ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 190, No. 2, October, 611-616, 1978
Reappraisal
of the Electron Spin Resonance Spectra of Maleimide lodoacetamide Spin Labels in Erythrocyte Ghosts GARY
L. JONES*
AND
DIXON
and
M. WOODBURYt
* Department of Pharmacology, North Texas State Uniuersity, Texas College of Osteopathic Medicine, Denton, Texas 76203and t Department of Pharmacology, University of Utah College of Medicine, Salt Lake City, Utah 84132 Received April 5, 1978; revised June 15, 1978 The effects of sulfhydryl inhibitors (iodoacetamide and N-ethylmaleimide) on the electron spin resonance spectra of two maleimide and two iodoacetamide spin labels in erythrocyte ghosts were found to correlate with their relative “lipid”/water partition coefficients. But the spectral characteristics of the maleirnide spin labels, and their ghost concentrations after iodoacetamide inhibition, are not consistent with the hypothesis that interprets their spectra solely on the basis of a heterogenous membrane distribution. An alternative hypothesis is suggested which is compatible with relative “lipid solubilities” and the iodoacetamide inhibition spectra.
A current hypothesis concerning the heterogenous esr spectrum of Mal-SL’ in red blood cell ghosts regards the “sharp-line” component as arising from partially immobilized Mal-SL bound to sulfhydryl groups near the membrane surface, and the broadline component due to strongly immobilized Mal-SL bound to sulfhydryls in the interior. Furthermore, the simple “sharp-line” spectrum of IA-SL is presumed the result of its reaction with surface sulfhydryls only (1, 2). However, in ghosts preincubated with IA (non-spin labeled), washed, and labeled with Mal-SL, a greater inhibition of MalSL incorporation occurred than could be accounted for by inhibition of surface sulfhydryl groups only; i.e., the broad-line component was substantially depressed. Thus, the sulfhydryls labeled by Mal-SL and IA-SL in ghosts are probably qualitatively more similar than has formerly been recognized. This preliminary observation
and the additional work reported herein form the basis of an alternative hypothesis that does not invoke a dissimilar topographical distribution of spin labels for the interpretation of their esr spectra. MATERIALS
AND
METHODS
The four protein-reactive spin labels (purchased from Syva Corp.) employed in the present study include, in addition to Mal-SL and IA-SL, Mal-CH2-SL and IA-CH,-SL. Erythrocyte ghosts were prepared from freshly acquired human blood by the method of Dodge et al. (3) and were used on the day of their preparation. The protein concentrations (4) of the ghost suspensions were adjusted to about 3 mg/ml (-4.5 x lo9 ghosts/ml). Two-milliliter aliquots of the protein-adjusted ghost suspensions were added to 20 ml of 10 mM NEM or IA in 50-n-d Erlenmeyer flasks. The NEM and IA solutions were made in the same buffer (PB) employed in the ghost preparation. The flasks were shaken on an orbital shaker (150 rpm) at room temperature for 4 h. Unreacted NEM (or IA) was removed by washing the ghosts four times in PB (27,000 g, 10 min). Two-milliliter aliquots of the protein-adjusted ghost suspensions were also added to 20 ml PB and processed in the above fashion simultaneously with those added to NEM or IA. After washing, the ghost suspensions were adjusted to about 2 mg/ml. Ethanolic solutions of the above spin labels were evaporated in separate 5-ml Erlenmeyer flasks with ground-glass joints for connection to a rotary evaporator. The protein-adjusted ghost
] Abbreviations used: Mal-SL, 4-maleimido-2,2,6,6tetramethylpiperidinooxyl; MaI-CH&SL, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-l-pyrrolidinyloxyl; IASL, 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl; IA-CHz-SL, 3-[(2-iodoacetamido)methyl]-2,2,5,5-tetramethyl-l-pyrrolidinyloxyl; IA, iodoacetamide; NEM, N-ethylmaleimide; TEMPO, 2,2,6,6-tetramethylpiperidine-I-oxyk PB, 20 m&m phosphate buffer, pH 7.4. 611
0003-9861/78/1902-0611$02.00/O Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.
