Abstract
A marked reactivation of tobacco mosaic virus protein that has been partially or completely inactivated by formaldehyde 1 has been obtained by dialysis at pH 3. In the experiments to be reported, inactivation was allowed to proceed at room temperature in reaction mixtures containing 2% purified virus protein and 2% formaldehyde in M/10 phosphate at pH7. Samples were removed after different periods of time and immediately dialyzed against cold distilled water for about 6 hours in order to stop the reaction by the removal of excess formaldehyde. Preliminary experiments indicated that such inactivated virus protein could be partially reactivated by incubation with dimethyldihydroresorcinol 2 or with histidine, 3 or by dialysis at pH3 or at pH7. Dialysis at pH3 gave the best results and, since very little change occurred after the third day, a 3-day period of dialysis was adopted as the reactivation procedure.
The dialyzed solutions of inactivated and of reactivated virus protein were adjusted to M/10 phosphate (pH7), analyzed for total nitrogen, and diluted with M/10 phosphate to concentrations suitable for inoculating against controls containing 10-5 or 10-6 gm. of active virus protein per cc.
Nicotiana glutinosa L. was chosen as test plant and the half-leaf method of inoculation was used, 4 with the exception that whole leaves were inoculated in tests for complete or nearly complete inactivation. The relative activities were interpreted after comparison with dilution curves of active virus and of mixtures of active and of inactive virus proteins. Inactive protein was found to reduce the lesion count by 40 to 50% when present at a concentration of gm./cc., but had very little effect at 10−3 gm./cc. This reduction was no greater than that caused by comparable amounts of hydrogen peroxide-inactivated virus protein or of egg albumin.
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