1H NMR relaxation dispersion profiles of solid BSA

POSTER by
aSykora S., bCremonini M.A., bLaghi L, cFantazzini P.

aStelar Srl, Via E. Fermi 4, 27035 Mede (PV), Italy.
bDept. of Food Science, University of Bologna, P.zza Goidanich 60, 47023 Cesena, Italy.
cDept. of Physics, University of Bologna, Viale Berti Pichat 6/2, 40127 Bologna, Italy.

presented at

XXXIV Congress on Magnetic Resonance, GIDRM, Porto Conte (Italy) 2004, Sept. 21-24.

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Abstract

Introduction: Bovine Serum Albumin is a representative of nearly globular proteins of intermediate molecular weight. During the last two decades, a considerable amount of research [1-5] has been dedicated to collecting and interpreting its NMR relaxation data, in particular T1 dispersion profiles of both solid BSA and of its solutions. Although there is so far no convincing consensus on the nature of the relaxation processes in BSA (and in proteins in general), one feels that an in-depth understanding might be in reach.

Here we present high quality proton NMRD profiles of solid BSA, measured over more than five decades of relaxation fields (Larmor frequencies ranging from 5 kHz to 600 MHz) which, hopefully, might contribute to reaching such an understanding.

Experimental: Lyophilized, ultra-pure (>99 %) and globulin-free BSA purchased from Sigma (product number A7638) was kept for 3 hours in a 10 mm NMR sample tube under high vacuum in order to remove all free/adsorbed oxygen and any traces of humidity before sealing. FID's, NMRD profiles, CPMG decays and other NMR signals of the sample were measured using the following instruments:

(a) Frequency range of 5 kHz - 30 MHz:
     fast-field-cycling (FFC) relaxometer (1T FFC SpinMaster produced by Stelar Srl, Mede, Italy).
(b) Frequency range 15 - 50 MHz:
     Stelar SpinMaster relaxometer of traditional type used with a recycled Jeol electromagnet.
(c) Fixed frequencies of 200, 400 and 600 MHz:
      respectively, Bruker AC200, Varian Mercury VX 400 and Varian INOVA 600.

Sample temperature has been carefully calibrated and rigorously controlled during all measurements. Since the FID's of the dry, solid BSA are very short and decay below the noise level in about 25 ms, we have used low Q-factor probes (~10) with very short dead-times (~7 ms) at frequencies below 100 MHz. Standard HR probes were used for experiments at higher resonance frequencies fields.

Experimental results:

The NMRD profiles show a steep dispersion extending from less than 10 kHz to over 300 MHz. Data acquired with completely different instruments are perfectly coherent with each other. The dispersion curves, plotted as log(R) against log(w), with R being the relaxation rate (inverse of T1) and w being the 1H Larmor frequency at relaxation field, exhibit two nearly linear regions, a low- and a high-frequency plateau and four superposed 14N glitches. These observations were combined into an empirical reference formula which describes the data with a precision better than the measurement errors.


Please, cite this online document as:
Sykora S., Cremonini M.A., Laghi L, Fantazzini P., 1H NMR relaxation dispersion profiles of solid BSA,
   Poster at XXXIV Congress on Magnetic Resonance, September 21-24, 2004, Porto Conte (Italy). DOI: 10.3247/SL1Nmr04.002.

References:

  • Kimmich R., Gneiting T., Kotitschke K., Schnur G., Fluctuations, exchange processes, and water diffusion in aqueous protein systems, Biophysical Journal 58, 1184 (1990).
  • Koenig S.H., Brown R.D., A Molecular Theory of Relaxation and Magnetization Transfer: Application to Cross-Linked BSA, a Model for Tissue, Magn.Resonance in Medicine 30, 685 (1993).
  • Halle B., Johannesson H., Venu K., Model-Free Analysis of Stretched Relaxation Dispersions, J.Magn.Reson. 135, 1 (1998).
  • Halle B., Denisov V.P., Venu K., Multinuclear Relaxation Dispersion Studies of Protein Hydration, in Biological Magnetic Resonance, Ed.s Krishna, Berliner, Vol.17, p.10, Kluwer Academic/Plenum Publishers 1999.
  • Bertini I., Fragai M., Luchinat C., Parigi G., 1H NMRD profiles of diamagnetic proteins: a model-free analysis, Magn.Reson.Chem. 38, 543 (2000).
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