- Email: [email protected]
Structural and functional degradation of Ca2+:Mg2+-ATPase rich sarcoplasmic reticulum vesicles photosensitized by Erythrosin B
Chem.-Biol. Interactions, 41 (1982) 313--325 Elsevier Scientific Publishers Ireland Ltd.
313
STRUCTURAL AND FUNCTIONAL DEGRADATION O F Ca2÷:Mg ~+ATPase R I C H S A R C O P L A S M I C RETICULUM VESICLES P H O T O SENSITIZED B Y E R Y T H R O S I N B
B.D. WATSON and D.H. HAYNES*
Department of Pharmacology, University of Miami School of Medicine, Miami, FL 33101 (U.S.A.) (Received March 10th, 1981) (Revision received March 12th, 1982) (Accepted March 23rd, 1982)
SUMMARY
Erythrosin B (Red Dye No. 3) and Rose Bengal photosensitize the destruction of the Ca2+: Mg2+-ATPase p u m p protein in sarcoplasmic reticulum (SR) vesicles with respective quantum efficiencies of (1.53 + 0.19) × 10 -3 and (1.25 -+ 0.18) × 10 -3. Damage to vesicle function was assayed by measurements of increases in passive Ca 2÷ permeability. Rates of passive Ca 2÷ movement into the SR lumen were increased b y dye photosensitization in proportion to radiation absorbed. Active Ca 2÷ transport into SR vesicles was blocked independent of radiation absorbed by Erythrosin B and Rose Bengal at free concentrations of 0.69 gM and 1.16 #M, respectively. The photochemical lability of the Ca 2÷ pump protein and alterations in passive and active Ca 2÷ transport may be dependent on the concentration of the dye in the membrane. The photosensitization results may have implications with respect to the suitability of Erythrosin B usage in vivo, since the brightness of our irradiation source is comparable to that of sunlight at 480 nm.
INTRODUCTION
Several recent studies have stimulated interest in possible mechanisms of Erythrosin B toxicity in brain and nervous tissue. In particular, Erythrosin B was found to inhibit neurotransmitter accumulation in rat brain [1] and dopamine transport in caudal synaptosomes [2]. Proper quantitation of these results [1,2] was later found to depend on the tissue concentration in the assay medium [3]. In addition, Erythrosin B increased the perme*To whom correspondence should be sent. Abbreviations: ANS-, 1-anilino-8-naphthalene sulfonate; SDS, sodium dodecyl sulfate; SR, sarcoplasmic reticulum.
0009--2797/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishers Ireland Ltd.
314 ability of mollusc buccal ganglia to K ÷ ions [4] and, when incubated with frog neuromuscular synapses, caused an irreversible increase [5] in transmitter release, which was said to be independent of ambient light. Contrasting with the foregoing examples of intrinsic effects of Erythrosin B on membrane function, photosensitization by Erythrosin B, and by xanthene dyes in general, has been demonstrated to induce functional damage in membrane systems. For example, active Ca 2÷ uptake in sarcoplasmic reticulum vesicles was inhibited, an effect attributed to photooxidation of tryptophan [6] or of histidine residues [7] of the Ca 2÷ pump protein. Another study showed that sodium channels [8] were inactivated in lobster giant axons. In this communication we present evidence for structural and functional degradation of the Ca2÷: Mg2÷-ATPase transport enzyme [9] of sarcoplasmic reticulum, photochemically induced by Erythrosin B incubated with sarcoplasmic reticulum vesicles rich in the Ca2÷: Mg2+-ATPase. For comparison, parallel experiments were run using Rose Bengal, a well-characterized xanthene photosensitizer. Inhibition of active Ca 2÷ transport by b o t h dyes was also observed, in the absence of irradiation.
MATERIALS AND METHODS Rose Bengal was obtained from Professor M. Kasha and was purified by column chromatography. Erythrosin B was obtained from the National Aniline Division through Dr. R.C. Leif and was used as received. The Erythrosin B sample was shown b y paper electrophoresis to be at least 96% pure. SR vesicles from rabbit skeletal muscle were isolated from the Ca2+: Mg2+-ATPase rich band (10) and resuspended in 50 mM borate buffer (pH 7.4) at an initial concentration of 3.2 mg (Lowry protein)/ml. The concentrations of Rose Bengal and Erythrosin B were adjusted to ensure equal rates of p h o t o n absorption (equal optical densities) at 480 nm. The molar extinction coefficients at 480 nm for Rose Bengal and Erythrosin B in water are 7700 and 14 800 1/M cm, respectively. A 25-pl aliquot of either dye was added to the irradiation cell first, followed b y dilution to 525 pl with borate buffer containing sucrose at a concentration equiosmolar to that in the SR suspension. A 50-pl aliquot of the SR suspension was added next in relative darkness and the irradiation proceeded after 1 min equilibration. The final irradiation medium contained 70 mM sucrose, 50 mM boric acid, 1.5 mM sodium borate, 90 gM Erythrosin B (or separately 173 pM Rose Bengal) and 0.29 mg/ml SR vesicles. The pH was 7.40. Samples were irradiated at 23°C at timed intervals of 0, 2, 5, 10, 18 and 30 min and then withdrawn for analysis. Optical densities of the irradiated SR suspensions remained constant throughout. The irradiation source was an Eimac/Varian VIX-300 Xenon arc coupled
315 by a fused silica lens to an Oriel 7204 holographic grating monochromator. The m o n o c h r o m a t o r dispersion is 3.2 nm/mm. The effective irradiation bandwidth was 10 nm. A fused silica cell of optical path length 2 mm and mounted at the exit slit contained the vesicle suspensions, which were magnetically stirred during exposure. A Laser Precision Corporation broadband detector (model kT-4020G) operated at 50% d u t y cycle was used to monitor the transmitted power through the irradiated suspensions. The transmitted irradiance at 480 nm was 60% of the incident irradiance of 1.30 mW/cm 2. At the m o n o c h r o m a t o r exit slit, the brightness of the irradiation source was calculated to be 0.33 mW/cm 2 nm. Photochemical damage to sarcoplasmic reticulum Ca2+: Mg2+-ATPase was assessed b y a structural test (SDS-polyacrylamide gel electrophoresis) and t w o functional tests (active and passive transport of Ca 2+) using 1anilino-8-naphthalene sulfonate (ANS-) as indicator [10]. In the onedimensional gel experiments, an acrylamide concentration of 8% was used. Approximately 0.1--0.4 pg protein was loaded per gel channel and bands stained with Coomassie Blue were recorded with a Gilson gel scanner and analyzed b y planimetry. Linearity of response was calibrated with bovine serum albumin. The Ca 2÷ passive transport assay [10] was used first to test functional integrity of the sarcoplasmic reticulum membranes. The use of an AmincoMorrow stopped-flow apparatus for this application has been described [10]. Concentrations of solution components were identical to those mentioned above, except that of Rose Bengal. Passive transport of Ca 2÷ was blocked in samples irradiated in the presence of 173 pM Rose Bengal. Reducing the Rose Bengal concentration to 17.3 pM allowed changes in passive transport rates of Ca 2÷ to be observed as a function of irradiation absorbed. Final concentrations of components in this assay were: CaC12, 15 mM; sucrose, 70 mM; boric acid, 50 mM; sodium borate, 1.5 mM; ANS-, 32 pM; valinomycin, 6 ~M; Rose Bengal, 0.86 pM; Erythrosin B, 4.5 pM; SR vesicles, 0.015 mg/ml. The active uptake assay [11,12] was also conducted on the AmincoMorrow apparatus. Final concentrations of components in this assay were: CaC12, 0.7 mM; EGTA, 1 mM; Tris, 30 mM: HEPES, 10 mM (pH 6.5); sucrose, 0.6 M; KC1, 0.1 M; MgC12, 0.6 mM; ATP, 0.5 mM; ANS-, 10 pM. valinomycin, 6 ~M; SR vesicles, 0.05 mg/ml. Dye concentrations tested were 1.73 gM and 17.3 #M for Rose Bengal, and 0.9 pM and 9.0 pM for Erythrosin B. Partitioning of the dyes in the SR membranes was studied by incubating the vesicles and dyes in darkness for times equivalent to the irradiation times, and then filtering the suspensions through a 0.22-pm millipore filter. Percentage of dye associated with membrane was deduced from optical absorbance measurements of filtrates of SR vesicles mixed with dye, compared to absorbancies o f filtrates of dye solutions not containing SR vesicles. The effect of attenuation of ANS- fluorescence caused by dye absorbance was also considered. For the experimental conditions used [10--12] in the
316 active and passive Ca 2÷ uptake experiments, the ANS- signal was attenuated less than 15% by Erythrosin B and less than 20% b y Rose Bengal. RESULTS
Figure 1 shows that irradiation in the presence of Erythrosin B causes an increase in passive Ca 2÷ permeability o f SR. Figure 1A is a control experi-
r'.l!