612
JUNL;s
ANlJ
0 Md-CH@
IA-CH2-Y
suspensions were added and incubation was conducted for at least 1 hat room temperature with gentle orbital motion. The spin-label concentration was 0.4 mM (Mal-SL, Mal-CHZ-SL) or 1.4 mM (IA-SL, IA-CH2SL). Unreacted spin label was removed by washing the ghosts four times in the manner already described. Protein concentrations were determined, and bound spin-label concentration was estimated by digesting aliquots of labeled suspension in 0.1 N NaOH for 6 h at room temperature. The resultant amplitude of the low-field esr line was compared to a standard curve constructed from known concentrations (h, = 243ma, E = 1870 &cm-‘, methanol; Ref. 5) of TEMPO. “Lipid”/water partition coefficients were estimated by permitting each compound to equilibrate between PB and n-butyl chloride. After rapid orbital shaking
(1 h, room temperature), the solvent phases were resolved and the concentrations in each were determined by comparison with standard curves for the respective solvent. Measured spectral parameters (Gary model 118C) were: h,, = 295rng, E = 733K’cm-’ for NEM in n-butyl chloride; h,, = 301.5 ny~, e = 619 M-‘cm-’ for NEM in PB; h,, = 259 rnp, E = 485 K’cm-’ for IA in n-butyl chloride; X,, = 266.5 rnp, l = 398 K’cm-1 for IA in PB. Spin-label concentration was determined by comparing the spectral intensity of the deoxygenated (to minimize oxygen broadening) sample with the TEMPO curve in the respective solvent. esr spectra were recorded in capillary tubes at ambient temperature in a Varian E-4 spectrometer. Instrumental parameters were optimized to avoid artifactual broadening. RESULTS
AND
DISCUSSION
An inhibition of Mal-SL binding to ghost membranes is depicted in Fig. 1. Pretreatment with 10 mM IA clearly reduced binding, but 10 mM NEM was significantly more effective. Double integration of the spectra yielded results compatible with the concentration data presented in Table I; IA reduced binding to about 28% of control,
I
I 10 Gauss
FIG. 1. esr spectra of erythrocyte ghosts spin labeled with Mal-SL, either with or without pretreatment with NEM or IA. PI and SI refer to the partially immobilized and strongly immobilized components, respectively, of the heterogenous esr spectra.
PROTEIN TABLE
SPIN-LABEL
SPECTRA
I
GHOST SPIN-LABEL CONCENTRATIONS UNDER SELECTED CONDITIONS~ IA No NEM preprepretreatmen treattreatmerit* ment Mal-SL Mal-CHs-SL IA-SL IA-CHz-SL
5.08 9.55 3.61 4.18
0.30 0.63 0.23 0.48
(6) (7) (6) (12)
1.40 4.34 0.41 0.60
(28) (45) (11) (14)
a Concentrations are expressed as nanomoles spin label per mg ghost protein. Data represent the average of measurements from duplicate experiments Figures in parentheses represent the percentage of the control (no pretreatment) value. * Pretreatment involved shaking erythrocyte ghosts at room temperature in 10 mru NEM or 10 mM IA. Abbreviations are those cited in the text.
whereas binding after NEM incubation was only 6% of control. The greater inhibition of binding by NEM is probably due simply to its relative “lipid solubility,” which more closely approximates that for Mal-SL than does IA (Table II). The inhibition of MalCH2-SL binding by pretreatment with IA and NEM is similar to that for Mal-SL (Fig. 2); IA inhibition again was not as complete as that for NEM, presumably due to the relatively greater “lipid solubility” of NEM (Table I). The same relationship was demonstrated for IA-SL (Fig. 3 and Table I) and for IA-CH2-SL (Table I). However, the difference in extent of inhibition by IA and NEM was in each case less than for the maleimide spin-label derivatives (the lower “lipid solubility” of the iodoacetamide spin labels likely enhances their susceptability to inhibition by IA). Thus, the horizontal comparison of data in Table I demonstrates a probable role of lipid solubility in determining the extent of membrane sulfhydryl inhibition. A vertical comparison likewise demonstrates this relationship. The control MalCH*-SL concentration (9.55 nmol/mg protein) was about 1.9 times that for Mal-SL (5.08 nmol/mg protein); and this approximates their ratio of partition coefficients (7.52/4.65 = 1.6; see Table II). The spin label IA-SL is less “lipid soluble” than is Mal-SL or Mal-CH2-SL, and the ghost concentration is correspondingly lower (even though the reaction concentration was sev-
