[73] Spectrophotometric and turbidimetric methods for measuring proteins
Publisher Summary
The turbidity produced when protein is mixed with low concentrations of any of the common protein precipitants can be used as an index of protein concentration. The resulting turbidity is maximum after about 10 minutes and may be measured spectrophotometrically in the wavelength region of 600 m. Standardization may be effected by comparison with the turbidity produced by a suspension of a dried protein precipitate, or reference may be had to the methyl acrylate-styrene polymer. Turbidimetric techniques are rapid and convenient, but they yield different values with different proteins. They do not permit differentiation between protein and acid-insoluble compounds such as nucleic acids. Protein estimation with the Folin-Ciocalteu reagent include (1) biuret reaction of protein with copper ion in alkali, and (2) reduction of the phosphomolybdic-phosphotungstic reagent by the tyrosine and tryptophan present in the treated protein. Protein estimation by ultraviolet absorption takes advantage of the fact that nucleic acid, however, absorbs much more strongly at 260 mμ than at 280 mμ, whereas with protein the reverse is true. This advantage is used to eliminate, by calculation, the interference of nucleic acids in the estimation of protein.
References (12)
- O.H. Lowry et al.
J. Biol. Chem.
(1951) - O. Folin et al.
J. Biol. Chem.
(1927) - A.G. Gornall et al.
J. Biol. Chem.
(1949) - H.W. Robinson et al.
J. Biol. Chem.
(1940) - H.m. Kalckar
J. Biol. Chem.
(1947) - M. Kunitz
J. Gen. Physiol.
(1952)
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