Interactions of myristic acid with bovine serum albumin:

Transcription

1 Proc. Natl. Acad. Sci. USA Vol. 81, pp , June 1984 Biophysics Interactions of myristic acid with bovine serum albumin: A 13C NMR study (fatty acid/molecular motion/chemical shift) JAMES A. HAMILTON*, DAVID P. CISTOLA*, JOEL D. MORRISETTt, JAMES T. SPARROWt, AND DONALD M. SMALL* *Biophysics Institute, Departments of Medicine and Biochemistry, Boston University School of Medicine, 80 East Concord Street, R111, Boston, MA 02118; and tdepartment of Medicine, Baylor College of Medicine, Methodist Hospital, Houston, TX Communicated by Alfred G. Redfield, February 24, 1984 ABSTRACT Interactions of myristic acid with bovine serum albumin were studied by 13C NMR spectroscoy at 50.3 MHz using 90% isotopically substituted [1-'3C1-, [3- C]-, and [14-'3Clmyristic acids, either individually or in a combination of all three with albumin. At ph 7.4, two or more resonances of different intensities were observed for each 13C-enriched myristic acid. Carboxyl and methylene C-3 resonances corresponding to the major myristic acid environment(s) exhibited ph-dependent chemical shift changes indicative of protonation below ph 6.7; in contrast, carboxyl groups in minor environments were resistant to protonation. 13C NMR spectra obtained as a function of the molar ratio of [3-13C]- and [14-13C]myristic acid to bovine serum albumin (from 0.7 to 5.6) revealed at least two narrow resonances for each carbon at all molar ratios. Thus, bovine serum albumin binding sites for myristic acid are heterogeneous with respect to titration behavior and with respect to the local magnetic environment at both the polar and the nonpolar ends of the fatty acid. The narrow resonances observed for the methylene and methyl carbons are inconsistent with complete immobilization of the protein-bound acid molecules. Together with spin-lattice relaxation times and nuclear Overhauser enhancements, the linewidth results indicate that bound myristic acid has internal motions that are rapid compared with overall protein tumbling and that the C-3 methylene carbon is more restricted than the terminal methyl carbon. Albumin is a single polypeptide chain of -580 amino acids that contains 17 disulfide bridges and putatively folds into an intricate looped structure with three domains and six subdomains (1, 2). It binds and transports fatty acids (FA) in the plasma; a structural model of this protein may provide a basis for understanding FA binding. FA of different chain lengths and degrees of unsaturation form tight complexes with albumin, although the Ka values differ (3). For example, those for the first five binding sites (-107_106 M-1) of the saturated 14-carbon FA, tetradecanoic acid or myristic acid (MyrOH), are -one-tenth of the corresponding constants for the monounsaturated 18-carbon FA, oleic acid, which is the most tightly bound of the long-chain FA (3). Recent 13C NMR studies have revealed multiple magnetic environments for the carboxyl carbon of oleic acid bound to bovine serum albumin (BSA), most likely because of different amino acids in the vicinity of the FA carboxyl group at various binding sites (4). In the present study, we examined complexes of MyrOH with BSA using 13C NMR spectroscopy. To study particular carbon atoms and to achieve adequate spectral sensitivity, we used three 90% isotopically substituted [13C]MyrOH species: (i) [1-13C]MyrOH, (ii) [3-3C]MyrOH, and (iii) [14-3C]MyrOH. The C-13 labels act The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C solely to indicate this fact. as nonperturbing probes of the microenvironment around the three different carbon atoms, and the relaxation behavior of the protonated carbons (methylene C-3 and methyl C-14) provides information about the molecular motions of the protein-bound FA. MATERIALS AND METHODS Materials. Essentially FA-free (crystalline, lyophilized) BSA from Sigma (lot 12E9340) was subjected to a charcoal defatting procedure (5, 6). Chromatographic analysis of Sigma FA-free BSA with 1-6 mol of oleic acid added showed that most (.65%) of the FA was bound to monomeric BSA (4). The BSA had.5% protein impurities as measured by 10% NaDodSO4/PAGE (7); 90% [1-'3C]MyrOH, 90% [3-13C]MyrOH, and 90% [14-13C]MyrOH were synthesized as described (8, 9). 