A reinvestigation of the hydration numbers of paramagnetic metalloenzymes by proton and deuteron NMR relaxation times

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Abstract

The analysis of the enhancement of water NMR relaxation rates by paramagnetic metal ions bound to macromolecules is reexamined. By measuring T1 and T2 for both protons and deuterons in the same solution and at one high magnetic field it is possible to obtain the hydration number, exchange lifetime, and correlation time of the bound water molecules as well as the magnitude of an additional contribution originating from outer-sphere water molecules. For Mn(II) bovine carboxypeptidase A and Mn(II) bovine carbonic anhydrase B, hydration numbers, q, of unity and exchange lifetimes τM = 1.0 × 10-7 and 0.75 X 10-7 sec at room temperature were found for the two enzymes, respectively. The same q and τM were obtained in independent measurements at different magnetic fields, and τM was found to be doubled upon reducing the temperature from 25 to 0°C. The outersphere relaxation could not be explained either by spin diffusion from the protein protons or by dipolar interaction between the Mn(II) ion and freely diffusing water molecules. A good account of this contribution could be obtained by a mechanism involving water molecules which are bound outside the first-hydration sphere and have an average exchange lifetime of about 3 × 10-10 sec.

Original languageEnglish
Pages (from-to)373-384
Number of pages12
JournalJournal of Magnetic Resonance (1969)
Volume56
Issue number3
DOIs
StatePublished - 15 Feb 1984

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