Differential scanning calorimetry of the irreversible thermal denaturation of thermolysin

Biochemistry ◽  
1988 ◽  
Vol 27 (5) ◽  
pp. 1648-1652 ◽  
Author(s):  
Jose M. Sanchez-Ruiz ◽  
Jose L. Lopez-Lacomba ◽  
Manuel Cortijo ◽  
Pedro L. Mateo
Keyword(s):  
Differential Scanning Calorimetry ◽  
Thermal Denaturation ◽  
Scanning Calorimetry

Thermal denaturation ofTrichoderma reesei cellulases studied by differential scanning calorimetry and tryptophan fluorescence

1992 ◽  
Vol 34-35 (1) ◽  
pp. 217-231 ◽  
Author(s):  
J. O. Baker ◽  
K. Tatsumoto ◽  
K. Grohmann ◽  
J. Woodward ◽  
J. M. Wichert ◽  
...  
Keyword(s):  
Differential Scanning Calorimetry ◽  
Thermal Denaturation ◽  
Tryptophan Fluorescence ◽  
Scanning Calorimetry

scholarly journals Comparative thermal denaturation of Thermus aquaticus and Escherichia coli type 1 DNA polymerases

2003 ◽  
Vol 374 (3) ◽  
pp. 785-792 ◽  
Author(s):  
Irene KARANTZENI ◽  
Carmen RUIZ ◽  
Chin-Chi LIU ◽  
Vince J. LiCATA
Keyword(s):  
Escherichia Coli ◽  
Differential Scanning Calorimetry ◽  
Thermal Denaturation ◽  
Dna Polymerases ◽  
Large Fragment ◽  
Scanning Calorimetry ◽  
Thermus Aquaticus ◽  
Taq Polymerase ◽  
Nuclease Domain

Thermal denaturations of the type 1 DNA polymerases from Thermus aquaticus (Taq polymerase) and Escherichia coli (Pol 1) have been examined using differential scanning calorimetry and CD spectroscopy. The full-length proteins are single-polypeptide chains comprising a polymerase domain, a proofreading domain (inactive in Taq) and a 5′ nuclease domain. Removal of the 5′ nuclease domains produces the ‘large fragment’ domains of Pol 1 and Taq, termed Klenow and Klentaq respectively. Although the high temperature stability of Taq polymerase is well known, its thermal denaturation has never been directly examined previously. Thermal denaturations of both species of polymerase are irreversible, precluding rigorous thermodynamic analysis. However, the comparative melting behaviour of the polymerases yields information regarding domain structure, domain interactions and also the similarities and differences in the stabilizing forces for the two species of polymerase. In differential scanning calorimetry, Klenow and Klentaq denature as single peaks, with a melting temperature Tm of 37 and 100 °C respectively at pH 9.5. Both full-length polymerases are found to be comprised of two thermodynamic unfolding domains with the 5′ nuclease domains of each melting separately. The 5′ nuclease domain of Taq denatures as a separate peak, 10 °C before the Klentaq domain. Melting of the 5′ nuclease domain of Pol 1 overlaps with the Klenow fragment. Presence of the 5′ nuclease domain stabilizes the large fragment in Pol 1, but destabilizes it in Taq. Both Klentaq and Klenow denaturations have a very similar dependence on pH and methanol, indicating similarities in the hydrophobic forces and protonation effects stabilizing the proteins. Melting monitored by CD yields slightly lower Tm values, but almost identical van't Hoff enthalpy ΔH values, consistent with two-state unfolding followed by an irreversible kinetic step. Analysis of the denaturation scan rate dependences with Arrhenius formalism estimates a kinetic barrier to irreversible denaturation for Klentaq that is significantly higher than that for Klenow.

scholarly journals Effect of irreversibility on the thermodynamic characterization of the thermal denaturation of Aspergillus saitoi acid proteinase

1995 ◽  
Vol 311 (3) ◽  
pp. 969-974 ◽  
Author(s):  
S R Tello-Solis ◽  
A Hernandez-Arana
Keyword(s):  
Molecular Structure ◽  
Differential Scanning Calorimetry ◽  
Equilibrium Constant ◽  
Thermal Denaturation ◽  
Acid Proteinase ◽  
Scanning Calorimetry ◽  
Thermodynamic Characterization ◽  
A Value ◽  
Van’T Hoff Enthalpy

The thermal denaturation of the acid proteinase from Aspergillus saitoi was studied by CD and differential scanning calorimetry (DSC). This process seemed to be completely irreversible, as protein samples that were heated to temperatures at which the transition had been completed and then cooled at 25 degrees C did not show any reversal of the change in the CD signal. Similar results were obtained with DSC. Nevertheless, we were able to detect the presence of reversibly unfolded species in experiments in which the enzyme solution was heated to a temperature within the transition region, followed by rapid cooling at 25 degrees C. Accordingly, the denaturation of behaviour of the acid proteinase seems to be consistent with the existence of one (or more) reversible unfolding transition followed by an irreversible step. The van't Hoff enthalpy, delta HvH, which corresponds to the reversible transition was calculated from extrapolation to infinite heating rate as 310 kJ.mol-1. This parameter was also determined from direct estimation of the equilibrium constant at several temperatures (delta HvH = 176 kJ.mol-1). Comparison of the average delta HvH with the calorimetric enthalpy (delta Hcal. = 770 kJ.mol-1) gave a value of 3.2 for the delta Hcal./delta HvH ratio, indicating that the molecular structure of the enzyme is probably formed by three or four cooperative regions, a number similar to that of the acid proteinase, pepsin. It should be noted that a completely different conclusion would be obtained from a straightforward analysis of the calorimetric curves, disregarding the effect of irreversibility on the denaturation process.

scholarly journals THE STUDY OF THERMAL DENATURATION OF BEEF, PORK, CHICKEN AND TURKEY MUSCLE PROTEINS USING DIFFERENTIAL SCANNING CALORIMETRY

2019 ◽  
Vol 4 (3) ◽  
pp. 19-23
Author(s):  
Irina V. Agafonkina ◽  
Igor A. Korolev ◽  
Taras A. Sarantsev
Keyword(s):  
Differential Scanning Calorimetry ◽  
Heat Capacity ◽  
Specific Heat ◽  
Specific Heat Capacity ◽  
Thermal Processing ◽  
Thermal Denaturation ◽  
Meat Product ◽  
Muscle Proteins ◽  
Scanning Calorimetry ◽  
Denaturation Process

In the temperature range from 45 °C to 90 °C the process of thermal denaturation of a whole complex of muscle proteins in meat takes place. An effective mode to register the thermal denaturation process is the method of differential scanning calorimetry (DSC). As a result of studies the differences during the process of thermal denaturation of muscle proteins of pork, beef, chicken and turkey were defined by the appearance of endothermic peaks in DSC thermograms. The main variances are associated with the process of denaturation of myosin and sacroplasmic proteins and indicate indirectly their quantitative ratio in meat. The values of effective specific heat capacity in the temperature range from 20 °C to 90 °C are obtained as well as those of heat spent on the denaturation process.At reheating, the values of specific heat capacity increased by 0.1 J/(g*K) on the average, and peaks of thermal denaturation were not detected, that certifies the irreversibility of the denaturation process and the decrease in the bound moisture proportion in meat after thermal processing. Knowledge of the nature of protein thermal denaturation of each kind of meat product is one of the necessary tools for developing the technology of meat product thermal processing.

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