th - 1987 - 51st ENC Conference
th - 1987 - 51st ENC Conference
th - 1987 - 51st ENC Conference
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MF7<br />
slV NMR: A NEW PROBE OF METAL ION BINDING<br />
IN METALLOPROTEINS<br />
Alison Butler~ Michael Danz|tz~ and Hel]mut Eckert*<br />
Department o[ Chemistry, University o[ CalJ[ornia at Santa Barbara and Division o[<br />
Chem~try and ChemJca/EnE/neering, Ca~/ornia/nstitute o[ TechnoloEy<br />
High detection sensitivity due to a large magnetic moment, high natural abun-<br />
dance (99.76 ~) and rapid quadrupolar relaxation in solution render 5iV one of <strong>th</strong>e<br />
most favorable nuclei for NIVIR studies. In addition, <strong>th</strong>e 51V NMR chemical shifts are<br />
extremely sensitive to changes in <strong>th</strong>e nature and <strong>th</strong>e symmetry of <strong>th</strong>e ligand coor-<br />
dination, <strong>th</strong>ereby providing an excellent diagnostic tool for detailed investigations of<br />
vanadium(V) bonding environments. However, in spite of <strong>th</strong>ese advantageous features<br />
and <strong>th</strong>e fact <strong>th</strong>at vanadium is widely recognized as a biologically important element,<br />
no 51V NMR data relating to vanadium bound to proteins have been published in<br />
<strong>th</strong>e literature. In <strong>th</strong>is poster, we report <strong>th</strong>e first 5]V NMR study of a V(V)-protein<br />
complex: V(V) 2-human transferrin.<br />
The 11.7T 51V NMR spectrum of a solution containing 2 equivalents of vanadate<br />
per protein is characterized by two partly resolved resonances at -529.5 and -531.5<br />
ppm (vs. VOCI3) wi<strong>th</strong> a total linewid<strong>th</strong> of 420 Hz. Linewid<strong>th</strong>s and chemical shifts are<br />
independent of concentration (range 10 -4 to 10-3M), pH (5-9), nature of <strong>th</strong>e buffer<br />
solution, and <strong>th</strong>e presence of excess free vanadate. On <strong>th</strong>is basis we assign <strong>th</strong>ese reso-<br />
nances to protein-bound vanadium which is present in two chemically distinct binding<br />
sites and which is in <strong>th</strong>e limit of slow metal ion exchange on <strong>th</strong>e NMR timescale (2.5,<br />
10 -4 s). Absolute intensity measurements indicate <strong>th</strong>at <strong>th</strong>e protein-bound vanadium<br />
is in <strong>th</strong>e slow-motion limit (wrc >> I), and <strong>th</strong>at only <strong>th</strong>e central (1/2 --* -1/2) tran-<br />
sition is observed. In consonance wi<strong>th</strong> <strong>th</strong>is interpretation, measurements at different<br />
magnetic field streng<strong>th</strong>s reveal <strong>th</strong>e presence of second-order frequency shifts. Several<br />
examples of <strong>th</strong>e use of 51V NMR to monitor chemical modifications at <strong>th</strong>e binding<br />
site of V(V)2-human transferrin are discussed.
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