Control B
30 min. Fig. 1. I n f l u e n c e o f i r r a d i a t i o n o n t h e t i m e c o u r s e o f passive Ca 2+ e q u i l i b r a t i o n across t h e S R m e m b r a n e s . Ca 2÷ i n f l u x i n t o t h e SR vesicle is m e a s u r e d b y a n increase in A N S f l u o r e s c e n c e (Y axis, 0.5 V / d i v i s i o n ) vs t i m e a f t e r m i x i n g o f Ca 2÷ w i t h S R ( X axis, 5 s/division). O t h e r details o f t h e assay are given in Materials a n d M e t h o d s . A : s h o w s t h e b e h a v i o r of t h e c o n t r o l sample (1 rain p r e i n c u b a t i o n f o l l o w e d b y 30 m i n i r r a d i a t i o n a t 4 8 0 n m in a b s e n c e of E r y t h r o s i n B). A t,/2 o f 75 s is observed. B: s h o w s t h e e f f e c t o f 30 rain i r r a d i a t i o n in t h e p r e s e n c e o f 90 pM E r y t h r o s i n B. T h e t,i 2 is r e d u c e d t o 8 s. T h e i r r a d i a t i o n i n t e n s i t y was 1.30 m W / c m 2.
317 TABLE I DEPENDENCE
O F tl/2 A N D ATPase B A N D I N T E N S I T Y ON I R R A D I A T I O N T I M E Dye added
Irradiation time (min) 0
2
5
10
18
30
60
59.0
k × 10 -2 (s -1)
EB RB a None
1.3 1.1 1.2
1.1 0.9
1.1 1.5
4.3 4.3
6.2 8.3
11.1 14.3 1.2
ATPase b a n d Intensity (area, c m 2)
EB RB a None
33.6 28.3 38.7
35.1 24.9 43.5
29.3 19.5 33.0
28.0 20.3 37.4
19.8 18.5 37.1
16.3 14.1 42.6
Full w i d t h (tool. wt.) At Half-maximum
EB RB a None
3100 3100 3010
3440 2750 3220
3270 1920 2800
3440 3100 3220
4000 3780 3220
4130 4130 3440
aThis assay was r u n a t a Rose Bengal c o n c e n t r a t i o n of 17.3 uM. N o r m a l c o n c e n t r a t i o n s are given in Materials a n d M e t h o d s .
men t showing the progress of passive Ca 2+ influx in untreated SR. An increase in ANS- fluorescence (owing to increased binding to the m em brane inner surface) is observed when Ca 2+ enters the sarcoplasmic reticulum vesicle lumen and shields surface charge on the inner membrane. From Figure 1A the time for half-maximal response (tl/2) in the absence of 480 nm irradiation is seen to be 75 s. Figure 1B depicts the shortening o f response time t ha t occurs when vesicles are incubated in the presence of Erythrosin B and are exposed to 30 min of irradiation, corresponding to (1.98 + 0.11) × 10 is phot ons absorbed at 480 nm. The t i n for this case has decreased to 8 s. The rates of passive Ca 2÷ transport as a f u n c t i o n of exposure time are given in Table I. Progressive increases in rates of passive Ca 2÷ transport into the SR vesicles are photochemically facilitated by b o t h Erythrosin B and Rose Bengal. No increases in transport rates were observed following timed incubations of SR vesicles in darkness with either dye. The effect of Erythrosin B and Rose Bengal on active Ca 2. transport was studied. Using dye concentrations described in Materials and Methods, it was found that active Ca 2÷ transport was com pl et el y inhibited, regardless o f irradiation fluence (including zero fluence). Therefore, attempts to use active transport of Ca 2÷ to test for p h o t o c h e m i c a l activity o f Erythrosin B and Rose Bengal were n o t pursued further. Dye-sensitized damage to protein was studied by assaying the a m o u n t of Ca2÷: Mg2+-ATPase remaining as a function o f irradiation time. The results are compiled in Table I in terms of peak area as observed by planimetric integration o f SDS-gel electrophoretograms. Table I shows t h a t the a m o u n t o f Ca 2÷ p u m p protein is observed t o decrease as a function of irradiation exposure for samples incubated with Erythrosin B as well as
318 Rose Bengal. Plots of integrated intensity versus irradiation absorbed over a 30-min period were characterized with straight line fits. The average losses of the Ca 2÷ p u m p protein due to 30-min periods of photosensitization by Erythrosin B or b y Rose Bengal were 60% and 46%, respectively. The possibility was also examined that loss of Ca 2÷ pump protein was artifactual, owing to inhibition of staining dye binding by the photosensitizing dyes. This is unlikely since the Erythrosin B and Rose Bengal effects were irradiation-dependent and since both dyes co-migrated with the tracking dye in the SDS gels. The fraction of dye concentration associated with the SR vesicles was determined b y millipore filtration. Referring to solution compositions in Materials and Methods, this fraction was 0.24 for Erythrosin B and 0.33 for Rose Bengal under conditions for active Ca 2÷ uptake. Under conditions for passive Ca 2÷ transport following irradiation, and during irradiation itself, this fraction was 0.17 for Erythrosin B and 0.16 for Rose Bengal. The binding fractions enable calculation of the quantum efficiencies for photodestruction for Erythrosin B and Rose Bengal, assuming that the loss of Ca 2÷ pump protein is solely mediated by dye associated with sarcoplasmic membranes. These quantum efficiencies are (1.53 + 0.19) × 10 -3 and (1.25 + 0.18) × 10 -3 for Erythrosin B and Rose Bengal, respectively. A
FWHM 26
,,
._~_~
B FWHM
Fig. 9.. 8DS-PAGE gel profiles of the Ca ~÷ pump protein from SR vesicles (0.3 mg/ml) irradiated at 480 nm in the presence of Erythrosin B (90 ~M) for 2 rain (A) and for 30 rain (B). The uncertainty in magnitude of each measurement is estimated at +10%. Note the increase in full width at half-maximum (FWHM). Dots indicate the points b e t w e e n which integration of area (intensity) was performed. T h e irradiation intensity was 1.30 mW/crn :.