IN ERYTHROCYTE
613
GHOSTS
eral times that for the maleimide derivatives). However, the data for IA-CHPSL are inconsistent with the above relationships. The membrane spin-label concentration was that expected from a relatively greater lipid solubility, but the measured partition coefficient was lower than that for IA-SL (Table II). A possible explanation for this inconsistency is that hydrogen bonding between water and the sterically less hindered amide hydrogen in IA-CH2SL perturbs its solubility relationship with IA-SL, which might otherwise be expected to parallel the comparison for Mal-SL and Mal-CH2-SL. Although the relatively greater IA-CH2-SL concentration in ghosts might demonstrate the lack of an ideal solvent model for lipid solubility, the same approximate relationship was observed when several other organic solvents were employed (e.g., octanol, hexane, and benzene). The above data are generally compatible with the notion that the more lipid-soluble spin labels react more extensively with membrane sulfhydryl groups (probably due to greater penetration of hydrophobic regions), and they are consistent with the current hypothesis already described. But the spectral characteristics depicted in Figs. 1 and 2 are not compatible with that hypothesis. Although IA inhibited spin-label binding to a degree consistent with relative lipid solubilities, the broad-line component was significantly depressed. This finding would not be expected if the broad-line component was indeed due to a selective reaction with a discrete class of sulfhydryl functions in the membrane interior. In the present study, the strongly immobilized (broad-line) Mal-SL population was at least TABLE
II
SUMMARY OF PARTITION COEFFICIENTS~ NEM* IA Mal-SL Mal-CHz-SL IA-SL IA-CHz-SL
8.49 0.014 4.65 7.52 1.64 0.54
+ * f f + +
0.02 0.003 0.40 0.80 0.16 0.06
a Partition coefficients were estimated using the nbutyl chloride/aqueous system described in the text. Data represent the means (k standard deviation) of measurements from four to five experiments. b Abbreviations are those cited in the text.
614
JONES
AND
WOODBURY
0
CONTROL
NEM
4 IA
I
I
10 Gauss
FIG. 2. esr spectra of erythrocyte ghosts spin labeled with Mal-CHZ-SL, either with or without pretreatment with NEM or IA. SI is not resolved from PI in these spectra.
30-fold greater than the partially immobilized (“sharp-line”) population. (The concentrations estimated from first derivative esr spectra are proportional to the square of the line width times the peak height.) According to the current hypothesis, an inhibitor blocking only surface sites should produce a considerable reduction of the “sharp-line” component without a substantial effect on the broad-line; and such an effect could not result in the significant (55%) reduction of total spin-label concentration observed in the present experiments. There is an approximate 16-22% relative decrease in the patially-immobilized population by IA pretreatment, but this still could not account for the substantial reduction of total spin-label concentration. Although transverse membrane sulfhydryl migration might permit exposure of “interior” functions to IA during the course of incubation, this cannot explain
the significant inhibition of Mal-SL incorporation and be in accord with the current hypothesis. Otherwise, IA-SL and IA-CH2SL should react with such functions and yield spectra similar to those for Mal-SL and Mal-CHz-SL. An alternative hypothesis grants that Mal-SL and Mal-CHZ-SL bind more extensively to membrane sulfhydryl groups because of their greater lipid solubility (hence, access to the more hydrophobic membrane interior), but a heterogenous spectrum is obtained regardless of their topographical distribution. The very low partition coefficient of IA likely dictates that only the sulfhydryl groups near the membraneaqueous interface are blocked by this compound. Thus, about 55% of the Mal-SL reactive sulfhydryl groups appear to be surface functions, but Mal-SL at these sites must also generate a complex spectrum. Likewise, IA-SL and IA-CH&L (which
PROTEIN
SPIN-LABEL
SPECTRA
FIG. 3.
IN ERYTHROCYTE
10 Gauss
esr spectra of erythrocyte ghosts spin labeled with IA-SL, either pretreatment with NEM or IA. Only PI peaks are apparent in these spectra.
