Proton Exchange as a Relaxation Mechanism for T1 in the Rotating Frame in Native and Immobilized Protein Solutions

Biochemical and Biophysical Research Communications 289, 813– 818 (2001) doi:10.1006/bbrc.2001.6058, available online at http://www.idealibrary.com on

Proton Exchange as a Relaxation Mechanism for T 1 in the Rotating Frame in Native and Immobilized Protein Solutions Heidi I. Ma¨kela¨, Olli H. J. Gro¨hn, Mikko I. Kettunen, and Risto A. Kauppinen 1 National Bio-NMR Facility, A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, FIN-70211 Kuopio, Finland

Received November 5, 2001

T 1 relaxation in the rotating frame (T 1␳) is a sensitive magnetic resonance imaging (MRI) contrast for acute brain insults. Biophysical mechanisms affecting T 1␳ relaxation rate (R 1␳) and R 1␳ dispersion (dependency of R 1␳ on the spin-lock field) were studied in protein solutions by varying their chemical environment and pH in native, heat-denatured, and glutaraldehyde (GA) cross-linked samples. Low pH strongly reduced R 1␳ in heat-denatured phantoms displaying proton resonances from a number of side-chain chemical groups in highresolution 1H NMR spectra. At pH of 5.5, R 1␳ dispersion was completely absent. In contrast, in the GA-treated phantoms with very few NMR visible side chain groups, acidic pH showed virtually no effect on R 1␳. The present data point to a crucial role of proton exchange on R 1␳ and R 1␳ dispersion in immobilized protein solution mimicking tissue relaxation properties. © 2001 Elsevier Science

Key Words: NMR; MRI; relaxation mechanisms; rotating frame relaxation; T 1␳; Z spectroscopy; proton exchange; molecular exchange.

It is well established that the nuclear magnetic relaxation of protons of bulk water and biological macromolecules proceed in an interconnected manner both in protein solutions and biological preparations. The interaction is one of the main sources of magnetic resonance imaging (MRI) contrast and thus mechanisms mediating the communication between the proton populations are of considerable interest. Our goal would be to reveal the mechanisms affecting longitudinal relaxation at very low fields or in the rotating frame, since our recent data demonstrate that T 1 in the rotating frame (T 1␳) provides novel information about brain 1 To whom correspondence and reprint requests should be addressed at A. I. Virtanen Institute for Molecular Sciences, University of Kuopio, P.O. Box 1627, FIN-70211 Kuopio, Finland. Fax: ⫹35817-163 030. E-mail: [email protected].

ischemia in vivo (1–3). Several physical mechanisms have been proposed to govern the relaxation in the rotating frame. Firstly, direct contribution by the macromolecular protons to the solvent protons has been suggested (4). This is reconciled by the fact that in the rotating frame the Larmor condition

␻1 ⫽ ␥B1

[1]

is fulfilled for the spins with substantially longer correlation times (␶ c) than that of bulk water. ␻ 1 corresponds to ␶ c in the millisecond range and thus events occurring at this time frame potentially contribute to T 1␳ relaxation. Furthermore, the static dipolar fields generated by the macromolecular tumbling (4) affect the effective locking field (␻ eff) leading to altered contribution by the laboratory frame spin-lattice effects to the R 1 in the rotating frame (R 1␳) (5). Secondly, the exchange of protons between solvent and macromolecules has been suggested as a means of relaxation for T 1␳ (6). On the other hand the physical mechanisms responsible for the transfer of “magnetization” include both proton and molecule exchange, the former taking place at the hydration shell (⫺NH, ⫺amide, ⫺OH protons) (7) and the latter within the protein molecule (8). Much of our understanding of water relaxation in biological systems stems from studies using aqueous solutions of bovine serum albumin (9, 10) or other proteins (11, 12). For relaxation quantification, proteins have often been dissolved in pure water (e.g., Refs. 10, 13). However, the number of available charged and dipolar sites on the protein surface as well as the strength of water–protein hydrogen bonds are strongly affected by the ionic environment and pH (14), thus the interpretation of NMR relaxation data from pure aqueous protein solutions in physiological equivalents may not be straightforward. Furthermore, protein conformation (i.e., native vs denatured state) influences protein–water interaction as a result of