319 Table I also includes a tabulation of the full width at half-maximal intensity of the Ca 2÷ pump protein band in terms of molecular weight. The widths of the intensity profiles increased by about 30% in 30 min of irradiation in the presence of either dye. This increase in width corresponds to an effective gain or loss of about 500 daltons with respect to the band widths of control samples. Neither the peak widths nor amplitudes were affected by timed incubation at 23°C in the dark with either dye. The phenomenon of band spreading is depicted in Fig. 2. The consequence of spreading is that the rate of disappearance of peak intensity exceeded that for integrated peak intensity. This observation may relate to the mechanism of damage, as described in the next section. DISCUSSION
Structural alterations in SR vesicles by dye molecules We have presented evidence for structural and functional damage of the Ca 2÷ pump protein of the sarcoplasmic reticulum photosensitized by Erythrosin B. This dye is similar in potency to Rose Bengal, a known photosensitizing agent. The activity was expressed as a dose-dependent decrease of integrated band intensity of the Ca2÷: Mg2÷-ATPase band as observed on SDS gels. The decrease in band intensity was not compensated by the appearance of new bands corresponding to lower or higher molecular weights than the Ca 2÷ pump protein. This suggests that pump protein fragments, if formed, must have been of random size. Alternatively, the decrease in integrated band intensity m a y be due to photopolymerization, such that the affected protein could not enter the gel. For samples irradiated in the presence of dye a band was observed at the beginning of gel channels but this band was n o t quantifiable. In addition, a progressive increase in width of the band intensity profile was observed. The Ca2÷: Mg2÷-ATPase band was symmetric and its position was unchanged with respect to non-irradiated control. The increase in spreading over a 30-min irradiation period, cf. Table I, amounts to molecular weights corresponding to the sum of several amino acids. This suggests that fragmentation of the Ca 2÷ pump protein may be occurring at the ends of its peptide chain with possible exchange and binding of small fragments to neighboring molecules. Such a process may be enhanced to the extent that the Ca 2÷ pump protein is situated in the SR membrane in tetrameric form [13,14]. On the other hand, similar increases in peak width were observed in electrophoretograms (not shown) of bovine serum albumin irradiated in the presence of Rose Bengal. This result suggests that modification of protein mobility in the gel is facilitated by photochemical damage to susceptible residues. Functional alterations in SR vesicles facilitated by dye molecules Correlated with the apparent damage to the pump protein is about a 10-fold increase in the passive permeability of the SR membrane for Ca 2÷.
320 It is probable that this is a direct effect of destruction of the pump molecule, although damage to other membrane components cannot be excluded. Increases in passive permeability (Table I) for Rose Bengal-photosensitized preparations were observed at a Rose Bengal concentration ten times lower (17.3 pM) than the concentration used for the photodestruction experiments. Accordingly, measurements of passive ion transport may afford a more sensitive measure of membrane damage than direct examination of protein loss. In addition to the photosensitizing capabilities described above, Rose Bengal and Erythrosin B were found to be strong inhibitors of Ca2÷: Mg2÷ATPase activity and of active Ca 2÷ uptake even in the absence of activating light [15]. Inhibition of Ca2÷ transport was observed in the present study at free dye concentrations as low as 0.69 ~M and 1.16 pM for Erythrosin B and Rose Bengal, respectively. Light-dependent blockage of Na+: K+-ATP-ase has been observed in preparations of rat brain incubated in darkness with Erythrosin B [16]. The critical concentration for half-maximal inhibition of [3H]ouabain binding was 500 nM. Our study complements these results, as well as previous in vitro work [5] which made explicit mention of the absence of Erythrosin B-induced photochemical processes in facilitating functional inhibition of membrane properties.
Mechanisms of photochemical damage processes For dyes of the xanthene class, of which Erythrosin B and Rose Bengal are examples, two general mechanisms for photosensitization are normally proposed [17,18]. Both involve the participation of molecular oxygen, and thus represent the kind of photosensitization known as photodynamic action, or photooxidation. Type I photooxidation [17] occurs via hydrogen atom abstraction from target molecules by triplet states of excited dye molecules. The target molecule is thereby radicalized, and may initiate chain reaction processes which include peroxidation and subsequent crosslinking with other protein and/or lipid molecules, or breakup into reactive fragments of lower molecular weight. Type II photooxidation [18] is mediated by singlet oxygen. Singlet oxygen results from oxygen quenching of dye triplet states, and is known to react strongly with proteins and lipids, producing peroxy compounds directly without the participation of freeradical intermediates. Because the comparison dye Rose Bengal is thought to function by pure Type II process [19] and Erythrosin B differs in structure only by substitution of hydrogen atoms for chlorine atoms in four positions, Erythrosin B is also likely to be a Type II photooxidizer. Evidence for Type II behavior is suggested by Erythrosin B photooxidation of 4-methoxynaphthol in a solid, but oxygen-permeable, medium [17]. In addition, previous studies on photooxidation of SR vesicles by Erythrosin B and Rose Bengal have shown that destruction of amino acid residues of the Ca2÷ pump protein was confined to those residues most susceptible to singlet oxygen attack, particularly
321 histidine [7] and tryptophan [6,7]. Photooxidation leading to loss of active Ca2÷ uptake [6,7] was correlated with amino acid loss from the Ca2+ pump protein, indicating the effect of structural damage on functional capability. This is similar to our observations of diminished total Ca2+ pump protein correlated with increase in passive transport rate of Ca2+. Contribution o f membrane-associated dye molecules to membrane alter-
atiotzs Since the reactive intermediate in Type II photooxidation is singlet oxygen, a diffusible entity having a lifetime [20] of 2 /as in water, it is possible that free dye molecules in solution may contribute to photooxidative damage in the sarcoplasmic membranes. The relative capabilities of free dye molecules and membrane-associated dye molecules may be compared by calculating their respective concentrations. This is conveniently done by examining the kinetics of dye binding with the Michaelis-Menten formalism. Denoting the concentration of membrane-dye complex referred to the volume of the total system by [MD]s , the total dye concentration by [D]t, the SR protein concentration by [SR], and the protein concentration for half-maximal dye binding by Kin, the appropriate Michaelis-Menten equation becomes:
[D]t[SR]
[MD]s [D] t
-f
K m + [SR]
(i)
where f is the dye binding fraction as reported in the Results section. The concentration ratio of membrane-associated dye molecules to free dye molecules, [MD]s/[D], may be derived by substituting [D]t = [D] + [MD]s to yield: [MD]s
[SR]
f
[D]
[Km]
1- f
(2)
The concentration of dye molecules in the membranes, [MD]m, may be obtained from the solution concentration of the dye-membrane complex, [MD]s~ by first relating the membrane volume Vrn to the solution volume Vs. Knowledge of the ratio [21] by weight of protein to lipid (3 : 5) and the densities [21] of protein (1.3 g/ml) and lipid (1.03 g/ml) enables calculation of the sum of protein and lipid volumes in the membranes as a function of SR protein concentration. The resultant scale factor is 2.387 X 10 -3 1 membrane per g vesicle protein. The ratio of Vm to vs is deduced by multiplying the SR protein concentration by the scale factor (or equivalently, dividing by its reciprocal): Vm
[SR]
[MD]s
vs
418
[MD]m
(3)
322 The latter relation holds since by definition the number of bound dye molecules is defined b y the equivalent products [MD]m vm or [MD]s Vs. It follows that: [MD]m [DI -
418 [Kin]-
418 [SR]
(~_ff) (4)