should partition more in the membrane interior than IA) must generate a simple (partially immobilized) spectrum regardless of their location. There does appear to be a limited (16-22s) selective inhibition of the partially immobilized component by IA, but the spectra cannot be interpreted solely on the basis of this action. The iodoacetamide spin labels likely bind for the most part with sulfhydryls identical to those bound by the maleimide spin-labels, but to a degree consistent with relative lipid solubilities. The different qualitative nature of the iodoacetamide spin-label spectra might, therefore, result from qualitatively different interactions with identical environments. A speculative explanation for such a phenomenon is depicted in Fig. 4. Inasmuch as imidazole is known to catalyze the hydrolysis (ring opening) of NEM (6), the possibility that histidine adjacent to a bound sulfhydryl might catalyze the hydrolysis of Mal-SL (and Mal-CH2-SL) must be considered. Such intramolecular catalysis has already been proposed for hemoglobin (7). Furthermore, the presence of an asymmetric carbon on the succinimide ring following sulfhydryl addition suggests the possibility of six diastereoisomeric complexes (two for
615
GHOSTS
with
or without
0NH i N-o b-WLH* Ao;c 8 G N-O sNH I--CH*-Ccb -R--5--cnyc-N” FIG. 4. Hypothetical course of spin-labeling reactions with erythrocyte ghost protein. Alternative MalSL hydrolysis schemes (A and B) depict products whose nitroxide spatial relationships with protein (Pr) differ. Product B is similar to that expected for the IASL reaction product.
each hydrolysis product and two for the unhydrolyzed addition product), each of which might interact differently with the same environment. By comparison with the probable protein-bound IA-SL product (which gives a “sharp-line” spectrum similar to the partially immobilized component of Mal-SL), the hydrolytic mechanism A is likely responsible for the broad-line com-
616
JONES
AND
ponent. Although this is not intuitively apparent (because frequently the farther removed the nitroxide moiety is from its binding site the greater rotational freedom is permitted), the relationship of the carbonyl oxygen to the protein might be significant in terms of a potentially stabilizing hydrogen-bonding interaction; or the amide hydrogen might be in a more favorable position for intramolecular hydrogen bonding. An alternative mechanism which cannot be ignored involves the possible nucleophilic attack of an amino or sulfhydryl group on one of the maleimide carbonyl groups (8). After ring opening the distance from the nitroxide to its protein binding site would be greater than that achieved by hydrolytic mechanisms A or B in Fig. 4, and this might be the source of the “sharpline” component. Although insignificant binding of Mal-SL to amino groups has been reported by others employing similar reaction conditions in ghosts (1, 2), a nucleophilic attack by sulfhydryl groups cannot presently be excluded. Barratt et al. (9) have demonstrated the potential for contamination of 3-maleimido - 2,2,5,5- tetramethyl- 1- pyrrolidinyloxyl (the pyrrolidinyl nitroxide counterpart of Mal-SL) with its isomaleimide isomer if reaction conditions are not rigorously controlled. Although their work did not involve Mal-SL, caution is nevertheless advised because the present authors have separated four additional products from Mal-SL supplied commercially as well as that prepared locally. The identity of the contaminants
WOODBURY
wasn’t established, but their reaction with ghosts yielded a simple “sharp-line” spectrum in each case. The contaminants were resolved from Mal-SL with thin-layer chromatography on alumina gel plates in chloroform. Alumina gel was judged to be superior to silica gel because only two contaminants were resolved using the latter. ACKNOWLEDGMENTS The technical help of Mrs. Mary C. Jones is gratefully acknowledged. This work wassupportedin part by each of the following: U. S. Public Health Service Pharmacology Training Grant GM-00153; PMA Foundation Research Starter Grant; Utah Heart Association Grant-in Aid Award; and University of Utah College of Medicine Faculty Research Award. REFERENCES 1. SANDBERG, H. E., AND PIETTE, L. H. (1968) Agressologie 9, 59-67. 2. HOLMES, D. E., AND PIETTE, L. H. (1970) J, Pharmacol. Exp. Ther. 173,78-a. 3. DoDGE,J.T.,MITcHELL,C., AND HANAHAN,D.J. (1963) Arch. B&hem. Biophys. 100, 119-130. 4. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL,R.J. (1951)J. Btil. Chem. 193,
265-275. 5. BRIERE, R., LEMAIRE, H., AND RASSAT, A. (1965) Bull. Sot. Chim. Fr. 11,3273-3283. 6. BRUICE, T. C., AND STURTEVANT, J. M. (1958)
Biochim. Biophys. Acta 30,208-209. 7. BENESCH, R., AND BENESCH, R. E. (1961) J. Bid.
Chem. 236,405-410. 8. MORRISETT, J. D. (976) in Spin Labeling, Theory and Applications (Berliner, L. J., ed.), p. 277, Academic Press, New York. 9. BARRATT, M. D., DAVIES, A. P., AND EVANS, M. T. A. (1971) Eur. J. Biochem. 24.280-283.
