Elsevier

Methods

Volume 35, Issue 2, February 2005, Pages 117-125
Methods

Measurement of protein stability and protein denaturation in cells using differential scanning calorimetry

https://doi.org/10.1016/j.ymeth.2004.08.002Get rights and content

Abstract

Many methods exist for measuring and studying protein denaturation in vitro. However, measuring protein denaturation in cells under conditions relevant to heat shock presents problems due to cellular complexity and high levels of light scattering that interfere with optical techniques. A general method for measuring protein denaturation in cells using high sensitivity differential scanning calorimetry (DSC) is given. Profiles of specific heat (cp vs. temperature) are obtained providing information about transitions in cellular components including the denaturation of proteins. The specific approaches employed with erythrocytes, bacteria, and mammalian cells are described, and an identification of several features of the DSC profiles is given. Protein denaturation on the level of roughly 7–20% occurs for commonly used heat shocks in mammalian cells.

Introduction

Cells and organisms respond to supra-optimal temperatures, referred to as hyperthermia or heat shock, in several ways. Cytotoxicity results if the heat shock is severe enough [1]. Milder heat shocks produce sub-lethal damage that can result in sensitization to other stressors. Thermal radiosensitization is of particular interest because of its potential in cancer therapy [2]. Heat shock also results in the induction of heat shock proteins, which correlate well, in general, with thermotolerance [3]. Each of these responses appear related and in large part due to a single initiating event. For nucleated cells, especially mammalian cells, temperatures only 3–5 °C above normal growth temperature are needed to induce a response to heat shock.

There is considerable evidence that this initiating event, occurring after a small increase in temperature, is the denaturation of thermolabile cellular proteins. Some of the earliest evidence implicating protein denaturation followed from the thermodynamics of cell killing. A high activation energy, usually in the range of 600–800 kJ/mol, is associated with hyperthermic cell killing [1]. In the simplest interpretation, the activation energy is just the temperature dependence of killing. However, the Arrhenius model predicts that the underlying mechanism of killing should have the same activation energy. Most enzymatic processes have activation energies in the range of 20–100 kJ/mol, while molecular transitions have much higher activation energies in the range of 400–800 kJ/mol. In addition, the thermal inactivation of enzymes also requires a high activation energy [4]. These observations are consistent with a temperature-induced transition such as protein denaturation being the initiating event during heat shock. The implication that the high Arrhenius activation energy of killing suggests that protein denaturation is the initial event in thermal damage was recognized independently by two groups as early as 1971 [1], [5].

A number of chemical agents, referred to as sensitizers or protectors, respectively, either sensitize cells to heat shock or protect when present during heating. Table 1 is a partial listing of the known cellular sensitizers and protectors. In addition to their effects on cell survival during heat shock, nearly all the sensitizers have been shown to induce heat shock protein (Hsp) synthesis at normal growth temperatures, while the protectors inhibit Hsp synthesis, and reduce the level of thermotolerance when present during heating (for a review see [6]). The agents given in Table 1 have little in common except that most, if not all, of the sensitizers destabilize or perturb protein structure. The protectors glycerol and D2O are protein stabilizers. The other two protective conditions, acquired thermotolerance and the tolerance induced by cycloheximide, are associated with protein stabilization in cells [6].

That numerous chemical agents sensitize cells to heat shock and induce Hsp synthesis and thermotolerance suggests that common initiating signals are responsible for these responses. Similar observations led Hightower to suggest in 1981 that Hsp induction is due to damaged or denatured protein [7]. Since then it has been shown that injection of denatured protein into cells is sufficient to induce Hsp synthesis [8], [9]. The correlation between Hsp synthesis and thermotolerance also suggests that protein denaturation is the initiating signal for both and also the initiating event for thermal damage resulting in death.

Section snippets

Assays for protein denaturation

If protein denaturation is the initial event occurring during heat shock that is ultimately responsible for thermal damage, then it is important to directly measure protein denaturation in cells during heat shock. Protein denaturation refers to a conformational transition resulting in a partial or total unfolding, depending on denaturing conditions, of a protein from its native state to a more disordered conformation. This transition is usually of first-order, and for thermal denaturation

Protein denaturation by differential scanning calorimetry

Differential scanning calorimetry (DSC) is a powerful technique for studying the thermodynamics of transitions in biological macromolecules [18]. The specific heat (cp) as a function of temperature can be determined for a protein solution or more complex biological structure. Differential scanning calorimeters ordinarily consist of two cells: one containing sample and the other the reference solvent. The heat flow into the cells is monitored as the temperature is increased at a constant rate.

Differential scanning calorimetry profiles of cells

Cells are a complex, interacting mixture of proteins, nucleic acids, and membrane lipids, each of which can undergo order–disorder, endothermic transitions detectable by DSC, and numerous small molecules and ions. A DSC scan should be the sum of the transitions of all components. In addition, other processes such as aggregation and metabolism, both exothermic events, can be detected by calorimetry and must be considered in interpreting a DSC profile. The human erythrocyte is a fairly simple

Summary

DSC is a technique that is sensitive to protein denaturation in intact cells including erythrocytes, bacteria, yeast [28], mammalian cells, and tissue [29]. Irreversible endothermic transitions are detectable in the relevant heat shock range for the above cells. These endothermic transitions can be identified as protein denaturation based on irreversibility, the values of ΔHcal obtained, the expected large contribution of protein denaturation due to the cellular content of proteins, the lack of

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