Calorimetric study of thermally induced conformational transitions of ribonuclease A and certain of its derivatives

A number of authors have reported observations on D-amino acid oxidase [D-amino acid: 02 oxidoreductase (deaminating), EC 1.4.3.3] that they have interpreted in terms of a temperature-dependent conformational transition having a van't Hoff enthalpy amounting to more than 1 cal per g of protein (1 cal = 4.184J). No indication of this transition is obtained by using a differential scanning calorimeter having a sensitivity considerably in excess of that required to detect such a transition. The implications of this discrepancy are discussed.Massey et al.(1) reported temperature dependencies for several properties of D-amino acid oxidase [DAAO, D-amino acid:02 oxidoreductase (deaminating), EC 1.4.3.3] that they interpreted in terms of a thermally induced conformational transition in the enzyme. The most significant of these for our present purposes was a 30% decrease in the tryptophan fluorescence of the enzyme as the temperature was raised from 8°to 22°. This decrease, which was reversible, followed the symmetrical sigmoidal curve expected for a two-state process with an enthalpy value, AHvH, The most direct means for observing a thermally induced change in a protein is differential scanning calorimetry (2). For any two-state equilibrium process not involving intermolecular cooperation-such as was assumed for the purported transition in DAAO in deriving a value for AHvH from the data on the fluorescence quenching-the apparent enthalpy, AHvH, must be equal to the actual enthalpy, AHA, as measured in the scanning calorimeter. Although no general proof has been given, a consideration of simple model systems (3) Details concerning the preparation and analysis of holo-and apoenzyme solutions and those concerning activity determinations were described in a previous publication (5). The appropriate dialysates were used as reference solutions in the differential scanning calorimetry experiments. It was ascertained that no significant losses of enzyme activity were incurred in any experiments, except for.fluorescence measurements on the apoenzyme in which photodegradation was a problem.Most of the calorimetric experiments were performed with a Privalov calorimeter (4)

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confidence: 99%

Proposed temperature-dependent conformational transition in D-amino acid oxidase: a differential scanning microcalorimetric study.

Sturtevant¹,

Mateo²

1978

“…Several types of equilibrium studies (6)(7)(8) have shown that ribonuclease A (RNase A) [bovine pancreatic ribonuclease A, with disulfide (-SS-) bridges intact] undergoes a typical 2-state thermal unfolding reaction in the pH range 1-2. However, at neutral pH, this reaction shows readily observable deviations from the 2-state behavior (8,9). Kinetic (6)(7)(8).…”

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confidence: 99%

“…However, at neutral pH, this reaction shows readily observable deviations from the 2-state behavior (8,9). Kinetic (6)(7)(8).…”

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confidence: 99%

The Sequential Unfolding of Ribonuclease A: Detection of a Fast Initial Phase in the Kinetics of Unfolding

Tsong

Baldwin

Elson

1971

Temperature-jump studies have been used to detect a rapid reaction in the thermal unfolding of ribonuclease A (RNase A). The fast reaction occurs over a wide range of pH, and the results of a detailed study at pH 1.3 are reported here. Although its amplitude is small, the reaction is easily measurable over the entire temperature range of thermal unfolding. It occurs in the millisecond time range, and is faster by 3-4 orders of magnitude than the slow unfolding reaction studied previously. Unfolding is measured here by the change in absorbance at 287 nm, which reflects the exposure to solvent of buried tyrosine groups. In an effort to understand the latter paradox, a simple sequential model of protein folding was introduced (II). It can approximate 2-state behavior by several tests, even when sizable concentrations of intermediate, partly-folded molecules are present. A basic prediction of this simple sequential model is the existence of a significant transient phase preceding steady-state kinetics of protein unfolding. If unfolding is highly cooperative, so that the reaction appears to be a 2-state reaction, the amplitude of the transient phase will be small and the separation in time scales between the transient phase and the steady-state phase will be large (e.g., 3-6 orders of magnitude in some hypothetical examples (1I). Nevertheless, temperature-jump studies (11) might detect the transient phases, since the sensitivity of modern instruments is excellent and the time range of observation extends down to a few microseconds.We have found a fast reaction in the thermal unfolding of RNase A over the entire pH range studied (from 1 to 7). Detailed measurements have been made only at pH 1.3, for the following reasons. (a) Unfolding occurs at relatively low temperatures (Fig. 1), which reduces the problem of cavitation (11) in measuring fast effects. (b) The reversibility of unfolding is good at this pH. (c) This is the pH range where an apparent 2-state unfolding of RNase A has been found (6-8).Oscilloscope records of the fast reaction observed at 286 nm are shown in Fig. 2. The reaction occurs in the millisecond time range over the entire temperature range of unfolding (Table 1). It is 3-4 orders of magnitude faster, depending on the temperature, than the slow unfolding reaction studied (11)

