METHOD 6 This fluorimetric method is based on the derivatisation of the protein with o-phthalaldehyde, which reacts with the primary amines of the protein (N-terminal amino acid and the -amino group of lysine residues). The sensitivity of the assay can be increased by hydrolysing the protein before adding o-phthalaldehyde. Hydrolysis makes the -amino group of the constituent amino acids available for reaction with the phthalaldehyde reagent. The method requires very small quantities of the protein. Primary amines, such as tris(hydroxymethyl) aminomethane and amino acid buffers, react with phthalaldehyde and must be avoided or removed. Ammonia at high concentrations reacts with phthalaldehyde. The fluorescence obtained when amine reacts with phthalaldehyde can be unstable. The use of automated procedures to standardise this procedure may improve the accuracy and precision of the test. Use distilled water R to prepare all buffers and reagents used for this method. Test solution. Dissolve a suitable quantity of the substance to be examined in a 9 g/l solution of sodium chloride R to obtain a solution having a concentration within the range of the concentrations of the reference solutions. Adjust the solution to pH 8 to 10.5 before addition of the phthalaldehyde reagent. Reference solutions. Dissolve the reference substance for the protein to be determined in a 9 g/l solution of sodium chloride R. Dilute portions of this solution with a 9 g/l solution of sodium chloride R to obtain not fewer than five reference solutions having protein concentrations evenly spaced over a suitable range situated between 10 μg/ml and 200 μg/ml. Adjust the solutions to pH 8 to 10.5 before addition of the phthalaldehyde reagent. Blank solution. Use a 9 g/l solution of sodium chloride R. Borate buffer solution. Dissolve 61.83 g of boric acid R in distilled water R and adjust to pH 10.4 with a solution of potassium hydroxide R. Dilute to 1000 ml with distilled water R and mix. Phthalaldehyde stock solution. Dissolve 1.20 g of phthalaldehyde R in 1.5 ml of methanol R, add 100 ml of borate buffer solution and mix. Add 0.6 ml of a 300 g/l solution of macrogol 23 lauryl ether R and mix. Store at room temperature and use within 3 weeks. Phthalaldehyde reagent. To 5 ml of phthalaldehyde stock solution add 15 μl of 2-mercaptoethanol R. Prepare at least 30 min before use. Use within 24 h. Procedure. Mix 10 μl of the test solution and of each of the reference solutions with 0.1 ml of phthalaldehyde reagent and allow to stand at room temperature for 15 min. Add 3 ml of 0.5 M sodium hydroxide and mix. Determine the fluorescent intensities (2.2.21) of solutions from the reference solutions and from the test solution at an excitation wavelength of 340 nm and an emission wavelength between 440 and 455 nm. Measure the fluorescent intensity of a given sample only once, since irradiation decreases the fluorescence intensity. Calculations. The relationship of fluorescence to protein concentration is linear. Plot the fluorescent intensities of the reference solutions against protein concentrations and use linear regression to establish the standard curve. From the standard curve and the fluorescent intensity of the test solution, determine the concentration of protein in the test solution.
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