Scanning microcalorimeters for studying macromolecules

Privalov, Peter L.

doi:10.1351/pac198052020479

Cited by 190 publications

(73 citation statements)

References 118 publications

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“…DSC-Calorimetry experiments were performed on the high sensitivity, differential adiabatic scanning microcalorimeter DASM-4 (Biobribor, Pushtchino, Russia) designed by Privalov (26). The scanning rate was 1°C/min; samples (1-2 mg/ml total LDL cholesterol) were loaded into the calorimeter sample cell at room temperature, and an equal amount of buffer was loaded into the reference cell.…”

Section: Methodsmentioning

confidence: 99%

Microphase Separation in Low Density Lipoproteins

Pregetter

Ruth

Schuster

et al. 1999

Journal of Biological Chemistry

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The structural organization of the neutral lipid core in human low density lipoproteins (LDL) was investigated in physicochemically defined, distinct human LDL subspecies in the density range of 1.0244 -1.0435 g/ml by evaluation of the core lipid transition temperature, chemical composition, and the behavior of spin-labeled core lipids. Calorimetric studies were performed on more than 60 LDL preparations, and the transition temperature, which varied between 19 and 32°C, was correlated to the chemical composition and revealed a discontinuity at a critical cholesteryl ester to triglyceride ratio of approximately 7:1. For electron spin resonance studies, several LDL preparations were probed with spin-labeled cholesteryl esters and triglycerides, respectively. In LDL with a high triglyceride content, both labels exhibited similar mobility behavior. In contrast, in LDL with only small concentrations of triglycerides, the behavior of labeled cholesteryl esters and labeled triglycerides differed distinctly. The cholesteryl esters were strongly immobilized below the transition temperature, whereas the triglycerides remained fluid throughout the measured temperatures. These results suggest that the critical cholesteryl ester to triglyceride mass ratio of 7:1 corresponds to two concentric compartments with a radial ratio of 2:1, where the liquid triglycerides occupy the core, and the cholesteryl esters form the frozen shell. At higher triglyceride contents, the triglyceride molecules insert into the cholesteryl ester shell and depress the peak transition temperature of the LDL core, whereas at lower triglyceride contents, excess cholesteryl esters are dissolved in the core. Low density lipoproteins (LDL)1 are the major carriers of cholesterol in the circulation and are intimately involved in atherogenesis (1 and references therein). These particles represent complex supramolecular assemblies of phospholipids (ϳ20% of the total mass), free (ϳ12%) and esterified (ϳ40%) cholesterol, triglycerides (ϳ5-10%), and a single copy of apolipoprotein B-100, a glycoprotein of 4,536 amino acids (2-4). In addition, LDL transports minor amounts of lipophilic vitamins and drugs (5). The structure of LDL can be described, in general terms, by a quasispherical core-shell model, in which the apolar constituents (cholesteryl esters (CE) and triglycerides (TG)) form a hydrophobic core of about 150 Å diameter, whereas the phospholipids, most of the unesterified cholesterol, and the apoprotein form an outer surface monolayer with a thickness of about 30 Å (6 -8).In this general sense, LDL would appear to fit readily into the structural core-shell scheme of circulating lipoproteins (9). However, LDL exhibits a distinct physical feature that makes it unique among all structural elements of blood: it is the only component to undergo a major structural transition just below physiological body temperature, in the range between 15 and 32°C (10, 11). The transition temperature varies among individual donors and is correlated to the core lipid compositi...

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Section: Methodsmentioning

confidence: 99%

Microphase Separation in Low Density Lipoproteins

Pregetter

Ruth

Schuster

et al. 1999

Journal of Biological Chemistry

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“…DSC experiments were performed with a high-sensitivity differential [19] with cell volumes of 0.48 ml. at a heating rate of l"C/min.…”

Section: Methodsmentioning

confidence: 99%

Thermal transitions in the purple membrane from Halobacterium halobium

Shnyrov

Mateo

1993

FEBS Letters

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The application of a successive annealing procedure to the scanning calorimetric endotherm of the purple membrane from H&bacterium halobium in phosphate buffer. pH 7.5, leads to five thermal transitions beneath the overall endotherm. Circular dichroism and fluorescence experiments have also been carried out with the natrve membrane heated at the same scan rate as in calorimetric runs (1"Clmin) as well as with previously heated membrane samples. These results, together with others from the literature, have been used to suggest a preliminary explanation of the five thermal transitions.

