Home	Minipurification

Thermal denaturation

Heat treatment as a plant virus diagnostic tool

Summary

This procedure is essentially two dimensional analysis of plant viruses with temperature (heat) as the first dimension and SDS gel electrophoresis of sedimentable macromolecules (minipurified virus) in the second dimension.

Thermal denaturation

Active proteins are precisely folded polypeptides. Heat unfolds proteins inactivating them in the process. Except under special conditions unfolding is irreversible. Unfolded proteins either precipitate or remain largely unfolded and inactive on cooling. Multisubunit structures (e.g. viruses) are generally more stable than isolated subunits. The temperature required to inactivate a virus is a measure of its stability.

History of thermal denaturation in virology

James Johnson (Phytopathology) in 1932 was the first to realize the value of thermal stability. Johnson measured infectivity of crude extracts heated at 10^oC intervals. Thermal stability proved to be a virus property independent of host. Though methodology was time consuming and imprecise, thermal stability was, at the time, a useful, readily measurable physical property. Thermal stability is commonly disparaged, because Johnson's methods are imprecise. This shouldn’t obscure the fact that it’s an informative property.

An alternative way of measuring thermal stability

Heating blocks are cheap. One can set a series of blocks at roughly 5^oC intervals. One then heats aliquots simultaneously. Virus denaturation either precipitates or solubilizes the coat protein. After cooling, samples are “minipurified” and analyzed on SDS gels. In either case denatured virus will no longer "minipurify". Minipurified samples contain rubisco and other host proteins which serve as internal standards.

Method

1 - Grind 5g tissue in ~120 ml of 0.2 M Na₃citrate adjusted to pH 6.5 with citric acid.
2- Filter through muslin. Add 12 ml aliquots to 30 ml Corex tubes and heat 20’ in a heating block. Each treatment consists of 2 tubes.
3- Cool aliquots on ice and proceed with minipurification (Pour 2 tubes into a 38 ml polycarbonate tube, centrifuge 12’ at 37 kRPM, pellet supernatants 40’ at 37 kRPM in a Ti50.2 Beckman rotor)
4- Dissolve pellets in ~100 µl of buffer, electrophorese aliquots on an SDS gel and stain with silver.

Results

Figure 1 shows that the methodology works. Virus and accompanying host proteins “disappear” as temperature increases. Mild heat treatment removes polydisperse impurities while rubisco and other host proteins are stable.

Considerations

Large volumes heat slowly. A few ml of water in heating block wells improves heat transfer. Nonetheless samples don’t reach steady state at high T. Therefore absolute T values can’t be taken literally.

This method measures physical disruption. Infectivity may be more thermolabile than physical integrity. For viruses with several particle types, different particle types may denature at different Ts. Virus particles may not denature in all or none fashion. For example, partially stripped particles could be intermediates in denaturation of elongated viruses.

Useful observations

Mild heat treatment removes polydisperse impurities. In no case do capsids proteolyze, therefore cooling extracts during purification is superfluous. Only the least stable viruses (alfalfa mosaic and tobacco streak) denature below 50^oC. Heating removes rubisco only from the most stable viruses.

Potential uses

Figure 2 shows the denaturation pattern of virus cultured from a Nicotiana glauca sample collected in Bisbee, AZ. The culture contains a potyvirus (PVY,denatures at low T), STMV (denatures at intermediate T) and TMGMV (survives the highest T). The thermal denaturation pattern shows that STMV and TMGMV capsid proteins are on different particles. One gets pure TMGMV by inoculating a high T aliquot to C. quinoa and transferring a single lesion to Turkish tobacco.

Heat treatments are convenient for testing how pH and salt concentration affect virus stability. Mild heat treatment is useful for clarifying. A simple clarification method is to incubate 38 ml polycarbonate tubes (with extracts) in hot tap water (~58^oC) for 10 min before centrifugation.