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changed rotational and dynamic properties of macromolecules. In the present work, we have studied R 1␳ in protein phantoms using native and immobilized solutions with an aim of quantifying the plausible contributions of proton and molecule exchange on the relaxation. Our data unambiguously show that proton exchange serves as a relaxation mechanism for T 1␳ at B 1 fields of 0.25–2.60 G (1.07–11.08 kHz), a range of frequencies used for in vivo T 1␳ MRI (1, 2), and under physiologically relevant conditions, proton exchange is the dominant mechanism for the R 1␳ dispersion, R 1␳(␻ 1). METHODS Relaxation measurements of the protein phantoms. Bovine serum albumin (BSA, fraction V, Sigma Chemicals, St. Louis, MO), was dissolved in either Millipore water, 0.1 M Tris (pH 7.4) or potassium phosphate buffer (KPB, pH 7.4) to yield an 8% solution. Four independent samples were analyzed by NMR from each group. To achieve different cross-linking densities as well as to block exchangeable NH and amide groups, BSA phantoms were supplemented with either 0 or 1% glutaraldehyde (GA), vigorously stirred and incubated at room temperature for 4 h prior to the NMR analyses (4). Heat exposure, which unfolds protein tertiary structures, aggregating them by forming intermolecular disulfide bonds, was used as an alternative means to denature BSA. BSA phantoms were exposed to 65–70°C for 3–5 min followed by incubation at room temperature for at least 4 h before NMR measurements. Since proton exchange is an acid-basecatalyzed reaction, the pH of BSA solution was lowered from 7.4 either to 6.3 or 5.5 by adding HCl before GA incubation or heat treatment. In pure aqueous BSA solutions, the lowest pH used in heat denaturation experiments was 6.0, since heat exposure caused precipitation seen as milk-like coloring of the phantoms below this pH. Relaxation rates (R 1, R 2 and R 1␳) and Z spectra (15) were measured using an SMIS spectrometer (Surrey Medical Imaging Ltd., Guildford, UK) interfaced to a 4.7 T horizontal magnet (Magnex Scientific Ltd, Abingdon, UK) and a custom-built three turn surface coil. A spectroscopic T 1␳ sequence consisted of an adiabatic spin-lock pulse followed by a crusher gradient in front of a 90° detection pulse (time-to-repetition ⫽ TR ⫽ 12 s, five spin lock times ranging from 30 to 70 ms, spectral width ⫽ SW ⫽ 5 kHz, 4096 data points). T 1␳ was quantified at 10 spin lock field strengths ranging from 0.25 to 2.6 G (1.07–11.08 kHz). T 1 was determined using an inversion recovery method (seven inversion times ranging from 100 to 3000 ms, TR ⫽ 10 s) and T 2 using a single spin echo sequence (TR ⫽ 2 s, 15 time-to-echo ⫽ TE ranging from 10 to 150 ms). Z spectra were acquired using an 8 s off-resonance saturation pulse (four B 2 fields ranging from 0.06 to 0.45 G (0.26 –1.92 kHz), off resonance frequencies incremented stepwise from 0.5 to 100 kHz) in front of a 90° detection pulse. The duration of B 2 field was long enough to allow for a steady state to be reached in the spin communication between bulk water and semi-solid pools (16). Since the Z spectra are symmetrical with respect to the water frequency (17), we have acquired data only from the upfield part of the spectrum. The high-resolution 1H NMR spectra (TR ⫽ 8 s, SW ⫽ 6 kHz) were acquired at 11.75 T (Bruker Avance 500, Karlsruhe, Germany) using a WATERGATE nonsaturating water-suppression scheme (18). Data analysis. The NMR spectroscopic data from protein phantoms were quantified as peak integrals (⫾2 kHz around the water peak) and the resulting peak areas were fitted into the standard equations for the given relaxation times. The Z spectra were analyzed by taking the water signal intensity obtained with most far off-resonance saturation as a reference value of unity (15). The data

obtained with four B 2 field strengths were fitted into the two-pool model by Henkelman et al. (16, 19, 20) to yield the characteristics of the proton pools and their interactions as follows: R, the rate constant for the magnetization exchange between the liquid and semisolid pools; M 0B, the number of spins in the semi-solid pool; R A, the longitudinal relaxation rate of protons in the liquid pool; T 2A, the transverse relaxation time of protons in the liquid pool and T 2B, the transverse relaxation time of protons in the semi-solid pool. In the computations, the transverse relaxation rate of protons in the semisolid pool (R B) was taken to be 1 s ⫺1 (16). The products RM 0B/R A and 1/R AT 2A were used as estimates of the amount of magnetization transfer and a measure of direct saturation, respectively. SuperLorenzian absorption line shape was used for Z spectral data fitting, since this lineshape has been shown to provide the best fit for the preparations containing a semi-solid component (see Ref. 19). The quality of each fit was evaluated as the average residual deviation per spectral point between the fit and the experiment (20).