Thus the desired ratio is fundamentally independent of vesicle concentration and may be deduced by either of t w o formulae. Under the conditions of the irradiation experiments ([SR] = 0.29 mg/ml) the ratio of bound dye concentration in the vesicle bilayers to free dye concentration was 285 for Erythrosin B and 275 for Rose Bengal. Substitutions of the parameters of the active Ca 2÷ uptake assay into Eqn. (4) shows that the ratio of dye concentration in the membrane to free dye concentration is 2600 for Erythrosin B and 4100 for Rose Bengal. Calculation of the b o u n d dye concentration in the membranes is conveniently carried out using the definition of dye binding fraction from Eqn. (1) and substituting Eqn. (3) into it to obtain: [MD]m -
418 f [D]t
[SR]
(5)
Membrane concentrations of dyes during the irradiation experiments were 22 mM and 40 mM for Erythrosin B and Rose Bengal, respectively. Membrane concentrations for dyes in the active uptake experiments were 1.8 mM and 18 mM for Erythrosin B and 4.8 mM and 48 mM for Rose Bengal. These results may be compared with the concentration of Ca2÷: Mg 2÷ATPase in the membrane. Assuming that the vesicle protein is composed entirely of the 102 000 dalton ATPase and transforming the protein concentration in water to membrane space with Eqn. (4) we obtain an ATPase membrane concentration of 4.17 mM. Free concentrations of dyes in solution are readily obtained from [D] = (1 - f) [D]t. Free dye concentrations for the active uptake case were 0.69 pM and 6.9 #M for Erythrosin B and 1.16 pM and 11.6 pM for Rose Bengal. Inhibition of active uptake was observed for both dyes at each concentration listed. The above calculations indicate that photochemical modification of structure and function of SR membranes is dominated by dye molecules dissolved in the membranes. In the case of photooxidation the contribution of membrane-associated dye molecules to structural and functional damage is further enhanced since the solubility of oxygen, as well as the lifetime of singlet oxygen [20] are enhanced by about an order of magnitude in nonpolar media compared to polar media. As noted above, Erythrosin B and Rose Bengal inhibit Ca 2÷ transport in a light-independent reaction [15]. The degree of inhibition has been shown to be dependent on the degree of binding of the dye to the membrane. Mailman et al. [3] found that the inhibitory effect of Erythrosin
323 B on synaptosomal dopamine uptake at fixed dye concentration was inversely proportional to tissue concentration. This observation can be explained directly by our considerations of partitioning since it is possible to express the dye concentration in the membranes as a function of tissue concentration. Substitution of Eqn. (3) into Eqn. (1) reveals that: [MD]m =
418[D] t [ g m ] + [SR]
(6)
This formula is of the required form, with respect to tissue concentration, to fit the data of Mailman et al. [3]. Although we did not experimentally investigate this formula as a function of tissue concentration, it would appear that, in evaluating possible toxic effects in organs and whole animals, the principal determinant of the inhibitory and photochemical effects of Erythrosin B (and Rose Bengal) is the concentration of these dyes in the target membrane preparation under study. Our results suggest a basis for understanding xanthene-dye induced modification of membrane function in the absence of light, as well as alterations of membrane structure and function in the presence of light. These results m a y have relevance to human concerns [22]. For example, Erythrosin B [23] and Rose Bengal [24] have been examined for use in retinal angiography. Calculated blood concentrations of these dyes after injection are comparable (10--100 gM} to the concentrations we have used in our in vitro experiments. Our results indicate that a possible contribution of membrane-associated dye molecules to abnormal structure and function deserves consideration in reported cases of dye sensitivity [3,22,24], although this will be modified to the extent to which the ingested dye enters the circulation [25]. Of relevance to possible photochemical considerations is the fact that our irradiation source is comparable in brightness [26] to the sun at the appropriate excitation wavelengths, and that human skin is known to transmit light [27] that is capable of exciting these dye molecules in dermal capillaries. ACKNOWLEDGEMENTS We wish to thank Donald Mouring for excellent technical assistance. We also wish to thank one of the reviewers for encouraging us to further examine the significance of d y e membrane concentrations. This research was supported b y NIH grants GM23990 and AM20086. REFERENCES 1 W.J. Logan and J.M. Swanson, Erytbrosin B inhibition of neurotransmitter accumulation by rat brain homogenate, Science, 206 (1979) 363. 2 J.A. Lafferman and E.K. Silbergeld, Erythrosin B inhibits dopamine transport in rat caudate synaptosomes, Science, 205 (1979) 410. 3 R.B. Mailman, R.M. Ferris, F.L.M. Tang, R.A. Vogel, C.D. Kilts, M.A. Lipton, D.A.
324
4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19 20 21 22 23 24
Smith, R.A. Mueller and G.R. Breese, Erythrosine (Red No. 3) and its nonspecific biochemical actions: what relation to behavioral changes?, Science, 207 (1980) 535. H. Levitan, Food, drug, and cosmetic dyes: biological effects related,to lipid solubility, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 2914. G.J. Augustine, Jr. and H. Levitan, Neurotransmitter release from a vertebrate neuromuscular synapse affected by a food dye, Science, 207 (1980) 1489. M. Kondo and M. Kasai, Photodynamic inactivation of sarcoplasmic reticulum vesicles by xanthene dyes, Photochem. Photobiol., 19 (1974) 35. B.P. Yu, E.J. Masoro and H.A. Bertrand, The functioning of histidine residues of sarcoplasmic reticulum in Ca 2÷ transport and related activities, Biochemistry, 13 (1974) 5083. J.P. Pooler and D.P. Valenzeno, Physiochemical determinants of the sensitizing effectiveness for photooxidation of nerve membranes by fluorescein derivatives, Photochem. Photobiol., 30 (1979) 491. D.H. MacLennan and P.C. Holland, Calcium transport in sarcoplasmic reticulum, Annu. Rev. Biophys. Bioeng., 4 (1975) 377. D.H. Haynes and V.C.K. Chiu, Kinetics of passive Ca ~÷ transport by skeletal sarcoplasmic reticulum, in: R.H. Wasserman, R.A. Corradino, E. Carafoli, R.H. Kretzinger, D.H. MacLennan and F.L. Siege, (Eds.), Calcium binding proteins and calcium function, North-Holland Publishing Co., New York, Amsterdam and Oxford, 1977, pp. 137--145. V.C.K. Chiu and D.H. Haynes, Rapid kinetic study of the passive permeability of a Ca2÷-ATPase rich fraction of the sarcoplasmic reticulum, J. Membr. Biol., 56 (1980) 203. V.C.K. Chiu and D.H. Haynes, Rapid kinetic studies of active Ca ~÷ transport in sarcoplasmic reticulum, J. Membr. Biol., 56 (1980) 219. A.J. Murphy, Cross-linking of the sarcoplasmic reticulum ATPase protein, Biochem. Biophys. Res. Commun., 70 (1976) 160. R.J. Baskin and S. Hanna, Cross-linking of the (Ca ~÷ + Mg~÷)-ATPase protein, Biochim. Biophys. Acts, 576 (1979) 61. S.J. Morris, E.K. Silbergeld, R.R. Brown and D.H. Haynes, Erythrosin B (USFD and C Red 3) inhibits calcium transport and ATPase activity of muscle sarcoplasmic reticulum, Biochem. Biophys. Res. Commun., 104 (1982) 1306. E.K. Silbergeld, Erythrosin B is a specific inhibitor of high affinity 3H-ouabain binding and ion transport in rat brain, Neuropharmacology, 20 (1981) 87. J. Bourdon and M. Durante, A study of the photodynamic inactivation of papaln in a solid matrix, Ann. N.Y. Acad. Sci., 171 (1970) 163. C.S. Foote, Photosensitized oxidation and singlet oxygen: consequences in biological systems, in: W.A. Pryor, (Ed.), Free Radicals in Biology, Vol. H, Academic Press, New York, 1976, pp. 85--134. K. Gollnick, T. Franken, G. Schade, and G. Dorhofer, Photosensitized oxygenation as a function of the triplet energy of sensitizers, Ann. N.Y. Acad. Sci., 171 (1970) 89. P.B. Merkel and D.R. Kearns, Radiationless decay of singlet molecular oxygen in solution. An experimental and theoretical study of electronic-to-vibrational energy transfer, J. Am. Chem. Soc., 94 (1972) 7244. Y.H. Lau, A.H. Caswell, J.P. Brunschwig, R.J. Baerwald, and M. Gareia, Lipid analysis and freeze-fracture studies on isolated transverse tubules and sarcoplasmic reticulum subfractions of skeletal muscle, J. Biol. Chem., 254 (1979) 540. D.P. Valenzeno and J.P. Pooler, Phototoxicity -- the neglected factor, J. Am. Med. Assoc., 242 (1979) 453. B.F. Hochheimer and S.A. D'Anna, Angiography with new dyes, Exp. Eye Res., 27 (1978) 1. W.H. Havener, Ocular Pharmacology, C.V. Moshy Co., St. Louis, 1974, pp. 412--413.
325 25 J.W. Daniel, The exczetion and metabolism of edible food colors, Toxicol. Appl. Pharmacol., 4 (1962) 572. 26 F.S. Johnson, The solar constant, J. Meteorol., 11 (1964) 431. 27 M.A. Everett, E. Yeargers, R.M. Sayre and R.L. Olson, Penetration of epidermis by ultraviolet rays, Photochem. Photobiol., 5 (1966) 533.