13C NMR spectra of each labeled MyrOH in C2HCl3 showed a single 13C resonance (with signal/noise ratios of -100:1) at the expected chemical shift (8) of each 13Cenriched nucleus (10). 1H NMR spectra showed the expected splitting pattern in the 1H multiplet next to the 13C-enriched nucleus. TLC showed a purity of -95% for each label; GLC showed that.96% of each label consisted of a saturated 14- carbon hydrocarbon chain. A portion of each sample was recrystallized to a purity of.98% by TLC and GLC criteria. FA-BSA complexes prepared with MyrOH containing these different levels of impurities showed no differences in their NMR spectra, including the minor FA resonances. Sample Preparation. MyrOH-BSA complexes were made from aqueous potassium myristate (KOMyr) essentially as described for oleic acid-bsa complexes (4). Samples (7.5% BSA, wt/vol) were equilibrated 12 hr at 250C prior to NMR analysis. Except as noted, samples were optically clear. Compositions of FA-BSA complexes are given as mol of MyrOH/mol of BSA. NMR Methods. Proton-decoupled Fourier-transform NMR spectra were obtained at 50.3 MHz with a Bruker WP200 spectrometer as described (4, 11). Internal 2H20 was used as a lock-and-shim signal. Except as noted, the sharpest protein peak, that at ppm relative to external (CH3)4Si in C2HC13, was used as an internal 8 reference (4) with an estimated uncertainty of +0.1 ppm. Spin-lattice relaxation times (T1) and nuclear Overhauser enhancement (NOE) were measured as before (4, 11). Peak areas (integrated intensities) were measured by planimetry. RESULTS The 13C NMR spectrum of a FA-BSA complex containing a total of 6 mol of MyrOH/mol of BSA (1 mol of [14-13C]- MyrOH, 2 mol of [3-13C]MyrOH, and 3 mol of [1-13C]- Abbreviations: FA, fatty acid(s); BSA, bovine serum albumin; MyrOH, myristic acid; KOMyr, potassium myristate; T1, spin-lattice relaxation time; NOE, nuclear Overhauser enhancement. 3718

2 Biophysics: Hamilton et al MyrOH) is shown in Fig. 1 (trace A). The MyrOH labels gave multiple narrow resonances in three spectral regions (Insets). Comparison with FA-free BSA (Fig. 1, trace B) shows that the FA carboxyl and methyl peaks occurred in regions that were almost devoid of resonances arising from protein carbons (at natural abundance), while the FA C-3 methylene peaks overlapped the protein aliphatic peaks. A difference spectrum (Fig. 1, trace C) obtained by digitally subtracting the FA-free BSA spectrum (trace B) from the BSA spectrum containing the three 13C labels (trace A) shows the MyrOH spectrum with the protein contribution removed. In the carboxyl region, a small contribution at ppm from protein carboxyl resonances was removed by subtraction. The MyrOH C-3 resonances at -26 ppm overlapped protein aliphatic resonances (compare traces A and B). Subtraction of protein resonances removed intensity under the C-3 resonances (trace C); however, the FA C-3 resonances were identifiable in the spectrum of the complex (compare Insets to traces A and C). The FA C-14 resonances were upfield from the protein methyl resonances at 15.9 ppm and contained no overlapping protein peaks. Trace C reveals at least two resonances (indicated by 8 values, Insets) for each FA label (C-1, C-3, and C-14). Protein resonances (trace B) contributed very little to the difference spectrum (trace C). Thus, there are no major conformational changes detectable by 13C NMR in BSA on binding MyrOH. A similar conclusion was reached based on BSA spectra taken before and after oleic acid binding (4). Molar Ratio Studies. 