Purification of Mouse Poliomyelitis Virus by Methanol Precipitation at Low Temperatures. ∗

Abstract

Conclusions

Although at first glance the data may appear rather inconsistent, closer inspection reveals that the results are consistent when consideration is given to the experimental error involved in some of the methods. The variation in the nitrogen determinations of the crude virus suspensions were thought to be due in turn to expected variation in the amount of protein removed in the freezing and thawing steps. Estimations of the degree of purity have been presented in terms of the amount of nitrogen eliminated from the crude sample. Since purified normal brain gave nitrogen values which bore a close similarity to those obtained with purified infected tissue, it can be seen that the so-called purified fractions still contain a large amount of normal brain protein. There is no doubt that the non-viral protein greatly overbalances the viral proteins in the purified fraction. However, a relatively large amount of protein is removed during the procedure, as compared with the amount removed in the ultracentrifugation methods of purification cited by Beard.¹ The activities of crude and purified virus fractions, with a few exceptions, remain fairly constant, indicating that most of the active virus is recovered. Although the titrations may not be accurate enough to determine the exact yield, they indicate that in many of the determinations 100% of the virus appeared to be recovered. The number of infectious units of virus per milligram of nitrogen depends, in part, on the original titer of the virus, and therefore is a relative value. When the methanol precipitate was held at −4°C for 4 hours the yield of virus was greater. Apparently no significant difference resulted from the use of 25% or 30% methanol. Also, the use of buffers at pH 5.1 or pH 5.6 to wash the precipitates did not seem to appreciably alter the results. One step that did result in the reduction of nitrogen was the immediate freezing of the final precipitate at pH 7 before it went into solution. Upon thawing, insoluble protein was removed by centrifugation.