OF BIOCHEMISTRY
AND
BIOPHYSICS
Vol. 190, No. 2, October, 611-616, 1978
Reappraisal
of the Electron Spin Resonance Spectra of Maleimide lodoacetamide Spin Labels in Erythrocyte Ghosts GARY
L. JONES*
AND
DIXON
and
M. WOODBURYt
* Department of Pharmacology, North Texas State Uniuersity, Texas College of Osteopathic Medicine, Denton, Texas 76203and t Department of Pharmacology, University of Utah College of Medicine, Salt Lake City, Utah 84132 Received April 5, 1978; revised June 15, 1978 The effects of sulfhydryl inhibitors (iodoacetamide and N-ethylmaleimide) on the electron spin resonance spectra of two maleimide and two iodoacetamide spin labels in erythrocyte ghosts were found to correlate with their relative “lipid”/water partition coefficients. But the spectral characteristics of the maleirnide spin labels, and their ghost concentrations after iodoacetamide inhibition, are not consistent with the hypothesis that interprets their spectra solely on the basis of a heterogenous membrane distribution. An alternative hypothesis is suggested which is compatible with relative “lipid solubilities” and the iodoacetamide inhibition spectra.
A current hypothesis concerning the heterogenous esr spectrum of Mal-SL’ in red blood cell ghosts regards the “sharp-line” component as arising from partially immobilized Mal-SL bound to sulfhydryl groups near the membrane surface, and the broadline component due to strongly immobilized Mal-SL bound to sulfhydryls in the interior. Furthermore, the simple “sharp-line” spectrum of IA-SL is presumed the result of its reaction with surface sulfhydryls only (1, 2). However, in ghosts preincubated with IA (non-spin labeled), washed, and labeled with Mal-SL, a greater inhibition of MalSL incorporation occurred than could be accounted for by inhibition of surface sulfhydryl groups only; i.e., the broad-line component was substantially depressed. Thus, the sulfhydryls labeled by Mal-SL and IA-SL in ghosts are probably qualitatively more similar than has formerly been recognized. This preliminary observation
and the additional work reported herein form the basis of an alternative hypothesis that does not invoke a dissimilar topographical distribution of spin labels for the interpretation of their esr spectra. MATERIALS
AND
METHODS
The four protein-reactive spin labels (purchased from Syva Corp.) employed in the present study include, in addition to Mal-SL and IA-SL, Mal-CH2-SL and IA-CH,-SL. Erythrocyte ghosts were prepared from freshly acquired human blood by the method of Dodge et al. (3) and were used on the day of their preparation. The protein concentrations (4) of the ghost suspensions were adjusted to about 3 mg/ml (-4.5 x lo9 ghosts/ml). Two-milliliter aliquots of the protein-adjusted ghost suspensions were added to 20 ml of 10 mM NEM or IA in 50-n-d Erlenmeyer flasks. The NEM and IA solutions were made in the same buffer (PB) employed in the ghost preparation. The flasks were shaken on an orbital shaker (150 rpm) at room temperature for 4 h. Unreacted NEM (or IA) was removed by washing the ghosts four times in PB (27,000 g, 10 min). Two-milliliter aliquots of the protein-adjusted ghost suspensions were also added to 20 ml PB and processed in the above fashion simultaneously with those added to NEM or IA. After washing, the ghost suspensions were adjusted to about 2 mg/ml. Ethanolic solutions of the above spin labels were evaporated in separate 5-ml Erlenmeyer flasks with ground-glass joints for connection to a rotary evaporator. The protein-adjusted ghost
] Abbreviations used: Mal-SL, 4-maleimido-2,2,6,6tetramethylpiperidinooxyl; MaI-CH&SL, 3-(maleimidomethyl)-2,2,5,5-tetramethyl-l-pyrrolidinyloxyl; IASL, 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl; IA-CHz-SL, 3-[(2-iodoacetamido)methyl]-2,2,5,5-tetramethyl-l-pyrrolidinyloxyl; IA, iodoacetamide; NEM, N-ethylmaleimide; TEMPO, 2,2,6,6-tetramethylpiperidine-I-oxyk PB, 20 m&m phosphate buffer, pH 7.4. 611
0003-9861/78/1902-0611$02.00/O Copyright 0 1978 by Academic Press, Inc. AU rights of reproduction in any form reserved.