13C NMR spectra of FA-BSA complexes with a single MyrOH label were obtained at different molar ratios of MyrOH to BSA at fixed ph (7.4) and concentration of BSA (7.5%, wt/vol). Samples were prepared at molar ratios of 1.4, 2.8, 4.2, and 5.6 with [3-13C]MyrOH or [14-13C]MyrOH, and a sample with a molar ratio of 0.7 was prepared with [14-13C]MyrOH; studies with [1-13C]MyrOH Proc. Natl. A cad. Sci. USA 81 (1984) 3719 will be reported elsewhere. The resonance envelope from the [3-13C]MyrOH after digital subtraction of a spectrum of FA-free BSA from a spectrum of the MyrOH-BSA complex at each molar ratio (indicated below each spectrum) is shown in Fig. 2. At least two resonances were detected at all molar ratios; the major one (:z-26.5 ppm) was asymmetric, and one or two small peaks were present on the upfield shoulder (25.6 ppm and 25.1 ppm). The width of the C-3 envelope at half the height of the major peak was -30 Hz in the 5.6-mol spectrum. The height of the 26.5-ppm peak increased linearly with increasing MyrOH/BSA ratio and the extrapolated line intercepted the origin; in contrast, the intensity of the peak at ppm first increased and then appeared to level off (Fig. 2 Inset). The integrated intensity of the entire envelope was proportional to the MyrOH/BSA ratio and the extrapolated value at 0 mol was slightly >0. The methyl resonance envelope from spectra of [14-13C]MyrOH-BSA complexes at the indicated molar ratios is shown in Fig. 3A. At least two methyl peaks were detected at all ratios. Three methyl peaks were detected at a ratio of 1.4, as shown by the main trace and in the resolution-enhanced convolution difference spectrum (12) above it. With increasing MyrOH/BSA ratio, the resolution of the two upfield peaks became progressively poorer concurrent with a change in 8 of the major peak (whether this change is a true 8 change or results from the merging of the two peaks is not clear). The height of the major peak (at ppm) and the integrated intensity of the entire envelope of resonances (from to ppm) were linearly proportional to the molar ratio (Fig. 3B) and both lines intercepted the origin. The height of the 14.3-ppm peak was relatively large at low molar ratios and showed a smaller increase with increasing molar ratio than the major resonance. The linewidth (v½,t) of the peak at ppm (molar ratio, 5.6) was 10 Hz. ph Studies. 13C NMR spectra were obtained as a function of ph (between ph 7.4 and 4.2) at fixed molar ratios for samples with [3-13C]MyrOH (4.2 mol/mol of BSA) or [14-3C]MyrOH (5.6 mol/mol of BSA) and at ph for a , 12.0.O L S 8.0 a (U U) 12.0 c co 8.0 s 4.0 N (5.6A Chemical shift from (CH3)4Si, ppm FIG C NMR spectra at 40 C of the complex of 6 mol of MyrOH (3 mol of 90% [1-13C], 2 mol of 90% [3-13C], and 1 mol of 90% [14-13C]) per mol of BSA (trace A) and of FA-free BSA (trace B). Spectra were recorded after 12,000 accumulations with a pulse interval of 2.0 s, 16,384 time-domain points, and a spectral width of 10,000 Hz. Digital line broadening of 3 Hz was used. Spectrum C was obtained by digital subtraction of spectrum B from spectrum A and shows the contribution of the three MyrOH labels, designated C-1, C-3, and C-14, to the complex spectrum (trace A). Insets are horizontal expansions of the indicated regions; 8 values (in ppm) of selected resonances are indicated. FIG. 2. The methylene C-3 region derived by subtraction of a spectrum of FA-free BSA from spectra of mixtures of 90% [3- '3C]MyrOH and BSA at four molar ratios (1.4, 2.8, 4.2, 5.6). Spectra were obtained as in Fig. 1 except for the number of accumulations (4000) and pulse interval (0.82 s). All processing conditions (line broadening of 3.0 Hz) and plotting conditions are identical for all spectra. (Inset) Plots of peak heights of the resonances at ppm and at ppm (A) and integrated intensity of the entire resonance envelope (w) as a function of the MyrOH/BSA ratio. The peak height was used as a relative measure of peak area for the unresolved resonances and is reported with the same qualifications as discussed before (4). Plots of peak area and peak height of the ppm peak are linear with r The intensity of the 25.1-ppm peak appeared to level off, and the points in the plot are not connected by a single straight line.