RESULTS R 1␳ as a function of ␻ 1 of native and denatured BSA phantoms in different buffers are shown (Fig. 1). It is evident that protein immobilization either by heat exposure (Figs. 1G and 1I) or GA cross-linking (Fig. 1K) strongly enhanced R 1␳. This is reconciled by the increased ␶ c of macromolecules upon immobilization (by 2–3 orders of magnitude) as well as elevated viscosity leading to solid-state broadening of the proton lines in the immobilized state (21). To determine the plausible contribution of proton exchange on the relaxation rates, pH was varied and the NMR relaxation measurements were carried out with BSA dissolved in (i) pure water, (ii) Tris buffer or (iii) KHB. Tris contains endogenous NH groups so that one would expect proton exchange also at the buffer sites adding to those on BSA. Phosphate buffer was used due to its well-known inherent ability to catalyze chemical exchange process at neutral pH. Proton exchange is catalyzed under alkaline conditions and inhibited under acidic ones, thus if the exchange would influence R 1␳ in protein solutions, one would expect to detect more efficient relaxation at alkaline and less efficient in acidic pHs. In native BSA water solutions, this was exactly our observation. Enhanced R 1␳ at pH 8 relative to pH 7.4 was seen, whereas at pH 6.3 or 5.5 it was reduced, and these effects were strongest at a ␻ 1 range of 0.7–3.5 kHz (Figs. 1A and 1B). A Tris buffer drop of pH from 7.4 to either 6.3 or 5.5, enhanced R 1␳ of water in the native BSA solution at ␻ 1 range from 0.7 to 2.7 kHz, but reduced it from 2.7 kHz at ␻ 1 above (Figs. 1C and 1D). R 2 increased under these conditions, but R 1 remained unchanged (Table 1). In native BSA phantoms dissolved in KHB, drop of pH from 7.4 to 5.5 resulted in a decrease of R 1␳ (Figs. 1E and 1F) and R 2 (Table 1), again in the ␻ 1 range of 0.8 –3.5 kHz, findings that were consistent with those seen in BSA dissolved in water. The effects of acidification on R 1␳ and R 2 were much greater in heat denatured than in native BSA phan-

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phantoms, pH had negligible influence on R 1␳ in the ␻ 1 range studied (Figs. 1K and 1L). Furthermore, neither R 1 nor R 2 changed in GA-treated phantoms in response to altered pH (Table 1). Magnetization transfer can be quantitatively probed by the Z spectroscopy (22). The magnetization transfer takes place through dipolar coupling made possible by chemical exchange (i.e., whole molecule exchange or proton exchange) (23). Molecule exchange is commonly considered to be the dominating mechanism for magnetization transfer as probed by the Z spectrum (8, 15, 23, 24). The Z spectra of native BSA phantoms (Fig. 2) with a very small volume of semi-solid pool (25) gave high residuals (around 0.05) for the super-Lorentzian fits and with a Lorentzian absorption lineshape fitting the R values were zero in many native phantoms (not shown). Therefore, we have not quantified magnetization transfer variables from these phantoms. It is evident that both heat-denatured and GA cross-linked BSA phantoms show substantial saturation at a large off-resonance frequency range consistent with a magnetization transfer rather than by direct saturation, seen in native protein phantoms (Fig. 2). The Z spectra yielded identical T 2B values for the immobile pool in heat denatured and GA-treated phantoms, while the magnetization transfer rates (R) were almost two times faster in the former than the latter preparation independent of buffer solution (Table 2). These data TABLE I

R 1 and R 2 in the BSA Phantom in Different Buffer Solutions

FIG. 1. Influence of pH on the T 1␳ relaxation rate, R 1␳. Absolute R 1␳ and ⌬R 1␳ at different pH are shown as a function of spin-lock (␻ 1) field amplitudes in native, heat-denatured, and cross-linked BSA phantoms dissolved in water, Tris, or KPB buffers. (A, B) Native BSA in water; (C, D) native BSA in Tris; (E, F) native BSA in KPB; (G, H) heat-denatured BSA in Tris; (I, J) heat-denatured BSA in KPB; (K, L) glutaraldehyde cross-linked BSA in Tris. Statistically significance differences (*P ⬍ 0.05) between given pH 5.5 and pH 7.4 were evaluated using unpaired t test. Fits to data are for visual aid only.

