Pressure effect on hydrogen isotope exchange kinetics in chymotrypsinogen investigated by FT-IR spectroscopy

1991 ◽  
Vol 69 (11) ◽  
pp. 1699-1704 ◽  
Author(s):  
P. T. T. Wong
Keyword(s):  
High Pressure ◽  
Exchange Rate ◽  
Rate Constant ◽  
Bound Water ◽  
Pressure Effects ◽  
Protein Molecules ◽  
Fast Exchange ◽  
Exchange Rate Constant ◽  
Hydrogen Deuterium ◽  
Exchange Kinetics

Hydrogen/deuterium (H/D) exchange rate constants in chymotrypsinogen have been determined at several pressures up to 28.9 kbar by FTIR spectroscopy. The secondary structure of the protein molecules was monitored simultaneously at the corresponding pressures by the intensity redistribution of the infrared amide I band at these pressures. As in other proteins, the labile protons on the amide groups in chymotrypsinogen can, to a good approximation, be separated into two classes, each with distinct first order H/D exchange rates constants in the time period from 10 min to ~24 h. The fast exchange rate constant increases while the slow exchange rate constant decreases with increasing pressure. The increase in the fast exchange rate constant at high pressure is largely associated with the pressure-induced unfolding of the protein molecules. At extremely high pressure (12.8 kbar), in addition to the unfolding of protein molecules, pressure induced a distortion and weakening of the hydrogen bonds of the fold protein segments also contribute to an increase in the overall H/D exchange rate. The present results confirm that when chymotrypsinogen is dissolved in D2O, a considerable amount of D2O molecules is bound to the protein molecules on the surface as well as in the interior cavities of the molecules. The H/D exchange takes place between these bound D2O and the protons in the protein molecules. The mechanism of the H/D exchange and the interior dynamics in proteins are discussed on the basis of the present results. Key words: hydrogen/deuterium exchange, exchange kinetics, rate constant, pressure effects, infrared spectroscopy, protein, conformation structure, bound water.

Transient Enzyme Kinetics at High Pressure

1996 ◽  
Author(s):  
Claude Balny
Keyword(s):  
High Pressure ◽  
Transition State ◽  
Rate Constant ◽  
Conformational Changes ◽  
Reaction Pathway ◽  
Enzyme Reaction ◽  
Pressure Effects ◽  
Complete Study ◽  
Temperature And Pressure ◽  
High Pressure Kinetics

In a detailed study of an enzyme reaction pathway, a measured composite rate constant, for example, kcat, can be interpreted in ways that lead to ambiguous conclusions. Two conditions must be met to solve this problem: (1) an elementary rate constant must be measured, and (2) a maximum number of physical-chemical parameters must be used to perturb the system under study. To gain access to elementary rate constants, cryobaroenzymology and/or transient methods, such as stopped-flow and flow-quench kinetics, can be used. Both perturbation and kinetics measurements performed under either high pressure or low temperatures can then be used to probe the thermodynamics of the interconversion of two successive intermediates to obtain parameters such as ΔG‡, ΔS‡, ΔH‡, and ΔV‡ The interdependence of the two major variables, namely temperature and pressure, is presented in this article, in which the role of organic cosolvents is considered as a third variable. During catalytic reactions, enzymes undergo a number of conformational changes related to their dynamic structural flexibility. This appears as a succession of different steps. A complete study of such processes, which generally are very rapid, consists of the exploration of the properties of these steps, including thermodynamic features obtained by the action of temperature and pressure. As long ago as 1950, Laidler (1950) formulated the first theoretical basis for explaining the responses of enzymes to high hydrostatic pressures. Chemists used this parameter extensively, and in the early stages of high-pressure kinetics they attempted to analyze the observed results on the basis of collision theory (Asano, 1991) or transition-state theory (Evans & Polanyi, 1935). These theories are still used to describe pressure effects on enzyme reactions. It is postulated that between two successive intermediates there is a labile transition state which governs the energetics of the reaction (Glastone et al., 1941). But we must remember that this theory was first applied only to simple homogeneous reactions in gases. For solutions, the treatment can require the introduction of other parameters such as the viscosity.

Dynamic Contrast-Enhanced MRI Identifies a Distinct Group of Asymptomatic Myeloma Patients with Increased Bone Marrow Microcirculation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5079-5079
Author(s):  
Jens Hillengass ◽  
Christian Zechmann ◽  
Andreas Nadler ◽  
Stefan Delorme ◽  
Axel Benner ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Exchange Rate ◽  
Rate Constant ◽  
Healthy Controls ◽  
Dynamic Contrast Enhanced ◽  
Contrast Enhanced ◽  
Significant Difference ◽  
Exchange Rate Constant ◽  
Focal Pattern ◽  
Contrast Enhanced Mri

Abstract Introduction: Dynamic contrast enhanced MRI (dceMRI) is an imaging technique detecting changes in local microcirculation reflecting increased angiogenesis. The dceMRI parameters Amplitude A and exchange rate constant kep have been shown to be significantly increased in patients with active multiple myeloma (MM) compared to healthy controls and to correlate with osteolytic bone involvement and prognosis. We now compared the parameters and infiltration patterns of dceMRI in patients with monoclonal gammopathy of undetermined significance (MGUS) as well as in patients with asymptomatic MM not requiring chemotherapy (NRC-group) with those in patients with symptomatic MM requiring chemotherapy according to international standards. Methods: The NRC group contained 71 patients: 29 patients with MGUS, 39 patients with MM stage I and 3 patients with MM stage IIA according to Durie and Salmon. 24 patients had a diagnosis of a MM in stage II in progression (n = 3) or stage III (n = 21). All patients underwent standardized dceMRI with high temporal resolution (T1w-turboFLASH) of the lumbar spine before start of therapy. Color coded pharmacokinetic maps of imaged area were classified according to three distinct patterns of microcirculation: “normal” (as in healthy controls), “diffuse” or “focal”. The contrast uptake was quantified using a two compartment model with the output parameters Amplitude A and distribution constant rate kep reflecting bone marrow microcirculation. Results: 63 % of patients in the NRC group were found to have changes in the microcirculation pattern with 26 patients (37%) displaying a normal, 43 (60%) a diffuse and 2 (3%) a focal pattern. Within the NRC group 11 MGUS patients (38%) were found to have a normal pattern, 17 patients (59%) had a diffuse and 1 patient (3%) presented with a focal pattern. 79 % of patients with symptomatic MM had an abnormal microcirculation pattern with 5 (21%) MM patients showing a normal, 12 (50%) a diffuse and 7 (29%) a focal pattern. Statistical comparison did not reveal a significant difference in the total incidence between NRC and symptomatic MM group. Comparison of quantitative microcirculation parameters did not show a significant difference of Amplitude A (p=0.87) and exchange rate constant kep (p=0.3) in MGUS patients compared to patients with asymptomatic MM. Comparing the NRC group with the symptomatic myeloma group revealed a significant higher Amplitude A in the symptomatic MM group (p=0.01). There was no significant difference in exchange rate constant kep. Conclusion: Our investigations revealed a group of patients with asymptomatic myeloma and MGUS that display significant increase in bone marrow microcirculation. Our findings could be the basis for stratified treatment of patients with novel therapeutics targeting the vascular system. Prognostic implications for systemic and local development of the malignant disease are topic of current investigations.

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