3 3720 Biophysics: Hamilton et al. FA-BSA complex containing all three labels at molar ratios of 1.0 ([14-1'3C]MyrOH), 2.0 ([3-13C]MyrOH), and 3.0 ([1-13C]MyrOH). In the absence of BSA, MyrOH is crystalline at ph -7.4, and no high-resolution 13C spectrum was observed (see below). The effect of added equivalents (,ul) of HCl on the 8 of MyrOH resonances is shown in Fig. 4. One resonance in each envelope (the C-1 peak at ppm, the C-3 peak at ppm, and the C-14 peak at ppm) exhibited a linear change in 8 (the C-1 and C-3 peaks exhibiting a break point at ph 6.7) with added equivalents of HCl; the C-1 peak showed the largest change (2.4 ppm), the C-3 peak showed a more modest change (0.8 ppm), and the C-14 peak showed a very small change (0.15 ppm) in the opposite direction of the C-1 and C-3 peaks. The minor MyrOH resonances became undetectable after the addition of gl of HCl, as indicated by the omission of points in the figure; they showed no 8 change before they became undetectable. The individual complexes with C-3 and C-14 MyrOH at molar ratios of 4.2 (13C-3) and 5.6 (13C-14) had a titration behavior (a in Fig. 4) that was the same as that for the BSA complex with all three MyrOH labels (o in Fig. 4). At ph < 4.7 (60,ul of HCl) the sample became increasingly turbid and, below ph 4.2, it gelled. Addition of sufficient KOH (while mixing with a Vortex) to bring the ph to 7.4 followed by incubation at 250C for 2 hr gave a clear liquid sample visually indistinguishable from the original sample at ph C NMR spectra of these samples resembled in all details the original spectra at ph 7.4. T1 and NOE Measurements. T1 and NOE values of the C-1, C-3, and C-14 MyrOH resonances were measured for several MyrOH-BSA complexes (Table 1). T1 and NOE values were not dependent on the MyrOH/BSA molar ratio; T1 values of the different resonances within each of the three resonance envelopes were not different. Both the T1 and NOE values of the C-14 resonance were much greater than those values for the C-3 resonance. A B Z) le I a-/ I id 1 9 la A r A C-14 acid/bsa ratio ff% I r% au.u 1, O cu 10.0 c ia N FIG. 3. (A) Methyl regions of 13C NMR spectra at 34 C of mixtures of 90% [14-13C]MyrOH and BSA at four molar ratios (1.4, 2.8, 4.2, and 5.6). Spectra were obtained as in Fig. 2 except for the pulse interval (1.8 s). All processing (line broadening of 0.5 Hz) and plotting conditions were identical for all spectra in the main figure. (Inset) Convolution difference spectrum derived from the same timedomain data as the bottom spectrum. (B) Plots of peak heights of the main resonance at =13.9 ppm and of the resonance at 14.3 ppm (A) and the integrated intensity of the entire resonance envelope (o) as a function of molar ratio. Plots were linear with r In the spectrum taken at a molar ratio of 0.7 (not shown), two methyl resonances (at and ppm) were detected. a E ' Q ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ E U 25.0 Proc. Nati. Acad Sci. USA 81 (1984) ph (514) (5-9)(6.7) (7.4) (4.2) (4.4)(47)(428) } C ~~~~~~~~ ~~~~~~~~ HOI (0.94 M),.il FIG. 4. Plot of 8 of the C-1, C-3, and C-14 resonances of MyrOH as a function of added HCl: o, 6 mol of MyrOH (3 mol of 90% [1- '3C], 2 mol of 90% [3-'3C], and 1 mol of 90% [14-13C])/mol of BSA; A, 4.2 mol of 90% [3-'3C]MyrOH/mol of BSA; o, 5.6 mol of 90% [14- '3C]MyrOH/mol of BSA. Measured ph values for each titration point are given in parentheses above the plot for the main C-3 peak. Spectral conditions for the titration sample containing all three labels (o) were the same as those given in Fig. 1, except for the number of accumulations (4000 per spectrum). Spectral conditions for the [3-'3C]MyrOH sample were the same as in Fig. 3; those for the [14-13C]MyrOH sample were the same as in Fig. 2, except for the number of accumulations (1000 per spectrum). For the titration series with all three labels (o), 8 was referenced to (CH3)4Si in C2HCl3 in a capillary insert to verify (4) that the 8 of the peak at ppm (used in the other titration series as an internal reference) did not change with ph and also to permit a more precise measurement of 8 (±0.05 ppm). The principle C-14 resonance (13.9 ppm at ph 7.4) and the principle C-3 resonance (26.4 ppm at ph 7.4) exhibited ph-dependent 8 changes without changes in intensity. In contrast, with the first addition of HCl (10,ul), the C-1 peak at ppm diminished in intensity; the C-1 peak at ppm became the most prominent C- 1 peak and then showed