toms (Figs. 1G–1J and Table 1). In Tris, R 1␳ severely reduced upon acidification in a dose-dependent manner peaking at ␻ 1 of 4 kHz (Figs. 1G and 1H). In KPB, where both R 1␳ (Fig. 1I vs 1G) and R 2 (Table 1) were smaller than in Tris at a pH of 7.4, a drop of pH caused a severe reduction of both relaxation rates (Figs. 1I, 1J, and Table 1). Interestingly, R 1␳ dispersion was almost completely absent at pH of 5.5 in heat-denatured BSA (Fig. 1I). In heat denatured aqueous BSA phantoms, similar changes upon pH reductions were detected as in those incubated in KPB (not shown). In GA-treated

Buffer

Treatment

pH

R 1 (s ⫺1)

R 2 (s ⫺1)

Tris Tris Tris Tris Tris Tris Tris Tris Tris KHB KHB KHB KHB KHB KHB Aqueous Aqueous Aqueous Aqueous

None None None GA (1%) GA (1%) GA (1%) Heat denatured Heat denatured Heat denatured None None None Heat denatured Heat denatured Heat denatured None None None None

7.4 6.3 5.5 7.4 6.3 5.5 7.4 6.3 5.5 7.4 6.3 5.5 7.4 6.3 5.5 8.0 7.4 6.3 5.5

0.60 ⫾ 0.02 0.58 ⫾ 0.02 0.57 ⫾ 0.03 0.57 ⫾ 0.03 0.55 ⫾ 0.02 0.55 ⫾ 0.02 0.66 ⫾ 0.01 0.61 ⫾ 0.00 a 0.61 ⫾ 0.01 a 0.60 ⫾ 0.02 0.59 ⫾ 0.06 0.60 ⫾ 0.07 0.65 ⫾ 0.04 0.63 ⫾ 0.03 0.63 ⫾ 0.04 0.53 ⫾ 0.01 0.54 ⫾ 0.01 0.57 ⫾ 0.01 0.61 ⫾ 0.01 a

5.1 ⫾ 0.1 8.3 ⫾ 0.9 a 9.2 ⫾ 0.5 a 11.3 ⫾ 0.4 11.6 ⫾ 0.2 b 11.8 ⫾ 0.4 b 10.9 ⫾ 0.7 9.1 ⫾ 0.7 b 11.6 ⫾ 0.5 5.6 ⫾ 0.1 4.6 ⫾ 0.0 a 3.7 ⫾ 0.0 a 8.1 ⫾ 0.6 6.0 ⫾ 0.3 b 4.6 ⫾ 0.2 b 5.3 ⫾ 0.2 5.2 ⫾ 0.1 3.6 ⫾ 0.1 a 3.1 ⫾ 0.1 a

Note. The R 1 and R 2 relaxation rates were determined for given conditions as described under Methods. Data are mean ⫾ SEM for 4 independent measurements. Statistically significance differences (P ⬍ 0.05) between given pH and pH 7.4 in native ( a) and immobilized phantoms ( b) were evaluated using unpaired t test. KHB, potassium phosphate buffer; GA, glutaraldehyde.

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FIG. 2. The Z-spectra from (A) native, (B) glutaraldehyde crosslinked and (C) heat denatured phantoms in Tris buffer fitted with super-Lorentzian lineshapes. The amplitudes of off-resonance saturation fields were as indicated.

show that the relaxation properties of solid components were identical, yet the magnetization transfer rates were different in the denatured phantoms. Interestingly, R did not change upon acidification in the GA-treated phantoms (Table 2) while negligible to moderate increases (11–13%) were detected in RM 0b/ Ra. In contrast, the R figures in the heat denatured BSA solutions were consistently lower by 13-37% at pH of 5.5 (pH 6.0 for aqueous) relative to those determined at pH of 7.4, independent of the buffering conditions (Table 2). The RM 0b/R A apparently decreased in heat denatured phantoms in all buffers as a function of decreased pH. Thus in heat denatured phantoms, Z spectral data directly argue for a contribution of pHsensitive proton exchange to the magnetization transfer between the liquid and semi-solid pool consistent with a study of multilamellar vesicles (26). To further characterize the influence of immobilization methods on the chemistry of BSA, high-resolution