313
STRUCTURAL AND FUNCTIONAL DEGRADATION O F Ca2÷:Mg ~+ATPase R I C H S A R C O P L A S M I C RETICULUM VESICLES P H O T O SENSITIZED B Y E R Y T H R O S I N B
B.D. WATSON and D.H. HAYNES*
Department of Pharmacology, University of Miami School of Medicine, Miami, FL 33101 (U.S.A.) (Received March 10th, 1981) (Revision received March 12th, 1982) (Accepted March 23rd, 1982)
SUMMARY
Erythrosin B (Red Dye No. 3) and Rose Bengal photosensitize the destruction of the Ca2+: Mg2+-ATPase p u m p protein in sarcoplasmic reticulum (SR) vesicles with respective quantum efficiencies of (1.53 + 0.19) × 10 -3 and (1.25 -+ 0.18) × 10 -3. Damage to vesicle function was assayed by measurements of increases in passive Ca 2÷ permeability. Rates of passive Ca 2÷ movement into the SR lumen were increased b y dye photosensitization in proportion to radiation absorbed. Active Ca 2÷ transport into SR vesicles was blocked independent of radiation absorbed by Erythrosin B and Rose Bengal at free concentrations of 0.69 gM and 1.16 #M, respectively. The photochemical lability of the Ca 2÷ pump protein and alterations in passive and active Ca 2÷ transport may be dependent on the concentration of the dye in the membrane. The photosensitization results may have implications with respect to the suitability of Erythrosin B usage in vivo, since the brightness of our irradiation source is comparable to that of sunlight at 480 nm.
INTRODUCTION
Several recent studies have stimulated interest in possible mechanisms of Erythrosin B toxicity in brain and nervous tissue. In particular, Erythrosin B was found to inhibit neurotransmitter accumulation in rat brain [1] and dopamine transport in caudal synaptosomes [2]. Proper quantitation of these results [1,2] was later found to depend on the tissue concentration in the assay medium [3]. In addition, Erythrosin B increased the perme*To whom correspondence should be sent. Abbreviations: ANS-, 1-anilino-8-naphthalene sulfonate; SDS, sodium dodecyl sulfate; SR, sarcoplasmic reticulum.
0009--2797/82/0000--0000/$02.75 © 1982 Elsevier Scientific Publishers Ireland Ltd.
314 ability of mollusc buccal ganglia to K ÷ ions [4] and, when incubated with frog neuromuscular synapses, caused an irreversible increase [5] in transmitter release, which was said to be independent of ambient light. Contrasting with the foregoing examples of intrinsic effects of Erythrosin B on membrane function, photosensitization by Erythrosin B, and by xanthene dyes in general, has been demonstrated to induce functional damage in membrane systems. For example, active Ca 2÷ uptake in sarcoplasmic reticulum vesicles was inhibited, an effect attributed to photooxidation of tryptophan [6] or of histidine residues [7] of the Ca 2÷ pump protein. Another study showed that sodium channels [8] were inactivated in lobster giant axons. In this communication we present evidence for structural and functional degradation of the Ca2÷: Mg2÷-ATPase transport enzyme [9] of sarcoplasmic reticulum, photochemically induced by Erythrosin B incubated with sarcoplasmic reticulum vesicles rich in the Ca2÷: Mg2+-ATPase. For comparison, parallel experiments were run using Rose Bengal, a well-characterized xanthene photosensitizer. Inhibition of active Ca 2÷ transport by b o t h dyes was also observed, in the absence of irradiation.
MATERIALS AND METHODS Rose Bengal was obtained from Professor M. Kasha and was purified by column chromatography. Erythrosin B was obtained from the National Aniline Division through Dr. R.C. Leif and was used as received. The Erythrosin B sample was shown b y paper electrophoresis to be at least 96% pure. SR vesicles from rabbit skeletal muscle were isolated from the Ca2+: Mg2+-ATPase rich band (10) and resuspended in 50 mM borate buffer (pH 7.4) at an initial concentration of 3.2 mg (Lowry protein)/ml. The concentrations of Rose Bengal and Erythrosin B were adjusted to ensure equal rates of p h o t o n absorption (equal optical densities) at 480 nm. The molar extinction coefficients at 480 nm for Rose Bengal and Erythrosin B in water are 7700 and 14 800 1/M cm, respectively. A 25-pl aliquot of either dye was added to the irradiation cell first, followed b y dilution to 525 pl with borate buffer containing sucrose at a concentration equiosmolar to that in the SR suspension. A 50-pl aliquot of the SR suspension was added next in relative darkness and the irradiation proceeded after 1 min equilibration. The final irradiation medium contained 70 mM sucrose, 50 mM boric acid, 1.5 mM sodium borate, 90 gM Erythrosin B (or separately 173 pM Rose Bengal) and 0.29 mg/ml SR vesicles. The pH was 7.40. Samples were irradiated at 23°C at timed intervals of 0, 2, 5, 10, 18 and 30 min and then withdrawn for analysis. Optical densities of the irradiated SR suspensions remained constant throughout. The irradiation source was an Eimac/Varian VIX-300 Xenon arc coupled
315 by a fused silica lens to an Oriel 7204 holographic grating monochromator. The m o n o c h r o m a t o r dispersion is 3.2 nm/mm. The effective irradiation bandwidth was 10 nm. A fused silica cell of optical path length 2 mm and mounted at the exit slit contained the vesicle suspensions, which were magnetically stirred during exposure. A Laser Precision Corporation broadband detector (model kT-4020G) operated at 50% d u t y cycle was used to monitor the transmitted power through the irradiated suspensions. The transmitted irradiance at 480 nm was 60% of the incident irradiance of 1.30 mW/cm 2. At the m o n o c h r o m a t o r exit slit, the brightness of the irradiation source was calculated to be 0.33 mW/cm 2 nm. Photochemical damage to sarcoplasmic reticulum Ca2+: Mg2+-ATPase was assessed b y a structural test (SDS-polyacrylamide gel electrophoresis) and t w o functional tests (active and passive transport of Ca 2+) using 1anilino-8-naphthalene sulfonate (ANS-) as indicator [10]. In the onedimensional gel experiments, an acrylamide concentration of 8% was used. Approximately 0.1--0.4 pg protein was loaded per gel channel and bands stained with Coomassie Blue were recorded with a Gilson gel scanner and analyzed b y planimetry. Linearity of response was calibrated with bovine serum albumin. The Ca 2÷ passive transport assay [10] was used first to test functional integrity of the sarcoplasmic reticulum membranes. The use of an AmincoMorrow stopped-flow apparatus for this application has been described [10]. Concentrations of solution components were identical to those mentioned above, except that of Rose Bengal. Passive transport of Ca 2÷ was blocked in samples irradiated in the presence of 173 pM Rose Bengal. Reducing the Rose Bengal concentration to 17.3 pM allowed changes in passive transport rates of Ca 2÷ to be observed as a function of irradiation absorbed. Final concentrations of components in this assay were: CaC12, 15 mM; sucrose, 70 mM; boric acid, 50 mM; sodium borate, 1.5 mM; ANS-, 32 pM; valinomycin, 6 ~M; Rose Bengal, 0.86 pM; Erythrosin B, 4.5 pM; SR vesicles, 0.015 mg/ml. The active uptake assay [11,12] was also conducted on the AmincoMorrow apparatus. Final concentrations of components in this assay were: CaC12, 0.7 mM; EGTA, 1 mM; Tris, 30 mM: HEPES, 10 mM (pH 6.5); sucrose, 0.6 M; KC1, 0.1 M; MgC12, 0.6 mM; ATP, 0.5 mM; ANS-, 10 pM. valinomycin, 6 ~M; SR vesicles, 0.05 mg/ml. Dye concentrations tested were 1.73 gM and 17.3 #M for Rose Bengal, and 0.9 pM and 9.0 pM for Erythrosin B. Partitioning of the dyes in the SR membranes was studied by incubating the vesicles and dyes in darkness for times equivalent to the irradiation times, and then filtering the suspensions through a 0.22-pm millipore filter. Percentage of dye associated with membrane was deduced from optical absorbance measurements of filtrates of SR vesicles mixed with dye, compared to absorbancies o f filtrates of dye solutions not containing SR vesicles. The effect of attenuation of ANS- fluorescence caused by dye absorbance was also considered. For the experimental conditions used [10--12] in the
316 active and passive Ca 2÷ uptake experiments, the ANS- signal was attenuated less than 15% by Erythrosin B and less than 20% b y Rose Bengal. RESULTS
Figure 1 shows that irradiation in the presence of Erythrosin B causes an increase in passive Ca 2÷ permeability o f SR. Figure 1A is a control experi-
r'.l!