the largest 8 changes with ph. Additional data (not shown) for a sample with 5 mol of [1-'3C]MyrOH showed that all three C-1 peaks had no significant change in 8 with added KOH between ph 7.4 and ph 9.5; also, the ppm peak was the most intense peak (by the criterion of peak height) and was the only C-1 peak that showed a titration curve (unpublished results). Aqueous KOMyr. An aqueous sample containing equimolar amounts of the C-1, C-3, and C-14 MyrOH labels as the K+ soap (ph > 10) was studied by 13C NMR. Spectra of micellar (c = 40 mm, 60 mm) KOMyr gave peaks at ppm (C-1), ppm (C-3), and ppm (C-14). Spectra (6000 accumulations) of monomeric (c = 1.5 mm) KOMyr gave peaks at ppm (C-1), ppm (C-3), and ppm (C-14). Each carbon gave a single resonance with P~, - 4 Hz. Lowering the ph of the clear and colorless 1.5 mm sample to 7.4 produced a turbid solution with 1:1 acid/soap crystals (unpublished data); this sample yielded no NMR resonances after 12,000 spectral accumulations. Therefore, the narrow MyrOH 13C resonances observed in the presence of BSA are attributable to protein-bound MyrOH. DISCUSSION Complexes of BSA with MyrOH were examined by 13C NMR spectroscopy. For preparation of FA-BSA complexes, two approaches were used: one was to make complexes containing a single type of 13C-enriched MyrOH, carboxyl (C-1), 8-methylene (C-3), or methyl (C-14); the other was to make complexes containing all three labeled species. The former approach had the advantage of permitting observation of 13C NMR signals from the FA label at low FA/BSA ratios (mol/mol). The latter approach diluted the signals

4 Table 1. Biophysics: Hamilton et al T1 and NOE values of MyrOH resonances in MyrOH- BSA complexes of different molar ratios Composition of MyrOH/BSA complex A* Bt C: D ET Car- 8, T1, T1, T1, T, bon ppm s1l NOE** NOE** s s NOE s NOE C NM NM C NM NM NM NM C NM NM, not measured. *3 mol of [1-13C]MyrOH, 2 mol of [3-'3C]MyrOH, and 1 mol of [14- '3CJMyrOH per mol of BSA, ph 7.4, 40'C. t5.6 mol of [3-13C]MyrOH/mol of BSA, ph 7.4, 340C. t4.2 mol of [3-13C]MyrOH/mol of BSA, ph 7.4, 340C. 2.8 mol of [3-13C]MyrOH/mol of BSA, ph 7.4, 340C. ~5.6 mol of [14-'3C]MyrOH/mol of BSA, ph 7.4, 340C. 1Estimated errors are ±20-25%, except for the methyl resonance at 13.8 ppm (±10%). **Maximum value = 3.0; estimated errors are ±20%. from each FA labelt but permitted simultaneous observation of all three labels. Because the BSA concentration, ph, sample temperature, and other variables were rigidly controlled in a given sample with the three labels, sources of variability in measurements of 8, linewidth, T1, and NOE values were eliminated. In all cases, NMR measurements made with three MyrOH labels present together agreed with measurements made with the labels present individually. The present study shows that the carboxyl carbon, the C-3 (methylene) carbon, and the C-14 (methyl) carbon of MyrOH all experience multiple environments in the presence of albumin. The carboxyl region of the MyrOH-BSA spectrum exhibited a cluster of resonances between and ppm (containing at least three narrow individual peaks at ph 7.4), along with a relatively weak resonance at ppm. The carboxyl region of oleic acid-bsa spectra at comparable molar ratios (5-6/1) was similar except for the presence in the oleic acid-bsa spectra of an intense narrow peak at ppm (4). The MyrOH C-3 resonances overlapped the protein aliphatic carbon resonances, but the lineshapes could be examined and reliable intensity measurements could be obtained following spectral subtraction of the protein component. The C-3 resonance was not a single lorentzian peak but had at least one or two partially resolved peaks lying on the upfield side of an intense peak. The MyrOH methyl carbon yielded two or three narrow resonances in a spectral region with no other peaks. The intensity of the major C-3 and C-14 resonances and the total peak areas reflected, with reasonable accuracy, the FA/BSA ratio. The environments corresponding to the minor C-3 and C-14 resonances were relatively more populated at low than at high molar ratios. The 8 of the carboxyl or the C-3 resonance can be used (14) to determine the ionization behavior of FA in a milieu tbecause there are only very small chemical and physical differences between MyrOH molecules containing the "3C enrichment in different carbons, the different labels will equilibrate randomly in the different binding environments. Thus, the spectrum