water-suppressed 1H NMR spectra were acquired (Fig. 3A). GA cross-links proteins from NH-groups and as a consequence all the NMR-visible NH- and amideproton peaks disappear from 1H spectra, whereas in heat denatured phantoms discernible peaks at this frequency range are present (Fig. 3A). The 1H NMR spectra show that the peaks from NH-/amide protons in heat denatured BSA moved upfield as a function of increased acidity (Fig. 3B), a finding consistent with an increased frequency difference between water and the chemical groups upon slow down of chemical exchange (and prolonged exchange lifetime, ␶ ex). In these phantoms, signal amplitudes of NH/amide protons increase at low pH (Fig. 3B) as a result of a reduced exchange rate consistent with a previous report (27). DISCUSSION The present study demonstrates that R 1␳ and R 1␳(␻ 1) both in native and in immobilized protein phantoms are strongly affected by pH. These results point to a crucial role of chemical exchange, mainly through proton exchange pathways, to the relaxation processes. In native BSA phantoms, the effect of pH-sensitive chemical exchange is in the ␻ 1 range below 3 kHz, however, the effect increases in the magnitude upon protein denaturation by heat exposure and extends to a much wider frequency range. Since the concept “chemical exchange” incorporates also molecule exchange (8, 23), separation between the exchange pathways is not always straightforward. Z spectroscopy probes chemical exchange as a whole, gaining contributions from both exchange pathways. Indeed, we show that in the heatdenatured BSA phantoms exchangeable groups in side chains are detected by the high-resolution 1H NMR. Moreover, in these preparations pH sensitive decrease in R 1␳, R 1␳(␻ 1) and magnetization transfer rate are sig-

TABLE II

Magnetization Transfer Variables from Immobilized BSA Phantoms Computed Using the Henkelman Model Buffer

Treatment

pH

T 2B (␮s)

R (s ⫺1)

RM 0B/R A

1/R AT2 A

Residual

Tris Tris Tris Tris KHB KHB KHB KHB Aqueous Aqueous Aqueous Aqueous

GA (1%) GA (1%) Heat denaturation Heat denaturation GA (1%) GA (1%) Heat denaturation Heat denaturation GA (1%) GA (1%) Heat denaturation Heat denaturation

7.4 5.5 7.4 5.5 7.4 5.5 7.4 5.5 7.4 5.5 7.4 6.0

7.5 ⫾ 0.5 8.4 ⫾ 0.1 7.5 ⫾ 0.1 8.3 ⫾ 0.1 a 8.1 ⫾ 0.5 8.3 ⫾ 0.1 8.0 ⫾ 0.1 8.2 ⫾ 0.1 8.0 ⫾ 0.1 8.1 ⫾ 0.003 7.8 ⫾ 0.3 8.2 ⫾ 0.1

40.0 ⫾ 7.2 37.6 ⫾ 0.8 76.3 ⫾ 4.4 47.9 ⫾ 0.4 a 31.5 ⫾ 7.2 34.6 ⫾ 0.6 73.6 ⫾ 5.2 64.4 ⫾ 6.4 32.3 ⫾ 4.3 35.7 ⫾ 4.9 73.3 ⫾ 0.6 62.4 ⫾ 4.1

3.5 ⫾ 0.4 3.9 ⫾ 0.2 3.0 ⫾ 0.1 1.3 ⫾ 0.2 a 3.8 ⫾ 0.4 4.3 ⫾ 0.0 a 3.3 ⫾ 0.5 1.4 ⫾ 0.3 a 3.6 ⫾ 0.1 4.0 ⫾ 0.1 a 3.5 ⫾ 0.1 2.2 ⫾ 0.1 a

19.1 ⫾ 0.8 16.5 ⫾ 0.3 17.8 ⫾ 0.4 12.7 ⫾ 0.9 a 13.4 ⫾ 0.8 14.8 ⫾ 0.2 16.3 ⫾ 2.0 7.5 ⫾ 0.6 a 12.5 ⫾ 1.2 13.9 ⫾ 2.0 13.8 ⫾ 0.9 8.5 ⫾ 0.4 a

0.02 0.03 0.02 0.02 0.03 0.04 0.02 0.02 0.03 0.03 0.02 0.02

Note. The parameters were computed from the Z spectra using the Henkelman model as described under Methods. The values shown are means ⫾ SEM. Statistically significance differences (P ⬍ 0.05) between given pH and pH 7.4 ( a) were evaluated using unpaired t test. 816

