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Effects of freezing rate on structural changes in L-lactate dehydrogenase during the freezing process.

ABSTRACT: Freezing is a common method for improving enzyme storage stability. During the freezing process, the freezing rate is an important parameter that can affect protein stability. However, there is limited information on the denaturation mechanisms and protein conformational changes associated with the freezing rate. In this study, the effects of freezing rate on activity loss and conformational changes in a model enzyme, L-lactate dehydrogenase, were evaluated. Enzyme solutions were frozen at various rates, from 0.2 to 70.6 °C/min, and ice seeding was conducted to reduce supercooling. The results demonstrated that fast freezing results in activity loss, structural changes, and aggregation. The residual activities at freezing rates of 0.2, 12.8, and 70.6 °C/min were 77.6 ± 0.9%, 64.1 ± 0.4%, and 44.8 ± 2.0%, respectively. As the freezing rate increased, the degree of dissociation and unfolding increased significantly, as determined using blue native-polyacrylamide gel electrophoresis and fluorescence spectroscopy. Moreover, a large number of amyloid aggregates were detected in samples frozen at a fast freezing rate (70.6 °C/min). The enzyme inactivation mechanism induced by fast freezing was proposed in terms of increased dehydration at the enzyme surface and an ice/unfroze solution interface, which could be helpful to establish a common understanding of enzyme inactivation during the freezing process.

SUBMITTER: Park H

PROVIDER: S-EPMC8249661 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:An adaptive response to threat requires optimized detection of critical sensory cues. This optimization is thought to be aided by freezing - an evolutionarily preserved defensive state of immobility characterized by parasympathetically mediated fear bradycardia and regulated by the amygdala-periaqueductal grey (PAG) circuit. Behavioral observations in humans and animals have suggested that freezing is also a state of enhanced visual sensitivity, particularly for coarse visual information, but the underlying neural mechanisms remain unclear. We induced a freezing-like state in healthy volunteers using threat of electrical shock and measured threat-related changes in both stimulus-independent (baseline) and stimulus-evoked visuocortical activity to low-vs. high-spatial frequency gratings, using functional MRI. As measuring immobility is not feasible in MRI environments, we used fear bradycardia and amygdala-PAG coupling in inferring a freezing-like state. An independent functional localizer and retinotopic mapping were used to assess the retinotopic specificity of visuocortical modulations. We found a threat-induced increase in baseline (stimulus-independent) visuocortical activity that was retinotopically nonspecific, which was accompanied by increased connectivity with the amygdala. A positive correlation between visuocortical activity and fear bradycardia (while controlling for sympathetic activation), and a concomitant increase in amygdala-PAG connectivity, confirmed the specificity of these findings for the parasympathetically dominated freezing-like state. Visuocortical responses to gratings were retinotopically specific, but did not differ between threat and safe conditions across participants. However, individuals who exhibited better discrimination of low-spatial frequency stimuli showed reduced stimulus-evoked V1 responses under threat. Our findings suggest that a defensive state of freezing involves an integration of preparatory defensive and perceptual changes which may be regulated by a common mechanism involving the amygdala.

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Effects of freezing rate on structural changes in L-lactate dehydrogenase during the freezing process.

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