J. Phys. Chem. 1995, 99, 17061
Comment on “Comparison of Water Relaxation Time in Serum Albumin Solutions Using Nuclear Magnetic Resonance and Time Domain Reflectometry” Peter S . Belton Institute of Food Research, Norwich Research Park, Colney Norwich NR47UA, UK Received: March 27, 1995; In Final Form: June 12, I995 The recent paper by Fukuzaki and co-workers’ is concerned with the reconciliation of the results obtained from dielectric dispersion and proton NMR relaxation studies on water in human serum albumin solutions. They measure the NMR relaxation times T I and T2. TZ is measured by the CarrPurcell-Meiboom-Gill (CPMG) pulse sequence, and the measured value is assumed to be a unique representation of the dipole-dipole correlation function of the water. There is strong evidence against this supposition.2 The time constant for exponential transverse proton relaxation in bovine serum albumin solution2 has been shown to be heavily dependent on the pulse spacing in the CPMG sequence and on the static magnetic field strength used in the experiment. The assumption in the paper’ that a meaningful correlation time can be calculated from a measurement taken at a single pulse spacing and field strength is untenable. The origins of the pulse spacing and field dependence phenomena can be explained by assuming that there is chemical exchange between the exchangeable protons on the bovine serum albumin and the protons in the water. The chemical shift difference between the water protons and the exchangeable protons on the protein results in a resonance frequency shift on exchange which acts as an additional dephasing mechanism for the spins. Further evidence for the importance of chemical exchange may be observed by the strong pH dependence of the transverse relaxation rates2 The details of the field and pulse spacing dependence can be quantified by a corrected form2 of the well-known CarverRichards3 equation. When the exchange is fast, the transverse relaxation rate is given by2
The transverse relaxation rate is R, and P is the relative population in sites a and b. The chemical shift difference between sites a and b is do;kb is the exchange rate. Clearly, any attempt to determine correlation times from such a relaxation would not result in meaningful values if account were not taken of exchange. In the limit of very fast pulse spacing, when the term in chemical shift can be ignored, the expression becomes that implied by Fukuzaki and co-workers. However, the exchanging species are the protons on exchangeable groups on the protein, not bound water. On the basis of this evidence, the correlation times calculated from the transverse relaxation rate cannot be considered to
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represent realistic estimates of correlation times for motion. A similar problem arises with the calculations of correlation times from spin-lattice relaxation. While it is true that the exchange between chemikally shifted sites does not contribute directly to the spin-lattice relaxation rate, the mixing of protons from protein and water by chemical exchange does mean that a simple interpretation of the data in terms of water mobility is not p ~ s s i b l e . It ~ must be concluded therefore that the neither of the Correlation times calculated in the paper really represents the values of the correlation times of water in the system. A much better route to the correlation times for water in protein solutions is the use of I7O relaxation,4g5which does not suffer from the exchange problem. A n a l y ~ i sof ~ .the ~ frequency dependence of I7O relaxation has shown that a two correlation time model must be used to describe the motion of the water at the protein interface. This implies that the simple BPP model used by the authors is not valid. The values of correlation time4 for water obtained by the analysis of the oxygen relaxation are in quite close agreement with the values of correlation time observed by Fukuzaki and co-workers’ using dielectric dispersion measurements. Typically, the I7O results find free water having correlation times of the order of 2.5 ps at 300 K, and the water close to the protein interface has correlation times of the order of 10-20 ns and 20-30 ps. By comparison, the values of Fukuzaki and co-workers are of the order of picoseconds and nanoseconds. The conclusion therefore is that, for the measurement of rotational correlation times for water, reliable NMR results are not in serious disagreement with the values obtained from dielectric measurements. However, the use of proton relaxation measurements to determine correlation times of water in systems where the solutes have protons which can exchange on the time scale of NMR relaxation times cannot be regarded as reliable unless very careful analyses of the effects of exchange are carried out. Note Added in Proof. A recent series of papers by Halle and co-workers6-s has suggested that even the concept of anisotropically rotating bound water at the protein surface is not valid and that NMR relaxation times of I7O may be explained by the exchange of internal slowly rotating waters with external water. As pointed out by Halle and co-workers, these interpretations deviate significantly from the interpretation of dielectric data and thus from the interpretation of Fukuzaki and co-workers.
References and Notes (1) Fukuzaki, M.; Miura, N.; Shinyashiki, N.; Kurita, D.; Shioya, S.; Haida, M.; Mashimo, S.J . Chem. Phys. 1995, 99, 431. (2) Hills, B. P.; Takacs, S. F.; Belton, P. S.Mol. Phys. 1989, 67, 903. (3) Carver, J. P.; Richards, R. E. J. Mugn. Reson. 1972, 6, 89. (4) Belton, P. S. Prog. Biophys. Mol. B i d . 1994, 61, 61. (5) Halle, B.; Anderson, T.; Forsen, S.; Lindman, B. J . Am. Chem. Soc. 1981, 103, 500. (6) Denisov, V. P.; Halle, B. J. Am. Chem. Soc. 1994, 116, 10324. (7) Denisov, V. P.; Halle, B. J . Mol. Biol. 1995, 254, 698. (8) Denisov, V. P.; Halle, B. J . Mol. Biol. 1995, 245, 682.
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0 1995 American Chemical Society