of all three labels resembles the spectrum for a high molar ratio (note the relative intensities within the C-3 and C-14 envelopes and compare with individual labels at a molar ratio of 5.8) with the total peak intensity of each envelope reduced in proportion to the molar ratio of each label. In contrast, a single FA carboxyl resonance was observed for [1-13C]octanoic acid in spectra of octanoic acid/bsa complexes at similar mol ratios (13). Proc. Natl. Acad. Sci. USA 81 (1984) 3721 containing many titratable groups (4). MyrOH C-1 and C-3 resonances showed no dependence of 8 on ph above ph 6.7. Between ph 6.7 and ph 4.7, the C-1 resonance at ppm and the C-3 resonance at 26.4 ppm showed a linear decrease in 8 with equivalents of HCI, indicative of titration in the acid ph range of the major carboxyl group (4, 14). T The total shift of the C-3 resonance (0.8 ppm) was -33% of that for the C-1 resonance (2.2 ppm), the same ratio found for titrations of water-miscible FA (14). This result rules out mechanisms that would affect the magnetic environment of the carboxyl carbon without affecting the f3-methylene carbon (such as H bonding, which has a significant effect on the C-1 8 value but very little on the C-3 8 value; ref. 14) as possible sources of ph dependence of 8. Note also that the titration curves are incomplete at the low ph end, as was the case for oleic acid-bsa titrations (14). Because the 8 are invariant with ph above ph 6.7, these carboxyl groups are fully ionized at physiological ph (and above). The minor C-1 and C-3 resonances have invariant 8 at all ph values, demonstrating a resistance to titration of FA molecules in those environments. Similar results were obtained for the ph dependence of the C-1 8 value of BSA-bound oleic acid (4). On the basis of peak intensities and titration behavior it is possible to assign resonances from the different MyrOH carbons to specific environments. Thus, the most intense resonance in each resonance envelope (the C-1 peak at and/or ppm, the C-3 peak at 26.4 ppm, and the C-14 peak at ppm) exhibits ph-dependent 8 changes (those for the C-14 peak are small and, if real, may represent a minor structural change in the vicinity of the methyl group). Based on the similarity of intensities, the minor C-14 and C-3 resonances (the C-3 peak at 25.0 ppm and the C-14 peak at 14.3 ppm) probably correspond to molecules with the C-1 peak at ppm. These three resonances were also resistant to titration effects, and thus together they represent MyrOH in a single type of binding environment. From the chemical shift results several important details about BSA binding environments emerge: (i) At FA/BSA ratios.1.4, MyrOH molecules are present in at least three environments and are in slow exchange on the NMR time scale (the exchange rate must be i15/s). Each NMR resonance represents at least one binding site of FA on BSA (4). Detection of three environments at a FA/BSA ratio of only 1.4 shows that there is a distribution of FA among binding sites of different affinities (15) rather than quantitative binding of the first FA molecule to a single high-affinity site. The NMR results are also compatible with the concept that there is an equilibration of FA among different BSA molecules, such that, for example, at a FA/BSA ratio of 1.0, an appreciable number of protein molecules bind no FA while others bind 2 mol of FA (16). (ii) At ph 7.4, the carboxyl group is bound in the ionic form, and the range of 8 may indicate ionic and/or H-bonding interactions between the carboxyl group and different basic amino acid groups (4). Most, but not all, of the carboxyl groups begin to titrate below ph 6.7. (iii) The range of 8 for the C-3 resonance at ph 7.4 is much larger than the KOMyr monomeric -* micellar 8 shift. The monomeric micellar shift reflects primarily differences in solvation and/or conformation (17). The 8 inhomogeneity may be a result of the different ionic interactions of the carboxyl group, and the resultant inductive effect on the C-3 resonance, and/or the heterogeneity of the protein environment in the vicinity of the FA C-3 methylene. (iv) The microenvironment of the myristic methyl group is heterogeneous. The WAlthough the peak height of the ppm peak is less than that for the ppm in Fig. 1, the true intensity may be greater. It is the ppm peak that is associated, via ph effects, with the most intense C-3 peak; thus, it appears to represent the principle MyrOH environment.