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FIG. 3. 1H spectra acquired with the WATERGATE sequence from (A) a native, heat-denatured and cross-linked BSA phantom; (B) heat denatured BSA phantoms at different pH as indicated. Chemical shift was referenced to 3-(trimethylsilyl)-propionic acid-d 4 at 0 ppm.

nificant. In contrast, the effect of pH in the GA-treated phantoms on the NMR relaxation and magnetization transfer is small and ambiguous. These observations argue for a dominant contribution of proton exchange at the labile side chain groups to T 1␳ relaxation and magnetization transfer in heat denatured protein solutions. This is feasible, since spin diffusion is very efficient under the conditions with the exchange rate Ⰶ tumbling rate (28) allowing the labile protons to reorient with the macromolecule (29). It should be emphasized, however, that numerous studies point to a negligible contribution of proton exchange to the magnetization transfer as probed by the Z spectrum in the biological preparations (15, 22–24). Proteins contain a number of chemically distinct exchangeable chemical groups such as ⫺NH, amides, ⫺OH and ⫺SH groups. The exchange rates of hydroxyl protons depend on the type of amino acid. The tyrosine hydroxyl proton exchange rate (⬃12000 s ⫺1) is faster than those of lysine (5000 –10000 s ⫺1) and arginine (1200 s ⫺1) amino protons, yet that of threonine hydroxyl (⬃1000 s ⫺1) is similar to the exchange rates of amines in the two amino acids, as determined in ␣-amino acetylated compounds (7). The exchange rate of sulfur protons under physiological conditions proceeds at a rate ⬎500 s ⫺1, whereas amide proton exchange occurs at a rate ⬍100 s ⫺1. It should be emphasized that these figures have been determined in amino acid derivatives or small peptides and those encountered in native proteins may be substantially slower than those above (30). The high-resolution 1H NMR spectra show a number of broad resonances in the amine/amide chemical shift region, which must be in a slow exchange regime to become detected, however, it is obvious that several kinds of chemical groups are available in proteins for chemical exchange. Interestingly, it has recently been shown that the intensities of the 1H NMR signals from the amine/amide groups in vivo (between 6.6 and 8.8 ppm), as acquired with a WATERGATE method, respond to tissue pH increasing upon acidification. These NMR signals show short T 2 and substantial saturation in a conventional watersuppressed brain 1H NMR spectrum due to exchange with bulk water. Thus spectroscopic use of amine/

amide protons as physiological indicators of intracellular pH by 1H NMR is technically demanding (27). The present data from protein phantoms suggest that R 1␳ and R 2 of water are sensitive to acid-base catalyzed proton exchange. Our results point to two important technical aspects in the interpretation of in vitro protein phantom relaxation data. Firstly, we show that the buffer substance, such as TRIS, can influence R 1␳ and R 2 of BSA solutions due to its endogenous NH-groups. Secondly, chemical cross-linking by glutaraldehyde, which is commonly used as a method for preparing phantoms mimicking tissue water relaxation (4, 31), blocks 1H NMR discernible NH-/amide groups. In the absence of these exchangeable groups much reduced effects of pH on R 1␳ and R 2 are determined, which makes this method dubious in regard to the physiological conditions. To conclude, the present data argue for a substantial contribution of proton exchange between the protein side chain groups and bulk water to the T 1␳ relaxation. There appears to be a striking difference between the mechanisms affecting magnetization transfer and T 1␳, since experimental evidence points to a dominant contribution of the dipolar interactions mediated through whole molecule exchange in the magnetization transfer (8, 15, 22–24, 32). The present data, taken together with previous evidence from multiexponential relaxation analysis (33) and isotope dilution studies (9), show a key role of a proton exchange pathway for the longitudinal relaxation at the rotating frame. The role of similar mechanisms under in vivo conditions should be explored in detail (1, 2). A recent report demonstrating tendency toward a pH-dependent decrease in R 1␳ in the ischemic rat brain (3) provides provocative evidence for a proton exchange pathway in this context. ACKNOWLEDGMENTS This study was supported by the grants from the Academy of Finland, the Sigrid Juselius Foundation, the North Savo Fund of Finnish Cultural Foundation and the EVO program of Kuopio University Hospital. Expert technical assistance by Ms. Niina Kuhmonen and Mrs. Tuula Salonen are greatly appreciated.

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