PHYSIOLOGICAL AND BIOCHEMICAL TECHNICS 179 



2 min in the presence of 0.5A^ HCL. The majority of the other proteins 

 are denatured and precipitated by this treatment and may be removed by 

 centrifugation. The myokinase remains in the supernatant fluid in a 

 nearly pure condition. It should be noted that not all denatured pro- 

 teins are precipitated by this and other methods. Thus, it would be as 

 useful in isolating an enzj^me activity to denature all other activities yet 

 allow the spurious proteins to remain in solution with the desired enzyme. 

 As pointed out above, the preparation of bacterial protein extracts often 

 requires somewhat more extensive methods than those needed for animal 

 protein solutions. In addition, certain special problems arise which are 

 peculiar to the fractionation of bacterial protein mixtures. The cell-free 

 extracts obtained by most of the methods cited above contain relatively 

 large amounts of nucleic acid. Because nucleic acid extends the range of 

 precipitation of a given protein from a mixture, it is highly desirable to 

 remove as much nucleic acid as possible from the mixture before proceed- 

 ing with the common fractionation procedures. Either one of two 

 methods is commonly used for precipitating nucleic acid. After the 

 crude cell-free extract is prepared, the solution is dialyzed for at least 

 4 hr at 0-5° C against a solution (distilled water or a suitable buffer at 

 about 0.01 M and pH 8 is often used) in order to remove as many small 

 molecules as possible. After dialysis, the protein solution is adjusted to 

 pH 6.0 by the dropwise addition of M CH3COOH. Nucleic acid is pre- 

 cipitated from the adjusted mixture by the dropwise addition of either 

 (1) 0.05 vol of M MnClo or (2) protamine sulfate. One milligram of 

 protamine sulfate (17 mg per ml, pH 5.0) is added for each 100 mg of 

 protein in the original cell-free extract. Protamine sulfate has a negative 

 temperature solubility coefficient, and the above concentration repre- 

 sents an approximately saturated solution at room temperature. The 

 additions are made at 0°C with gentle stirring. After 20 min, the mixture 

 is centrifuged and the supernatant solution, with a greatly decreased 

 concentration of nucleic acid, may be fractionated with conventional 

 procedures. For these and many other specific protein fractionation 

 procedures, the treatise of Colo wick and Kaplan (1955) is recommended. 



Protein Estimation 



The optical method of Warburg and Christian (1941) for the estima- 

 tion of protein is simple and rapid but is unfortunately less precise when 

 protein solutions contain high concentrations of nucleic acid. Thus, this 

 technic cannot be used reliably with crude cell-free bacterial extracts. 

 The method depends upon the relative optical densities of the protein 

 solution at 260 mju (due to the purine and pyrimidine components of 

 nucleic acid) and at 280 m/x (due to the aromatic amino acids of the 



