Abstract
A purification procedure has been devised for a highly potent inhibitor of virus infection occurring in spinach, Spinacia oleracea L.. The frozen leaves and stems of spinach plants were homogenized with 0.1M, pH 7.0 phosphate buffer (neutral PB) and pressed through gauze. The expressed juice was centrifuged at low speed (at 10,000×g for 20 minutes). The supernatant was heated at 100°C for 1-3 minutes in a water bath and centrifuged after cooling. The supernatant was then brought to 50% saturation by slowly adding ammonium sulphate with stirring. After centrifugation, the inhibitor was precipitated from the obtained supernatant by 100%saturated ammonium sulphate. The resulted precipitate was collected by centrifugation and resuspended in neutral PB.
The above partially purified inhibitor was further purified by gel filtration and ion exchange column chromatographies. Sephadex G50 gel filtration chromatography of the inhibitor solution showed that most inhibitory activity was eluted in the void volume. The inhibitor-containing fractions were combined and subjected to Sephadex G100 gel filtration. Three peaks were obtained and the inhibitory activity was fined in the second peak. These gel filtration experiments suggested that the inhibitor has molecular weight of 20,000-40,000.
Anion exchange chromatography on ECTEOLA cellulose showed that inhibitory activity was not adsorbed by the adsorbent at neutral pH. Cation exchange column chromatography on cellulose phosphate showed that the inhibitor was adsorbed at pH 5.9 and eluted by 1M NaCl.
When the inhibitor was adjusted to pH 0.5 for 0.5-3 hours and the reaction readjusted to pH 7, there was rather small change in the inhibitive effect. However, the greater part of inhibitive activity was destroyed in alkaline solution at pH 13.3 for 0.5-3 hours. The activity was also completely destroyed with phenol treatment.
The ultraviolet absorption spectrum of the purified inhibitor solution showed the typical curve of a protein with a maximum absorption at about 280 nm.
The separation of virus from the virus-inhibitor mixture by gel filtration showed that no permanent effect occurred between virus and inhibitor.
