The rate of exchange of the labile hydrogens of lysozyme was measured by out-exchange of tritium from solution samples and powder samples at varied hydration levels for pH 2, 3, 5, 7 and 10. The dependence of exchange of powder samples on the degree of hydration was the same at all pH, reaching rates of exchange equivalent to solution samples at 0.2 g H₂0/g protein (160 mol H₂0/mol protein), which corresponds to coverage of one-half the protein surface with monolayer water (Yang and Rupley, 1979). No additional hydrogen exchange was observed for protein powders equilibrated with higher water content. Considered in conjunction with other lysozyme hydration data (Rupley et al, 1983), this observation indicated that internal protein dynamics are not strongly coupled with surface properties. The use of powder samples offered control of water activity through regulation of water vapor pressure. The dependence of exchange rate on water activity was of low order, 2.9 average, and pH independent. This value, observed from 95 to 7 mol hydrogen remaining unexchanched/mol lysozyme, indicated that the rate determining step for protein hydrogen exchange is similar for all backbone amides and involves few water lilolecules. Powder samples were hydrated either by isopiestic equilibration against H₂S0₄ or NaOH solutions or by addition and mixing of solvent to rapidly reach final hydration. Samples hydrated slowly by isopiestic equilibration exhibited more exchange than was observed for samples of the same water content that had been hydrated rapidly by solvent addition. Conformational change with hydration was ruled out as an explanation of this difference by a powder to solution jump experiment at pH 5, which proved the rank order of exchange was preserved. The difference can be explained by salt and pH effects expected to contribute to exchange of the nearly dry protein. Solution hydrogen exchange measured using the same lyophilized protein as the hydration experiments was in good agreement with published data. Rank order was proven the same for all pH by solution pH jump experiments. The effect of ionic strength on hydrogen exchange was examined at pH 2 and pH 5 for protein solutions containing up to 1.0 M added salt. The influence of ionic strength was similar for both pH and exhibited a complex character, in contrast to the dependence of exchange observed for positively charged polypeptide models.
| Identifer | oai:union.ndltd.org:arizona.edu/oai:arizona.openrepository.com:10150/186579 |
| Date | January 1983 |
| Creators | SCHINKEL, JEFFREY ERICH. |
| Publisher | The University of Arizona. |
| Source Sets | University of Arizona |
| Language | English |
| Detected Language | English |
| Type | text, Dissertation-Reproduction (electronic) |
| Rights | Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. |
Page generated in 0.0025 seconds