th  - 1988 - 51st ENC Conference

I~ 17 J QUANTITATIVE 2D NMR STUDIES OF PROTON EXCHANGE IN AMMONIUM ION.
Charles L. Perrin and Tammy J. Dwyer,* Department of Chemistry, University-of
California, San Diego, La Jolla, California 92093.
To investigate kinetic isotope effects on proton exchange, we have studied
ammonium ion by a combination of isotopic substitution and quantitative twodimensional
NMR. Solutions of ISNHANO~ in a 3:2 mixture of D20:H20 result in five
isotopomers of the ammonium ion, four of which are distinguishable in the IH NMR
spectrum. At pH < 1 the protons exchange only with water. This is detected as
crosspeaks connecting each isotopomer with its nearest neighbor(s). Base-catalyzed
exchange involves transfer of a proton from an ammonium ion to an ammonia. In this
case, crosspeaks are observed which connect the different spin states of the nitrogen
nucleus as well as the individual isotopomers of the ammonium ion. It has been
shown (Perrin and Gipe, J. Am. Chem. Soc. 1984, 106, 4036) that each crosspeak can
be integrated to determine the site-to-site rate constants for the individual
exchange processes. The rate constant for base-catalyzed proton exchange obtained
by this method is 2.7 x 108 M -1 sec -1. This agrees nicely with a value obtained
previously. More importantly, an isotope effect of kD/k H = 0.56 was observed for
this process.
18 J TWO-DIMENSIONAL NMR STUDIES OF THE CONFORMATIONS OF BRADYKININ IN
AQUEOUS SOLUTION AND IN THE PRESENCE OF MICELLES: Susannie C. Lee and Anne F.
Russell, Procter and Gamble Co., Miami Valley Laboratories, P.O. Box 398707,
Cincinnati, Ohio 45239
The conformational properties of a nonapeptide hormone, bradykinin, have been
determined by two-dimensional NMR techniques at 500 MHz. In particular, homonuclear
Hart_mann-Hahn (HOHAHA) and rotating frame cross-relaxation (ROESY) experiments were
essential in the assignment of resonances and the elucidation of the structure of
this 1280 Da polypeptide. Our studies indicate that bradykinin exists, in aqueous
solution, either as a completely disordered structure or as an average of several
conformations in fast exchange. To gain a better understanding of the structural
properties of bradykinin in a cell membrane receptor environment, various micellar
systems were examined for their ability to stabilize a preferred conformation.
Three membrane mimetic systems were studied: sodium dodecyl sulfate (SDS),
myristoyl-lysophosphatidyl choline, and dodecyl phosphocholine. The optimal system
for this investigation was a mixture of bradykinin and sodium dodecyl sulfate
(perdeuterated) at a 1:5 molar ratio, as confirmed by the temperature-dependent
behavior of the amide protons. Under these conditions, we were able to detect the
presence of a gamma turn at residues 7-9 of bradykinin. Detailed structural
information, in the presence of SDS, was obtained from quantitative 2-D NOE analyses
and distance geometry calculations.
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