5 3722 Biophysics: Hamilton et al. C-14 resonance exhibits a 8 variation that is much larger than the monomer -+ micelle shift. It is possible that a group such as an aromatic ring system, which can induce substantial 8 changes in nearby carbons (18), is present in one environment (at 14.3 ppm), which shows a large downfield shift relative to the principle environments. These NMR results are compatible with several structural features of FA binding sites proposed from three-dimensional modeling of BSA (2). Based on this model, there is one specific binding site in each of the six subdomains; of these sites, three or four are specific for long-chain FA. Each site consists of a narrow channel that runs through the molecule and is lined primarily with hydrophobic amino acid side chains. At the ends of each channel, on the protein surface, there are clusters of basic amino acid residues. Although the general features of the different sites appear to be similar, large sequence differences between domains (2) suggest that the FA microenvironment is different in each binding site. A primary reason for using a FA molecule with C-13 enrichment at sites other than the carboxyl carbon was to obtain information about the molecular motions of FA bound to BSA. At present it is not possible to assess the molecular motions from relaxation measurements of carboxyl resonances because the 13C relaxation mechanism of the carboxyl carbon is not known (4). However, '3C relaxation of protonated carbons occurs via 13C-1H dipolar interactions at magnetic fields comparable with that used in this study (19). If the MyrOH molecule is rigidly bound to BSA then the overall tumbling of the protein molecule will be the dominant motion determining the relaxation of protonated carbons. The rotational reorientation of the BSA molecule is anisotropic, with correlation times in the range of ns (20, 21). When the shortest correlation time (25 ns) and the theoretical analysis for isotropic reorientation are used, the predicted vi, of a methylene or methyl resonance is.100 Hz and the predicted NOE is the minimum value of 1.15 (22). The observed Pi'½ values for the C-3 and C-14 MyrOH resonances (which include a significant contribution from 8 inhomogeneity) are much smaller, and the observed NOE values much larger, than these predicted values. Therefore, both the C-3 and C-14 carbons must have internal motions that are rapid compared with protein tumbling. A comparison of T1 and NOE values of the C-3 and C-14 resonances suggests that the motions of the C-3 carbon are relatively restricted compared with those of the methyl carbon. In support of this conclusion, the methyl peaks appear to be much narrower than the C-3 peaks.11 Thus, the polar end of the FA chain may be more rigidly attached to the binding site than the nonpolar end, consistent with the hypothesis that strong and specific ionic interactions between FA and BSA play a significant role in FA binding (4). In addition, T1, NOE, and vi½h values do not differ markedly for different peaks within a resonance envelope, so that the molecular motions appear to be similar in different binding environments. Molecular motions of FA bound to BSA have previously been assessed by using spin-labeled FA. ESR studies have produced conflicting ideas concerning (i) whether the (derivatized) FA is rigidly or loosely attached to BSA and (ii) IBecause the methyl group has an extra degree of rotational freedom, it is not possible to compare the relaxation data for the methyl and (C-3) methylene carbons strictly. The ratio of NT1 values for the methyl and methylene resonance (-8:1, Table 1) is about onehalf the ratio of the NT1 values (at the same magnetic field) of the corresponding resonances of the oleic acid moiety of liquid cholesteryl oleate (23). The NOE values of corresponding resonances in both systems are very similar. In the case of neat cholesteryl oleate, the oleic acid is anchored at the carboxyl group to the steroid ring system by a covalent ester linkage and the terminal methyl is present in a highly viscous lipid milieu with no known stringent restrictions on its molecular motions. Proc. Natl. Acad ScL USA 81 (1984) which portion of the FA molecule is most rigidly bound (see, e.g., refs ). In contrast to the 13C results, stearic acid molecules containing nitroxide probes near the carboxyl group often appear to be immobilized (25, 27, 28). However, nitroxide probes may significantly alter both the FA binding environment and FA molecular motions whereas the 13C probes are nonperturbing. This study shows that the high-affinity ("strong") binding of MyrOH to BSA does not result in complete immobilization of FA molecules in the binding sites. MyrOH bound to BSA at physiological ph exhibits rapid internal reorientations. In the absence of BSA, MyrOH at similar concentrations (e.g., 1.5,uM) at ph 7.4 and 380C is a crystalline acid/ soap that gives no high-resolution 13C NMR spectrum. We thank Dr. David Croll for helpful comments and Anne Gibbons for preparation of the manuscript. This work was supported by Public Health Service Grant HL26335 (J.A.H., D.P.C., D.M.S.) and Grant Q-837 from the Robert Welch Foundation (J.D.M.). 1. Brown, J. R. (1975) Fed. Proc. Fed. Am. Soc. Exp. Biol. 34, 591 (abstr.). 2. Brown, J. R. & Shockley, P. (1982) in Lipid-Protein Interactions, eds. Jost, P. C. & Griffith, 0. H. 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