Hydrogen exchange hydration

This genera] scheme could be used to explain hydrogen exchange in the 5-position, providing a new alternative for the reaction (466). This leads us also to ask whether some reactions described as typically electrophilic cannot also be rationalized by a preliminary hydration of the C2=N bond. The nitration reaction of 2-dialkylaminothiazoles could occur, for example, on the enamine-like intermediate (229) (Scheme 141). This scheme would explain why alkyl groups on the exocyclic nitrogen may drastically change the reaction pathway (see Section rV.l.A). Kinetic studies and careful analysis of by-products would enable a check of this hypothesis. 

Rosenberg, A. and Somogyi, B. (1986) Conformational fluctuations, thermal stability and hydration of proteins, studies by hydrogen exchange kinetics. n Dynamic of Biochemical systems, edited by S. Damjanovich, T.Keleti and L.Tron, pp. 101-112. Amsterdam Elsevier. 

Hydrogen-exchange rates as a function of level of hydration can be calculated from data such as those of Fig. 22. Results of this kind, for pH 2-10, are given in Fig. 23. The slope of the exchange rate-water... 

Fig. 22. Hydration dependence of amide hydrogen exchange in lysozyme powder at pH 5. Individual samples of pH 5 fully labeled (with H O) lysozyme were equilibrated at 25°C for 24 hr at various water contents obtained by isopiestic equilibration ( ) or by the addition and mixing of solvent (A). The samples were then dissolved to a concentration of 20 mg/ml and 100-/U.1 aliquots were analyzed by gel filtration. The arrow indicates the 24-hr solution H . end point. H, represents the number of hydrogens remaining unexchanged. From Schinkel el al. (1985).

The activities of several enzymes have been studied in partially hydrated powders as a funcuon of water activity or water content. Experiments of this type are not difficult to perform. Solutions of substrate and enzyme are mixed quickly, and the mixture is immediately frozen and lyophilized, which stops the reaction and gives a stable dry powder. If appropriately high concentrations of enzyme and substrate are mixed, the powder is of the enzyme-substrate complex. The sample is rehydrated under a controlled atmosphere to give the desired final hydration level. Conditions, particularly the pH of the sample, are set such that the hydration equilibrium is substantially complete (within several hours) before appreciable enzyme reaction has taken place. The problem of defining pH in partially hydrated powders was discussed in Section II,D in connection with hydrogen-exchange measurements. The pH of a powder appears to equal the nominal pH (that of the solution from which the powder was lyophilized) above about 0.15 A. 

Fig. 30. Comparison of ESR and enzyme activity changes with hydration. Effect of hydration on lysozyme dynamic properties. (Curve f) Log rate of peptide hydrogen exchange. (Curve g) , Enzyme activity (log uo) O, rotational relaxation time (log t ) of the ESR probe TEMPONE. From Rupley et al. (1983).

Amide hydrogen exchange in protein powders depends weakly on water activity, and its hydration dependence is complete within the low-hydration region (0.15 A). Apparently, the rate-determining step for the exchange of buried hydrogens is not much influenced by the protein surface. This is unexplained. 

Concurrently, a series of papers began to appear on the isomerization and hydrogen-exchange reactions of hydrocarbons with acid catalysts. Over slightly hydrated aluminum bromide at room temperature propane containing carbon-13 at one end (C Hq-I-C )was found to isomerize toward a statistical mixture with... 

Scfainkel, J.E., Downer, N.W., Rupley, J.A. (1985) Hydrogen exchange of lysozyme powders. Hydration dependence of internal motions. Biochemistry, 24 (2), 352—366. 

Internal dynamics of biomolecules is practically frozen without water. Upon increasing hydration level, it develops in a stepwise fashion [508]. At h K 0.15 g/g, internal protein motion, monitored by hydrogen exchange, achieves its solution rate [509]. Full internal dynamics of lysozyme is restored at /t 0.38 g/g [510]. Mossbauer spectroscopy studies evidence restoration of the internal dynamics of lysozyme... 

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