PARENT SESSION
3:45 PM to 5:15 PM
Monday, April 22, 2002
NAHS Mini-Symposium 9
NAHS Heat Denaturation and Protein Stability
Room: Nevada 1-2
Chair: Lepock, James²²Dept. of Physics, Waterloo, Ontario
Speakers: Borrelli, Michael ³; Lepock, James⁴; Weber, Lee⁵; Kampinga, Harm⁶³Dept. of Radiation Oncology, Royal Oak, MI⁴Dept of Physics, Waterloo, ON, CA⁵Dept of Biology, Reno, NV⁶Dept of Radiation/Stress Cell Biology, Groningen, NL

(MS09-) Quantification of protein denaturation and aggregation during hyperthermia.

Lepock, James^*,1, ¹ Departments of Physics and Biology, Waterloo, Ontario

ABSTRACT-
There is considerable evidence that the initial event occurring during heat shock is the thermal denaturation of a set of thermolabile proteins whose stability determines the intrinsic resistance of cells to hyperthermia. Denatured protein is the primary inducing signal for heat shock protein synthesis and appears to be the primary cause of thermal damage and cytotoxicity. Protein denaturation induces protein insolubilization due to aggregation. The identity of the thermolabile proteins and the amount of protein denaturation necessary for killing are unknown. Protein denaturation was quantitated in a number of cell lines using differential scanning calorimetry. Fractional denaturation was related to cell killing for CHL V79 and HeLa cells which differ in their intrinsic resistance to hyperthermia. To a rough approximation, mammalian cells can withstand approximately 5% denaturation with little killing. Virtually all cells are killed when the level of protein denaturation exceeds approximately 10%. Thus, cells can accommodate a small amount of denaturation before death ensues, probably because the pathways for removal or refolding of denaturated protein become saturated. The denaturation of this small amount of protein causes the insolubilization of a much larger quantity of protein. Studies using Triton X-100 and digitonin to measure protein insolubilization in the nucleus and the cytoplasm, respectively, imply that similar heat exposures cause the insolubilization of approximately 70% of total cell protein. A large fraction of this aggregated protein is native and potentially functional. Thus, denaturation and aggregation of proteins are early events during hyperthermia which may be responsible for later cellular responses such as killing and radiosensitization.

KEYWORDS: protein denaturation, hyperthermia, protein aggregation