Author:

Keywords:

transform infrared-spectroscopy, egg-white lysozyme, ribonuclease-a, thermal-denaturation, secondary structure, high-pressure, cytochrome-c, hydrostatic-pressure, alpha-lactalbumin, disulfide bonds, Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, RIBONUCLEASE-A, THERMAL-DENATURATION, SECONDARY STRUCTURE, HUMAN LYSOZYME, CYTOCHROME-C, CORRELATION SPECTROSCOPY, HYDROSTATIC-PRESSURE, DISULFIDE BONDS, DISAGGREGATION, FTIR, Animals, Cattle, Chickens, Cold Temperature, Deuterium Exchange Measurement, Enzyme Stability, Hot Temperature, Macromolecular Substances, Muramidase, Oxidation-Reduction, Protein Conformation, Protein Denaturation, Ribonuclease, Pancreatic, Spectroscopy, Fourier Transform Infrared, Thermodynamics, 0304 Medicinal and Biomolecular Chemistry, 0601 Biochemistry and Cell Biology, 1101 Medical Biochemistry and Metabolomics, 3101 Biochemistry and cell biology, 3205 Medical biochemistry and metabolomics, 3404 Medicinal and biomolecular chemistry

Abstract:

The thermal denaturation of lysozyme and fibonculease A (RNase A) under reducing and nonreducing conditions at neutral pH has been monitored by Fourier transform infrared spectroscopy. In the absence of the reductant, lysozyme and RNase A undergo apparent three- and two-state denaturation, respectively, as observed from the conformation-sensitive amide I' band. For both proteins the hydrogen-deuterium exchange takes place at lower temperatures than the main denaturation temperatures, suggesting that a transient denaturation mechanism occurs. The observed transition at 51.2 degreesC during the denaturation of lysozyme is attributed to this transient effect, rather than to the loss of tertiary structure. Under reducing conditions lysozyme aggregates during the heating phase, whereas RNase A shows only a minor aggregation, which further increases during the cooling step. The reduced stability of both proteins can be correlated with the transient denaturation behavior, which is also suggested to be involved in protein aggregation at physiologically relevant temperatures. In addition, it is shown that when the temperature is further increased, the amorphous aggregates dissociate. Comparison of the dissociated states with the denatured states obtained under nonreducing conditions indicates that these states have the same conformation. By using a two-dimensional correlation analysis we were able to show that the dissociation is preceded by a conformational change. It is argued that this extends to other types of perturbation.