A MODIFIED METHOD FOR ESTIMATION OF FISH MUSCLE PROTEIN BY BIURET METHOD

Abstract

Colorimetric method for protein determination of fish muscle based on biuret reaction had been reported by SNOW1), MATSUMOTO2) and TORTEN3). In case of opaque turbid protein solutions, however, some erratic estimates are occasionally obtained by the biuret method. To eliminate this error, the author intended to modify the MATSUMOTO'S method. It was found that the linearity between the protein concentration and the corrected color intensity of the biuret reaction (“biuret color intensity”) obeys BEER's law (Fig. 3), and this allows the estimation of protein. The modified method is as follows: The reagents and procedure are shown also in Table 1. To prepare A solution, 5.00ml of sample protein solution containing 0.1 to 0.5mg of protein nitrogen per ml and 5.00ml of biuret reagent I are well mixed. In the same way, B solution is prepared with each 5.00ml of sample solution and turbidity correction reagent II. For optical density measurement, reference solutions which contain 5.00ml of water instead of sample protein solution in each of A and B solution respectively are also prepared. After two hours standing at room temperature, the optical densities of A and B solutions are read at the wavelength of 545mμ with one cm of length of light path. The “biuret color intensity” is obtained by subtracting optical density of B solution from that of A solution, where optical density of B solution is the correction term for turbidity. The protein nitrogen concentration is calculated by the following equation. protein nitrogen mg/ml=0.94×biuret color intensity With respect to the above method some observations were made and the obtained results are as follows: Protein concentrations of 0.1 to 0.5mg of nitrogen per ml were determined rapidly with an error less than 4 per cent (Table 3). The conversion factors were almost same for vari-ous kinds of fish muscle and also for the proteins of myosins and myogens fractions (Fig. 3). But for shark or whale muscle protein, other conversion factors should be used. The reagent were kept for several months in refrigerator without any change in color developing ability for protein (Table 4). The “biuret color intensity” was not affected by the presence of such salts in the sample solution as KCl, NaCl, NaHCO3 and potassium or sodium phosphates (Table 5a), while the interferences were encountered by the presence of reducing sugars, large amount of peptides, and also of such substances as listed in Table 5b. When the sample solution contained ammonium sulfate as interfering substance, it was necessary to separate the protein from the interfering substance by precipitation with trichloroacetic acid and centrifugation. Thus obtained protein precipitate was dissolved with 0.2N sodium hydroxide solution. This pretreatment with trichloroacetic acid made it possible to estimate the protein by the modified biuret method (Table 6).