612
JUNL;s
ANlJ
0 Md-CH@
IA-CH2-Y
suspensions were added and incubation was conducted for at least 1 hat room temperature with gentle orbital motion. The spin-label concentration was 0.4 mM (Mal-SL, Mal-CHZ-SL) or 1.4 mM (IA-SL, IA-CH2SL). Unreacted spin label was removed by washing the ghosts four times in the manner already described. Protein concentrations were determined, and bound spin-label concentration was estimated by digesting aliquots of labeled suspension in 0.1 N NaOH for 6 h at room temperature. The resultant amplitude of the low-field esr line was compared to a standard curve constructed from known concentrations (h, = 243ma, E = 1870 &cm-‘, methanol; Ref. 5) of TEMPO. “Lipid”/water partition coefficients were estimated by permitting each compound to equilibrate between PB and n-butyl chloride. After rapid orbital shaking
(1 h, room temperature), the solvent phases were resolved and the concentrations in each were determined by comparison with standard curves for the respective solvent. Measured spectral parameters (Gary model 118C) were: h,, = 295rng, E = 733K’cm-’ for NEM in n-butyl chloride; h,, = 301.5 ny~, e = 619 M-‘cm-’ for NEM in PB; h,, = 259 rnp, E = 485 K’cm-’ for IA in n-butyl chloride; X,, = 266.5 rnp, l = 398 K’cm-1 for IA in PB. Spin-label concentration was determined by comparing the spectral intensity of the deoxygenated (to minimize oxygen broadening) sample with the TEMPO curve in the respective solvent. esr spectra were recorded in capillary tubes at ambient temperature in a Varian E-4 spectrometer. Instrumental parameters were optimized to avoid artifactual broadening. RESULTS
AND
DISCUSSION
An inhibition of Mal-SL binding to ghost membranes is depicted in Fig. 1. Pretreatment with 10 mM IA clearly reduced binding, but 10 mM NEM was significantly more effective. Double integration of the spectra yielded results compatible with the concentration data presented in Table I; IA reduced binding to about 28% of control,
I
I 10 Gauss
FIG. 1. esr spectra of erythrocyte ghosts spin labeled with Mal-SL, either with or without pretreatment with NEM or IA. PI and SI refer to the partially immobilized and strongly immobilized components, respectively, of the heterogenous esr spectra.
PROTEIN TABLE
SPIN-LABEL
SPECTRA
I
GHOST SPIN-LABEL CONCENTRATIONS UNDER SELECTED CONDITIONS~ IA No NEM preprepretreatmen treattreatmerit* ment Mal-SL Mal-CHs-SL IA-SL IA-CHz-SL
5.08 9.55 3.61 4.18
0.30 0.63 0.23 0.48
(6) (7) (6) (12)
1.40 4.34 0.41 0.60
(28) (45) (11) (14)
a Concentrations are expressed as nanomoles spin label per mg ghost protein. Data represent the average of measurements from duplicate experiments Figures in parentheses represent the percentage of the control (no pretreatment) value. * Pretreatment involved shaking erythrocyte ghosts at room temperature in 10 mru NEM or 10 mM IA. Abbreviations are those cited in the text.
whereas binding after NEM incubation was only 6% of control. The greater inhibition of binding by NEM is probably due simply to its relative “lipid solubility,” which more closely approximates that for Mal-SL than does IA (Table II). The inhibition of MalCH2-SL binding by pretreatment with IA and NEM is similar to that for Mal-SL (Fig. 2); IA inhibition again was not as complete as that for NEM, presumably due to the relatively greater “lipid solubility” of NEM (Table I). The same relationship was demonstrated for IA-SL (Fig. 3 and Table I) and for IA-CH2-SL (Table I). However, the difference in extent of inhibition by IA and NEM was in each case less than for the maleimide spin-label derivatives (the lower “lipid solubility” of the iodoacetamide spin labels likely enhances their susceptability to inhibition by IA). Thus, the horizontal comparison of data in Table I demonstrates a probable role of lipid solubility in determining the extent of membrane sulfhydryl inhibition. A vertical comparison likewise demonstrates this relationship. The control MalCH*-SL concentration (9.55 nmol/mg protein) was about 1.9 times that for Mal-SL (5.08 nmol/mg protein); and this approximates their ratio of partition coefficients (7.52/4.65 = 1.6; see Table II). The spin label IA-SL is less “lipid soluble” than is Mal-SL or Mal-CH2-SL, and the ghost concentration is correspondingly lower (even though the reaction concentration was sev-