Control B
30 min. Fig. 1. I n f l u e n c e o f i r r a d i a t i o n o n t h e t i m e c o u r s e o f passive Ca 2+ e q u i l i b r a t i o n across t h e S R m e m b r a n e s . Ca 2÷ i n f l u x i n t o t h e SR vesicle is m e a s u r e d b y a n increase in A N S f l u o r e s c e n c e (Y axis, 0.5 V / d i v i s i o n ) vs t i m e a f t e r m i x i n g o f Ca 2÷ w i t h S R ( X axis, 5 s/division). O t h e r details o f t h e assay are given in Materials a n d M e t h o d s . A : s h o w s t h e b e h a v i o r of t h e c o n t r o l sample (1 rain p r e i n c u b a t i o n f o l l o w e d b y 30 m i n i r r a d i a t i o n a t 4 8 0 n m in a b s e n c e of E r y t h r o s i n B). A t,/2 o f 75 s is observed. B: s h o w s t h e e f f e c t o f 30 rain i r r a d i a t i o n in t h e p r e s e n c e o f 90 pM E r y t h r o s i n B. T h e t,i 2 is r e d u c e d t o 8 s. T h e i r r a d i a t i o n i n t e n s i t y was 1.30 m W / c m 2.
317 TABLE I DEPENDENCE
O F tl/2 A N D ATPase B A N D I N T E N S I T Y ON I R R A D I A T I O N T I M E Dye added
Irradiation time (min) 0
2
5
10
18
30
60
59.0
k × 10 -2 (s -1)
EB RB a None
1.3 1.1 1.2
1.1 0.9
1.1 1.5
4.3 4.3
6.2 8.3
11.1 14.3 1.2
ATPase b a n d Intensity (area, c m 2)
EB RB a None
33.6 28.3 38.7
35.1 24.9 43.5
29.3 19.5 33.0
28.0 20.3 37.4
19.8 18.5 37.1
16.3 14.1 42.6
Full w i d t h (tool. wt.) At Half-maximum
EB RB a None
3100 3100 3010
3440 2750 3220
3270 1920 2800
3440 3100 3220
4000 3780 3220
4130 4130 3440
aThis assay was r u n a t a Rose Bengal c o n c e n t r a t i o n of 17.3 uM. N o r m a l c o n c e n t r a t i o n s are given in Materials a n d M e t h o d s .
men t showing the progress of passive Ca 2+ influx in untreated SR. An increase in ANS- fluorescence (owing to increased binding to the m em brane inner surface) is observed when Ca 2+ enters the sarcoplasmic reticulum vesicle lumen and shields surface charge on the inner membrane. From Figure 1A the time for half-maximal response (tl/2) in the absence of 480 nm irradiation is seen to be 75 s. Figure 1B depicts the shortening o f response time t ha t occurs when vesicles are incubated in the presence of Erythrosin B and are exposed to 30 min of irradiation, corresponding to (1.98 + 0.11) × 10 is phot ons absorbed at 480 nm. The t i n for this case has decreased to 8 s. The rates of passive Ca 2÷ transport as a f u n c t i o n of exposure time are given in Table I. Progressive increases in rates of passive Ca 2÷ transport into the SR vesicles are photochemically facilitated by b o t h Erythrosin B and Rose Bengal. No increases in transport rates were observed following timed incubations of SR vesicles in darkness with either dye. The effect of Erythrosin B and Rose Bengal on active Ca 2. transport was studied. Using dye concentrations described in Materials and Methods, it was found that active Ca 2÷ transport was com pl et el y inhibited, regardless o f irradiation fluence (including zero fluence). Therefore, attempts to use active transport of Ca 2÷ to test for p h o t o c h e m i c a l activity o f Erythrosin B and Rose Bengal were n o t pursued further. Dye-sensitized damage to protein was studied by assaying the a m o u n t of Ca2÷: Mg2+-ATPase remaining as a function o f irradiation time. The results are compiled in Table I in terms of peak area as observed by planimetric integration o f SDS-gel electrophoretograms. Table I shows t h a t the a m o u n t o f Ca 2÷ p u m p protein is observed t o decrease as a function of irradiation exposure for samples incubated with Erythrosin B as well as
318 Rose Bengal. Plots of integrated intensity versus irradiation absorbed over a 30-min period were characterized with straight line fits. The average losses of the Ca 2÷ p u m p protein due to 30-min periods of photosensitization by Erythrosin B or b y Rose Bengal were 60% and 46%, respectively. The possibility was also examined that loss of Ca 2÷ pump protein was artifactual, owing to inhibition of staining dye binding by the photosensitizing dyes. This is unlikely since the Erythrosin B and Rose Bengal effects were irradiation-dependent and since both dyes co-migrated with the tracking dye in the SDS gels. The fraction of dye concentration associated with the SR vesicles was determined b y millipore filtration. Referring to solution compositions in Materials and Methods, this fraction was 0.24 for Erythrosin B and 0.33 for Rose Bengal under conditions for active Ca 2÷ uptake. Under conditions for passive Ca 2÷ transport following irradiation, and during irradiation itself, this fraction was 0.17 for Erythrosin B and 0.16 for Rose Bengal. The binding fractions enable calculation of the quantum efficiencies for photodestruction for Erythrosin B and Rose Bengal, assuming that the loss of Ca 2÷ pump protein is solely mediated by dye associated with sarcoplasmic membranes. These quantum efficiencies are (1.53 + 0.19) × 10 -3 and (1.25 + 0.18) × 10 -3 for Erythrosin B and Rose Bengal, respectively. A
FWHM 26
,,
._~_~
B FWHM
Fig. 9.. 8DS-PAGE gel profiles of the Ca ~÷ pump protein from SR vesicles (0.3 mg/ml) irradiated at 480 nm in the presence of Erythrosin B (90 ~M) for 2 rain (A) and for 30 rain (B). The uncertainty in magnitude of each measurement is estimated at +10%. Note the increase in full width at half-maximum (FWHM). Dots indicate the points b e t w e e n which integration of area (intensity) was performed. T h e irradiation intensity was 1.30 mW/crn :.
