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| Protein |
To calculate specific activity of the enzyme, protein content was detrmined using the biuret method (Layne, 1957). Substances containing two or more peptide bonds form a purple complex with copper salt in alkaline.
Reagents used in the assay were as follows:
1. Bovine serum albumin standard (Albumin, Bovine Fraction V. Powder, No. A-4503 Lot 23 C-1630, Sigma Chemical Co., Sigma International, Ltd., P.O. Box 14508, St. Louis, Missouri 63178). Protein (100 mg) was dissolved in 10 ml of distilled water.
2. Biuret solution. CuS04* 5H20 (1.50g) and 6.0 g of NaKC4H4)6*4H20 were dissolved in 500 mL of distilled water.Ten percent NaOH (300 ml) was added with constant swirling. The solution was diluted to one liter with distilled water and stored in a paraffin-lined bottle.
The procedure used in the assay was as follows:
1. One ml of bovine serum albumin standard or 1 ml of supernatant from the enzyme extraction was pipetted into a cuvette.
2. Biuret solution (4.0 ml) was added. The contents of the cuvette were mixed and allowed to stand for 30 minutes at room temperature (20 to 25C). Optical density was recorded at 550 nm (Spectronic-20 Spectrophotometer). A reagent blank of 1.0 ml of distilled water and 4.0 ml of biuret solution was used and a blank of 1.0 mL of supernatant from the enzyme extraction plus 4.0 ml of water was used.
Concentration of protein in a sample was obtained by reference to a standard curve made with bovine standard albumin at concentrations of 1.0, 2.5, 5.0, 7.5 and 10.0 mg/ml. The standard curve was made by plotting optical density against the concentration of bovine serum albumin standard.
Updated: Wednesday, June 20, 2007.
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