IN ERYTHROCYTE
613
GHOSTS
eral times that for the maleimide derivatives). However, the data for IA-CHPSL are inconsistent with the above relationships. The membrane spin-label concentration was that expected from a relatively greater lipid solubility, but the measured partition coefficient was lower than that for IA-SL (Table II). A possible explanation for this inconsistency is that hydrogen bonding between water and the sterically less hindered amide hydrogen in IA-CH2SL perturbs its solubility relationship with IA-SL, which might otherwise be expected to parallel the comparison for Mal-SL and Mal-CH2-SL. Although the relatively greater IA-CH2-SL concentration in ghosts might demonstrate the lack of an ideal solvent model for lipid solubility, the same approximate relationship was observed when several other organic solvents were employed (e.g., octanol, hexane, and benzene). The above data are generally compatible with the notion that the more lipid-soluble spin labels react more extensively with membrane sulfhydryl groups (probably due to greater penetration of hydrophobic regions), and they are consistent with the current hypothesis already described. But the spectral characteristics depicted in Figs. 1 and 2 are not compatible with that hypothesis. Although IA inhibited spin-label binding to a degree consistent with relative lipid solubilities, the broad-line component was significantly depressed. This finding would not be expected if the broad-line component was indeed due to a selective reaction with a discrete class of sulfhydryl functions in the membrane interior. In the present study, the strongly immobilized (broad-line) Mal-SL population was at least TABLE
II
SUMMARY OF PARTITION COEFFICIENTS~ NEM* IA Mal-SL Mal-CHz-SL IA-SL IA-CHz-SL
8.49 0.014 4.65 7.52 1.64 0.54
+ * f f + +
0.02 0.003 0.40 0.80 0.16 0.06
a Partition coefficients were estimated using the nbutyl chloride/aqueous system described in the text. Data represent the means (k standard deviation) of measurements from four to five experiments. b Abbreviations are those cited in the text.
614
JONES
AND
WOODBURY
0
CONTROL
NEM
4 IA
I
I
10 Gauss
FIG. 2. esr spectra of erythrocyte ghosts spin labeled with Mal-CHZ-SL, either with or without pretreatment with NEM or IA. SI is not resolved from PI in these spectra.
30-fold greater than the partially immobilized (“sharp-line”) population. (The concentrations estimated from first derivative esr spectra are proportional to the square of the line width times the peak height.) According to the current hypothesis, an inhibitor blocking only surface sites should produce a considerable reduction of the “sharp-line” component without a substantial effect on the broad-line; and such an effect could not result in the significant (55%) reduction of total spin-label concentration observed in the present experiments. There is an approximate 16-22% relative decrease in the patially-immobilized population by IA pretreatment, but this still could not account for the substantial reduction of total spin-label concentration. Although transverse membrane sulfhydryl migration might permit exposure of “interior” functions to IA during the course of incubation, this cannot explain
the significant inhibition of Mal-SL incorporation and be in accord with the current hypothesis. Otherwise, IA-SL and IA-CH2SL should react with such functions and yield spectra similar to those for Mal-SL and Mal-CHz-SL. An alternative hypothesis grants that Mal-SL and Mal-CHZ-SL bind more extensively to membrane sulfhydryl groups because of their greater lipid solubility (hence, access to the more hydrophobic membrane interior), but a heterogenous spectrum is obtained regardless of their topographical distribution. The very low partition coefficient of IA likely dictates that only the sulfhydryl groups near the membraneaqueous interface are blocked by this compound. Thus, about 55% of the Mal-SL reactive sulfhydryl groups appear to be surface functions, but Mal-SL at these sites must also generate a complex spectrum. Likewise, IA-SL and IA-CH&L (which
PROTEIN
SPIN-LABEL
SPECTRA
FIG. 3.
IN ERYTHROCYTE
10 Gauss
esr spectra of erythrocyte ghosts spin labeled with IA-SL, either pretreatment with NEM or IA. Only PI peaks are apparent in these spectra.