319 Table I also includes a tabulation of the full width at half-maximal intensity of the Ca 2÷ pump protein band in terms of molecular weight. The widths of the intensity profiles increased by about 30% in 30 min of irradiation in the presence of either dye. This increase in width corresponds to an effective gain or loss of about 500 daltons with respect to the band widths of control samples. Neither the peak widths nor amplitudes were affected by timed incubation at 23°C in the dark with either dye. The phenomenon of band spreading is depicted in Fig. 2. The consequence of spreading is that the rate of disappearance of peak intensity exceeded that for integrated peak intensity. This observation may relate to the mechanism of damage, as described in the next section. DISCUSSION
Structural alterations in SR vesicles by dye molecules We have presented evidence for structural and functional damage of the Ca 2÷ pump protein of the sarcoplasmic reticulum photosensitized by Erythrosin B. This dye is similar in potency to Rose Bengal, a known photosensitizing agent. The activity was expressed as a dose-dependent decrease of integrated band intensity of the Ca2÷: Mg2÷-ATPase band as observed on SDS gels. The decrease in band intensity was not compensated by the appearance of new bands corresponding to lower or higher molecular weights than the Ca 2÷ pump protein. This suggests that pump protein fragments, if formed, must have been of random size. Alternatively, the decrease in integrated band intensity m a y be due to photopolymerization, such that the affected protein could not enter the gel. For samples irradiated in the presence of dye a band was observed at the beginning of gel channels but this band was n o t quantifiable. In addition, a progressive increase in width of the band intensity profile was observed. The Ca2÷: Mg2÷-ATPase band was symmetric and its position was unchanged with respect to non-irradiated control. The increase in spreading over a 30-min irradiation period, cf. Table I, amounts to molecular weights corresponding to the sum of several amino acids. This suggests that fragmentation of the Ca 2÷ pump protein may be occurring at the ends of its peptide chain with possible exchange and binding of small fragments to neighboring molecules. Such a process may be enhanced to the extent that the Ca 2÷ pump protein is situated in the SR membrane in tetrameric form [13,14]. On the other hand, similar increases in peak width were observed in electrophoretograms (not shown) of bovine serum albumin irradiated in the presence of Rose Bengal. This result suggests that modification of protein mobility in the gel is facilitated by photochemical damage to susceptible residues. Functional alterations in SR vesicles facilitated by dye molecules Correlated with the apparent damage to the pump protein is about a 10-fold increase in the passive permeability of the SR membrane for Ca 2÷.
320 It is probable that this is a direct effect of destruction of the pump molecule, although damage to other membrane components cannot be excluded. Increases in passive permeability (Table I) for Rose Bengal-photosensitized preparations were observed at a Rose Bengal concentration ten times lower (17.3 pM) than the concentration used for the photodestruction experiments. Accordingly, measurements of passive ion transport may afford a more sensitive measure of membrane damage than direct examination of protein loss. In addition to the photosensitizing capabilities described above, Rose Bengal and Erythrosin B were found to be strong inhibitors of Ca2÷: Mg2÷ATPase activity and of active Ca 2÷ uptake even in the absence of activating light [15]. Inhibition of Ca2÷ transport was observed in the present study at free dye concentrations as low as 0.69 ~M and 1.16 pM for Erythrosin B and Rose Bengal, respectively. Light-dependent blockage of Na+: K+-ATP-ase has been observed in preparations of rat brain incubated in darkness with Erythrosin B [16]. The critical concentration for half-maximal inhibition of [3H]ouabain binding was 500 nM. Our study complements these results, as well as previous in vitro work [5] which made explicit mention of the absence of Erythrosin B-induced photochemical processes in facilitating functional inhibition of membrane properties.
Mechanisms of photochemical damage processes For dyes of the xanthene class, of which Erythrosin B and Rose Bengal are examples, two general mechanisms for photosensitization are normally proposed [17,18]. Both involve the participation of molecular oxygen, and thus represent the kind of photosensitization known as photodynamic action, or photooxidation. Type I photooxidation [17] occurs via hydrogen atom abstraction from target molecules by triplet states of excited dye molecules. The target molecule is thereby radicalized, and may initiate chain reaction processes which include peroxidation and subsequent crosslinking with other protein and/or lipid molecules, or breakup into reactive fragments of lower molecular weight. Type II photooxidation [18] is mediated by singlet oxygen. Singlet oxygen results from oxygen quenching of dye triplet states, and is known to react strongly with proteins and lipids, producing peroxy compounds directly without the participation of freeradical intermediates. Because the comparison dye Rose Bengal is thought to function by pure Type II process [19] and Erythrosin B differs in structure only by substitution of hydrogen atoms for chlorine atoms in four positions, Erythrosin B is also likely to be a Type II photooxidizer. Evidence for Type II behavior is suggested by Erythrosin B photooxidation of 4-methoxynaphthol in a solid, but oxygen-permeable, medium [17]. In addition, previous studies on photooxidation of SR vesicles by Erythrosin B and Rose Bengal have shown that destruction of amino acid residues of the Ca2÷ pump protein was confined to those residues most susceptible to singlet oxygen attack, particularly
321 histidine [7] and tryptophan [6,7]. Photooxidation leading to loss of active Ca2÷ uptake [6,7] was correlated with amino acid loss from the Ca2+ pump protein, indicating the effect of structural damage on functional capability. This is similar to our observations of diminished total Ca2+ pump protein correlated with increase in passive transport rate of Ca2+. Contribution o f membrane-associated dye molecules to membrane alter-
atiotzs Since the reactive intermediate in Type II photooxidation is singlet oxygen, a diffusible entity having a lifetime [20] of 2 /as in water, it is possible that free dye molecules in solution may contribute to photooxidative damage in the sarcoplasmic membranes. The relative capabilities of free dye molecules and membrane-associated dye molecules may be compared by calculating their respective concentrations. This is conveniently done by examining the kinetics of dye binding with the Michaelis-Menten formalism. Denoting the concentration of membrane-dye complex referred to the volume of the total system by [MD]s , the total dye concentration by [D]t, the SR protein concentration by [SR], and the protein concentration for half-maximal dye binding by Kin, the appropriate Michaelis-Menten equation becomes:
[D]t[SR]
[MD]s [D] t
-f
K m + [SR]
(i)
where f is the dye binding fraction as reported in the Results section. The concentration ratio of membrane-associated dye molecules to free dye molecules, [MD]s/[D], may be derived by substituting [D]t = [D] + [MD]s to yield: [MD]s
[SR]
f
[D]
[Km]
1- f
(2)
The concentration of dye molecules in the membranes, [MD]m, may be obtained from the solution concentration of the dye-membrane complex, [MD]s~ by first relating the membrane volume Vrn to the solution volume Vs. Knowledge of the ratio [21] by weight of protein to lipid (3 : 5) and the densities [21] of protein (1.3 g/ml) and lipid (1.03 g/ml) enables calculation of the sum of protein and lipid volumes in the membranes as a function of SR protein concentration. The resultant scale factor is 2.387 X 10 -3 1 membrane per g vesicle protein. The ratio of Vm to vs is deduced by multiplying the SR protein concentration by the scale factor (or equivalently, dividing by its reciprocal): Vm
[SR]
[MD]s
vs
418
[MD]m
(3)
322 The latter relation holds since by definition the number of bound dye molecules is defined b y the equivalent products [MD]m vm or [MD]s Vs. It follows that: [MD]m [DI -
418 [Kin]-
418 [SR]
(~_ff) (4)
