply). Let cool, dilute to 1 L with distilled water, filtered (filtrate greenish). Before use, dilute one part of the filtrate with 5 parts distilled water.

Standard:

Use standard stock solution of protein (eg albumin) containing 4 mg / mL protein in distilled water, stored at-20oC. Prepare a standard solution by diluting stock solution as follows:
Stock solution (μL) 0 1.25 2.50 6.25 12.5 25.0 62.5 125 250
Distilled water (μL) 500 499 498 494 488 475 438 375 250
Protein concentration (tg / mL) 0 10 20 50,100,200,500 1000 2000

Procedure:

1. Take one mL of sample or standard, add 1 mL of NaOH 2N. Hydrolysis at 100oC for 10 minutes in a water bath.
2. Chill at ambient temperature, add 5 mL of reagent complex formation. Let the solution for 10 minutes at room temperature.
3. Add 0.5 mL of Folin-Ciocalteu reagent, homogenkan with a vortex, let dive 30-60 minutes (do not let more than 60 minutes)
4. Read absorbance at 660 nm when the concentration of proteins below 500 tg / mL or 550 nm when the protein concentration between 100-2000 tg / mL.

Note:

If the shape of sediment samples, dilute first with 2N NaOH.
Reactions that occur in the Lowry method is highly dependent on pH. Therefore, arrange for pH 10 – 10.5

Interference:

Some substances that could interfere with determination of protein content with Lowry method for this distribution are: buffer, nuklet acids, and sugars / carbohydrates, detergents, glycerol, Tricine, EDTA, Tris, potassium compounds, sulfhydryl, disulfide, phenolics, uric acid, guanine , xanthine, magnesium and calcium. Interference these agents can be minimized by eliminating these interferens. Highly recommended to use a blank to correct the absorbance. Interference caused by detergents, sucrose and EDTA can be eliminated with the addition of SDS or performing sample preparation with protein deposition.

Reference:

Dennison, C., 2002, A Guide to Protein Isolation, Kluwer Academic Publishers, New York
Lowry, O.H., Rosenbrough, N.J., Farr, A.L. & Randall, RJ, 1951, Protein Measurement with the Folin Phenol Reagent, J. Biol. Chem, 193: 265-275