should partition more in the membrane interior than IA) must generate a simple (partially immobilized) spectrum regardless of their location. There does appear to be a limited (16-22s) selective inhibition of the partially immobilized component by IA, but the spectra cannot be interpreted solely on the basis of this action. The iodoacetamide spin labels likely bind for the most part with sulfhydryls identical to those bound by the maleimide spin-labels, but to a degree consistent with relative lipid solubilities. The different qualitative nature of the iodoacetamide spin-label spectra might, therefore, result from qualitatively different interactions with identical environments. A speculative explanation for such a phenomenon is depicted in Fig. 4. Inasmuch as imidazole is known to catalyze the hydrolysis (ring opening) of NEM (6), the possibility that histidine adjacent to a bound sulfhydryl might catalyze the hydrolysis of Mal-SL (and Mal-CH2-SL) must be considered. Such intramolecular catalysis has already been proposed for hemoglobin (7). Furthermore, the presence of an asymmetric carbon on the succinimide ring following sulfhydryl addition suggests the possibility of six diastereoisomeric complexes (two for
615
GHOSTS
with
or without
0NH i N-o b-WLH* Ao;c 8 G N-O sNH I--CH*-Ccb -R--5--cnyc-N” FIG. 4. Hypothetical course of spin-labeling reactions with erythrocyte ghost protein. Alternative MalSL hydrolysis schemes (A and B) depict products whose nitroxide spatial relationships with protein (Pr) differ. Product B is similar to that expected for the IASL reaction product.
each hydrolysis product and two for the unhydrolyzed addition product), each of which might interact differently with the same environment. By comparison with the probable protein-bound IA-SL product (which gives a “sharp-line” spectrum similar to the partially immobilized component of Mal-SL), the hydrolytic mechanism A is likely responsible for the broad-line com-
616
JONES
AND
ponent. Although this is not intuitively apparent (because frequently the farther removed the nitroxide moiety is from its binding site the greater rotational freedom is permitted), the relationship of the carbonyl oxygen to the protein might be significant in terms of a potentially stabilizing hydrogen-bonding interaction; or the amide hydrogen might be in a more favorable position for intramolecular hydrogen bonding. An alternative mechanism which cannot be ignored involves the possible nucleophilic attack of an amino or sulfhydryl group on one of the maleimide carbonyl groups (8). After ring opening the distance from the nitroxide to its protein binding site would be greater than that achieved by hydrolytic mechanisms A or B in Fig. 4, and this might be the source of the “sharpline” component. Although insignificant binding of Mal-SL to amino groups has been reported by others employing similar reaction conditions in ghosts (1, 2), a nucleophilic attack by sulfhydryl groups cannot presently be excluded. Barratt et al. (9) have demonstrated the potential for contamination of 3-maleimido - 2,2,5,5- tetramethyl- 1- pyrrolidinyloxyl (the pyrrolidinyl nitroxide counterpart of Mal-SL) with its isomaleimide isomer if reaction conditions are not rigorously controlled. Although their work did not involve Mal-SL, caution is nevertheless advised because the present authors have separated four additional products from Mal-SL supplied commercially as well as that prepared locally. The identity of the contaminants
WOODBURY
wasn’t established, but their reaction with ghosts yielded a simple “sharp-line” spectrum in each case. The contaminants were resolved from Mal-SL with thin-layer chromatography on alumina gel plates in chloroform. Alumina gel was judged to be superior to silica gel because only two contaminants were resolved using the latter. ACKNOWLEDGMENTS The technical help of Mrs. Mary C. Jones is gratefully acknowledged. This work wassupportedin part by each of the following: U. S. Public Health Service Pharmacology Training Grant GM-00153; PMA Foundation Research Starter Grant; Utah Heart Association Grant-in Aid Award; and University of Utah College of Medicine Faculty Research Award. REFERENCES 1. SANDBERG, H. E., AND PIETTE, L. H. (1968) Agressologie 9, 59-67. 2. HOLMES, D. E., AND PIETTE, L. H. (1970) J, Pharmacol. Exp. Ther. 173,78-a. 3. DoDGE,J.T.,MITcHELL,C., AND HANAHAN,D.J. (1963) Arch. B&hem. Biophys. 100, 119-130. 4. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL,R.J. (1951)J. Btil. Chem. 193,
265-275. 5. BRIERE, R., LEMAIRE, H., AND RASSAT, A. (1965) Bull. Sot. Chim. Fr. 11,3273-3283. 6. BRUICE, T. C., AND STURTEVANT, J. M. (1958)
Biochim. Biophys. Acta 30,208-209. 7. BENESCH, R., AND BENESCH, R. E. (1961) J. Bid.
Chem. 236,405-410. 8. MORRISETT, J. D. (976) in Spin Labeling, Theory and Applications (Berliner, L. J., ed.), p. 277, Academic Press, New York. 9. BARRATT, M. D., DAVIES, A. P., AND EVANS, M. T. A. (1971) Eur. J. Biochem. 24.280-283.