“…Differential scanning calorimetry (DSC) would afford a useful means for determining the enthalpy change in a thermally-induced polymerization. We have employed two sensitive scanning calorimeters in our study of tubulin, one constructed in this laboratory (8,9), and the other designed and constructed in Russia (10). Although various experimental difficulties prevented our obtaining clearcut results from numerous DSC experiments, we never saw any indication of a significant enthalpy change.…”

Section: Methodsmentioning

confidence: 99%

Calorimetric studies of the in vitro polymerization of brain tubulin.

Sutherland¹,

Sturtevant²

1976

The enthalpy change for chain propagation in the polymerization of bovine tubulin has been studied directly by stopped-flow microcalorimetry at 170 and 250, and found to be 0 + 1 kcal per mol of 6S tubulin dimer at both temperatures.Substantial heat evolution with a half-time of decay of approximately 1 hr was observed when tubulin was injected into the calorimeter. This heat was shown to result from contamination of the tubulin by small amounts of some material from the crude brain homogenate from which the tubulin was prepared, and to be totally unconnected with microtubule assembly. Model In the following discussion, it should be remembered that thermodynamics strictly applies only to reversible processes between well defined states. Microtubule assembly is only quasi-reversible (3) and may involve nucleotide hydrolysis (4). Moreover, the intermediate species and even the polymers may vary with the biological system or from one preparation to another. We therefore attempted to keep our experimental procedure as nearly constant as possible. Of course, the same restrictions apply as well to van't Hoff measurements but for these measurements the interpretation is complicated by the existence of states intermediate between the polymerizing unit and the final polymer, as will be discussed below.Although the measurement discussed herein applies primarily to the chain lengthening process and not to the nucleation process, the amount of tubulin in the form of nuclei is likely to comprise at most only a small percentage of the total mass of tubulin in any microtubule preparation. Therefore, the overall enthalpy change involved in the formation of microtubules reflects primarily this enthalpy and not the enthalpy of nucleation. MATERIALS AND METHODSTubulin, twice polymerized and depolymerized, was prepared by the method of Shelanski et al. (5) The GTP was purchased from Sigma (Type II-S) and fl,ymethyleneguanosine triphosphate (GMPPCP) was from P-L Biochemicals. Protein concentrations were determined by a modified Lowry procedure (6), using a bovine serum albumin standard.Flow Calorimetry. Our most reliable thermochemical data were obtained by means of flow calorimetry. The instrument employed is a modification of the Beckman model 190 batch calorimeter, and was previously described (7). The flow tubing is platinum of 1 mm internal diameter, and a total volume of 1 ml in thermal contact with the measuring thermopile. The volume of delivery tubing from each syringe to the mixer is 0.58 ml; part of this tubing is Teflon in good thermal contact with the constant temperature water bath surrounding the calorimeter submarine, and part of it is platinum in good thermal contact with the massive aluminum heat sink of the calorimeter.Various experimental formats were tried with this instrument, of which the only successful one may be described as a 3565 Abbreviations: DSC, differential scanning calorimetry; GMPPCP, f3, 'y-methyleneguanosine triphosphate.