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“…Calorimetric experiments were carried out at 2"C/min in a DASM-4 model with 0.47-ml cell volume, as described by Privalov [15]. Protein concentrations, determined using the Lowry method [16], were in the range of 1.0-2.5 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

“…The bleached membrane was regenerated by adding retinal dissolved in ethanol. The samples were checked for polypeptide integrity using SDS/PAGE.Calorimetric experiments were carried out at 2"C/min in a DASM-4 model with 0.47-ml cell volume, as described by Privalov [15]. Protein concentrations, determined using the Lowry method [16], were in the range of 1.0-2.5 mg/ml.…”

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confidence: 99%

The role of retinal in the thermal stability of the purple membrane

Cladera

Galisteo²,

Sabés

et al. 1992

European Journal of Biochemistry

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Differential scanning calorimetry demonstrates that the bleached form of the purple membrane does not possess any measurable thermal transition in water, up to 105"C, whereas in 0.1 M phosphate pH 7.5 it shows a transition at about 8 2 T , with an enthalpy of 110 kJ/mol. In the latter medium, the native membrane shows the main transition at 97"C, with an enthalpy of 390 kJ/mol. The reduced form of the purple membrane shows two small transitions in water, as well as in 0.1 M phosphate, which do not seem to be related to the main thermal transition of the native membrane. Fouriertransform infrared spectra in D 2 0 show that the two modified samples, as well as the native one, undergo similar secondary structural changes upon thermal denaturation. These changes appear to extend through a wide temperature range for both modified forms, particularly for the bleached one. The results suggest that the main thermal transition in the purple membrane is due to a cooperative conformational change involving the disruption of the network of electrostatic and hydrogen-bonding interactions which originate from the protonated Schiff base. In the two modified membranes, these conformational changes appear to proceed smoothly through a rather low or non-cooperative process. The thermal behaviour of the bleached membrane in water resembles that of the molten globule state described for several globular proteins.Bacteriorhodopsin is a retinylidene protein found in the purple membrane patches of Halobacterium halobium cells. It translocates protons from the inside to the outside of the cell upon light absorption, thus forming a proton gradient across the celular membrane. The protein is arranged in seven transmembrane a-helices, and possess a retinal molecule attached to a lysine residue through a protonated Schiff base [I -31. The bacteriorhodopsin molecules are distributed through the bilayer in trimers, which in turn are ordered in a two-dimensional hexagonal array. Thermal denaturation studies of bacteriorhodopsin have shown the high stability of the purple complex. At neutral pH, the melting temperature (t,,,) is found near 95°C for the major transition of the native complex, which is found to be an irreversible process [4]. Extreme pH values [5], deionization [6], delipidation [7], or incorporation of bacteriorhodopsin in vesicles [8] lowers the t,, but in all cases the enthalpy of denaturation is about four times lower than those found for globular proteins [9]. This lower denaturation enthalpy seems to be the general situation for all the intrinsic membrane proteins so far investigated, a fact related to the lack of protein/water interactions in their denatured state [lo].The thermal transition is accompanied by decoloration due to the hydrolysis of the retinal Schiff base. On the other hand, it is known that the retinal molecule occupies a central position in the seven a-helical rods [l 11 and thus it can act as a tightening or clamping element. These features prompted us to investigate the influence of the retinal molecule...

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Scanning microcalorimeters for studying macromolecules

Abstract: -Principles of scanning microcalorimeter design, their basic characteristics and microcalorimetric data processing in studying biological macromolecules in solution are discussed.

Cited by 190 publications

References 118 publications

Microphase Separation in Low Density Lipoproteins

Microphase Separation in Low Density Lipoproteins

Thermal transitions in the purple membrane from Halobacterium halobium

The role of retinal in the thermal stability of the purple membrane

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