Thus the desired ratio is fundamentally independent of vesicle concentration and may be deduced by either of t w o formulae. Under the conditions of the irradiation experiments ([SR] = 0.29 mg/ml) the ratio of bound dye concentration in the vesicle bilayers to free dye concentration was 285 for Erythrosin B and 275 for Rose Bengal. Substitutions of the parameters of the active Ca 2÷ uptake assay into Eqn. (4) shows that the ratio of dye concentration in the membrane to free dye concentration is 2600 for Erythrosin B and 4100 for Rose Bengal. Calculation of the b o u n d dye concentration in the membranes is conveniently carried out using the definition of dye binding fraction from Eqn. (1) and substituting Eqn. (3) into it to obtain: [MD]m -
418 f [D]t
[SR]
(5)
Membrane concentrations of dyes during the irradiation experiments were 22 mM and 40 mM for Erythrosin B and Rose Bengal, respectively. Membrane concentrations for dyes in the active uptake experiments were 1.8 mM and 18 mM for Erythrosin B and 4.8 mM and 48 mM for Rose Bengal. These results may be compared with the concentration of Ca2÷: Mg 2÷ATPase in the membrane. Assuming that the vesicle protein is composed entirely of the 102 000 dalton ATPase and transforming the protein concentration in water to membrane space with Eqn. (4) we obtain an ATPase membrane concentration of 4.17 mM. Free concentrations of dyes in solution are readily obtained from [D] = (1 - f) [D]t. Free dye concentrations for the active uptake case were 0.69 pM and 6.9 #M for Erythrosin B and 1.16 pM and 11.6 pM for Rose Bengal. Inhibition of active uptake was observed for both dyes at each concentration listed. The above calculations indicate that photochemical modification of structure and function of SR membranes is dominated by dye molecules dissolved in the membranes. In the case of photooxidation the contribution of membrane-associated dye molecules to structural and functional damage is further enhanced since the solubility of oxygen, as well as the lifetime of singlet oxygen [20] are enhanced by about an order of magnitude in nonpolar media compared to polar media. As noted above, Erythrosin B and Rose Bengal inhibit Ca 2÷ transport in a light-independent reaction [15]. The degree of inhibition has been shown to be dependent on the degree of binding of the dye to the membrane. Mailman et al. [3] found that the inhibitory effect of Erythrosin
323 B on synaptosomal dopamine uptake at fixed dye concentration was inversely proportional to tissue concentration. This observation can be explained directly by our considerations of partitioning since it is possible to express the dye concentration in the membranes as a function of tissue concentration. Substitution of Eqn. (3) into Eqn. (1) reveals that: [MD]m =
418[D] t [ g m ] + [SR]
(6)
This formula is of the required form, with respect to tissue concentration, to fit the data of Mailman et al. [3]. Although we did not experimentally investigate this formula as a function of tissue concentration, it would appear that, in evaluating possible toxic effects in organs and whole animals, the principal determinant of the inhibitory and photochemical effects of Erythrosin B (and Rose Bengal) is the concentration of these dyes in the target membrane preparation under study. Our results suggest a basis for understanding xanthene-dye induced modification of membrane function in the absence of light, as well as alterations of membrane structure and function in the presence of light. These results m a y have relevance to human concerns [22]. For example, Erythrosin B [23] and Rose Bengal [24] have been examined for use in retinal angiography. Calculated blood concentrations of these dyes after injection are comparable (10--100 gM} to the concentrations we have used in our in vitro experiments. Our results indicate that a possible contribution of membrane-associated dye molecules to abnormal structure and function deserves consideration in reported cases of dye sensitivity [3,22,24], although this will be modified to the extent to which the ingested dye enters the circulation [25]. Of relevance to possible photochemical considerations is the fact that our irradiation source is comparable in brightness [26] to the sun at the appropriate excitation wavelengths, and that human skin is known to transmit light [27] that is capable of exciting these dye molecules in dermal capillaries. ACKNOWLEDGEMENTS We wish to thank Donald Mouring for excellent technical assistance. We also wish to thank one of the reviewers for encouraging us to further examine the significance of d y e membrane concentrations. This research was supported b y NIH grants GM23990 and AM20086. REFERENCES 1 W.J. Logan and J.M. Swanson, Erytbrosin B inhibition of neurotransmitter accumulation by rat brain homogenate, Science, 206 (1979) 363. 2 J.A. Lafferman and E.K. Silbergeld, Erythrosin B inhibits dopamine transport in rat caudate synaptosomes, Science, 205 (1979) 410. 3 R.B. Mailman, R.M. Ferris, F.L.M. Tang, R.A. Vogel, C.D. Kilts, M.A. Lipton, D.A.
324
4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19 20 21 22 23 24
Smith, R.A. Mueller and G.R. Breese, Erythrosine (Red No. 3) and its nonspecific biochemical actions: what relation to behavioral changes?, Science, 207 (1980) 535. H. Levitan, Food, drug, and cosmetic dyes: biological effects related,to lipid solubility, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 2914. G.J. Augustine, Jr. and H. Levitan, Neurotransmitter release from a vertebrate neuromuscular synapse affected by a food dye, Science, 207 (1980) 1489. M. Kondo and M. Kasai, Photodynamic inactivation of sarcoplasmic reticulum vesicles by xanthene dyes, Photochem. Photobiol., 19 (1974) 35. B.P. Yu, E.J. Masoro and H.A. Bertrand, The functioning of histidine residues of sarcoplasmic reticulum in Ca 2÷ transport and related activities, Biochemistry, 13 (1974) 5083. J.P. Pooler and D.P. Valenzeno, Physiochemical determinants of the sensitizing effectiveness for photooxidation of nerve membranes by fluorescein derivatives, Photochem. Photobiol., 30 (1979) 491. D.H. MacLennan and P.C. Holland, Calcium transport in sarcoplasmic reticulum, Annu. Rev. Biophys. Bioeng., 4 (1975) 377. D.H. Haynes and V.C.K. Chiu, Kinetics of passive Ca ~÷ transport by skeletal sarcoplasmic reticulum, in: R.H. Wasserman, R.A. Corradino, E. Carafoli, R.H. Kretzinger, D.H. MacLennan and F.L. Siege, (Eds.), Calcium binding proteins and calcium function, North-Holland Publishing Co., New York, Amsterdam and Oxford, 1977, pp. 137--145. V.C.K. Chiu and D.H. Haynes, Rapid kinetic study of the passive permeability of a Ca2÷-ATPase rich fraction of the sarcoplasmic reticulum, J. Membr. Biol., 56 (1980) 203. V.C.K. Chiu and D.H. Haynes, Rapid kinetic studies of active Ca ~÷ transport in sarcoplasmic reticulum, J. Membr. Biol., 56 (1980) 219. A.J. Murphy, Cross-linking of the sarcoplasmic reticulum ATPase protein, Biochem. Biophys. Res. Commun., 70 (1976) 160. R.J. Baskin and S. Hanna, Cross-linking of the (Ca ~÷ + Mg~÷)-ATPase protein, Biochim. Biophys. Acts, 576 (1979) 61. S.J. Morris, E.K. Silbergeld, R.R. Brown and D.H. Haynes, Erythrosin B (USFD and C Red 3) inhibits calcium transport and ATPase activity of muscle sarcoplasmic reticulum, Biochem. Biophys. Res. Commun., 104 (1982) 1306. E.K. Silbergeld, Erythrosin B is a specific inhibitor of high affinity 3H-ouabain binding and ion transport in rat brain, Neuropharmacology, 20 (1981) 87. J. Bourdon and M. Durante, A study of the photodynamic inactivation of papaln in a solid matrix, Ann. N.Y. Acad. Sci., 171 (1970) 163. C.S. Foote, Photosensitized oxidation and singlet oxygen: consequences in biological systems, in: W.A. Pryor, (Ed.), Free Radicals in Biology, Vol. H, Academic Press, New York, 1976, pp. 85--134. K. Gollnick, T. Franken, G. Schade, and G. Dorhofer, Photosensitized oxygenation as a function of the triplet energy of sensitizers, Ann. N.Y. Acad. Sci., 171 (1970) 89. P.B. Merkel and D.R. Kearns, Radiationless decay of singlet molecular oxygen in solution. An experimental and theoretical study of electronic-to-vibrational energy transfer, J. Am. Chem. Soc., 94 (1972) 7244. Y.H. Lau, A.H. Caswell, J.P. Brunschwig, R.J. Baerwald, and M. Gareia, Lipid analysis and freeze-fracture studies on isolated transverse tubules and sarcoplasmic reticulum subfractions of skeletal muscle, J. Biol. Chem., 254 (1979) 540. D.P. Valenzeno and J.P. Pooler, Phototoxicity -- the neglected factor, J. Am. Med. Assoc., 242 (1979) 453. B.F. Hochheimer and S.A. D'Anna, Angiography with new dyes, Exp. Eye Res., 27 (1978) 1. W.H. Havener, Ocular Pharmacology, C.V. Moshy Co., St. Louis, 1974, pp. 412--413.
325 25 J.W. Daniel, The exczetion and metabolism of edible food colors, Toxicol. Appl. Pharmacol., 4 (1962) 572. 26 F.S. Johnson, The solar constant, J. Meteorol., 11 (1964) 431. 27 M.A. Everett, E. Yeargers, R.M. Sayre and R.L. Olson, Penetration of epidermis by ultraviolet rays, Photochem. Photobiol., 5 (1966) 533.