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Biochimica et Biophysica Acta, 511 (1978) 141--151 © Elsevier/North-Holland Biomedical Press

BBA 78094

PHOTODYNAMIC EFFECTS OF PROTOPORPHYRIN ON HUMAN ERYTHROCYTES

NATURE OF THE CROSS-LINKING OF MEMBRANE PROTEINS

T.M.A.R. DUBBELMAN, A.F.P.M. DE GOEIJ and J. VAN 8TEVENINCK

Sylvius Laboratories, Laboratory for Medical Chemistry, Wassenaarseweg 72, Leiden (The Netherlands)

(Received January 20th, 1978)

Summary

Protoporphyrin-sensitized photooxidat ion in human red blood cell mem- branes leads to severe deterioration of membrane structure and function. The membrane damage is caused by direct oxidation of amino acid residues, with subsequent cross-linking of membrane proteins.

The chemical nature of these cross-links was studied in model systems, isolated spectrin and red cell ghosts. Cysteine and methionine are not involved in the cross-linking reaction. Further it could be shown that dityrosine forma- tion, the crucial mechanism in oxidative cross-linking of proteins by peroxidase-H202 treatment, plays no role in photodynamic cross-linking.

Experimental evidence indicated that a secondary reaction between free amino groups and a photooxidat ion product of histidine, tyrosine or t rypto- phan is involved in photodynamic cross-linking. This was deduced from the reaction observed between compounds containing a free amino group and photooxida t ion products of these amino acids, both in model systems, isolated spectrin and ery throcyte ghosts. In accordance, succinylation of free amino groups of membrane proteins or addition of compounds with free amino groups protec ted against cross-linking.

Quantitative data and consideration of the reaction mechanisms of photo- dynamic oxidation of amino acids make it highly probable that an oxidation product of histidine rather than of tyrosine or t ryptophan is involved in the cross-linking reaction, via a nucleophilic addition by free amino groups.

Introduction

The photodynamic effects of protoporphyr in on human erythrocytes have been described in preceding papers [ 1--8]. These effects can be observed when

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protoporphyr in is present inside the cells, as in erythropoietic protoporphyria , but also when protoporphyrin is added to the medium of normal erythrocytes [2,4]. It is highly probable that the photodynamic action involves the partici- pation of singlet oxygen [9--12].

Irradiation of red blood cells in the presence of protoporphyrin leads among others to inhibition of several membrane-bound enzymes [ 13,14] and increased passive cation permeabili ty of the membrane with subsequent osmotic hemo- lysis [2]. The membrane deterioration can be visualized by freeze-etch electron microscopy [4]. It has been shown that the membrane damage cannot be rationalized on the basis of peroxidation, of unsaturated fat ty acid residues of cholesterol in the membrane [5,6] bu t should be ascribed to photooxidat ion of amino acid residues of membrane proteins [6]. This photooxidat ion of amino acid residues leads to extensive cross-linking of membrane proteins. Especially spectrin is very sensitive to this protoporphyrin-induced cross-linking [4,6,8].

The amino acids, sensitive to protoporphyrin-induced photooxidat ion are cysteine, histidine, tyrosine, t ryptophan and methionine [3]. Although several studies have been performed on the photooxidat ion of these individual amino acids, the chemical nature of the photodynamical ly formed cross-links of the membrane proteins is not clear. It has only been shown that this cross-linking is not caused by formation of disulfide bridges [6,8].

The present studies were designed to elucidate the chemical nature of the protoporphyrin- induced cross-linkages.

Methods

Heparinized human blood was centrifuged shortly after collection and washed three times in phosphate-buffered isotonic NaC1 solution. Red cell ghosts were prepared according to the method of Weed et al. [15]. Spectrin was eluted from ghosts as described by Fairbanks et al. [16], precipitated at pH 5.1 [17] and further purified by gel chromatography on Sephadex G-200. Amino acid analysis was performed according to D~v~nyi [18] on a Beckman 120C amino acid analyzer equipped with a 6 mm sensitivity cell and a scale expander. Further analytical methods were: free sulfhydryl groups: following the method of Sedlak and Lindsay [19]; histidine: according to Sokolovsky and Vallee [20]; tyrosine: as described by Uehara et al. [21]; t ryptophan: by the method of Spies and Chambers [22] and total protein: according to Lowry et al. [23]. Polyacrylamide gel electrophoresis of mem- brane proteins was performed as described by Fairbanks et al. [16] after dis- solving the proteins in a solution containing 10 mM Tris, pH 8.0, 1 mM EDTA, 40 mM dithiothreitol and 1% sodium dodecyl sulphate.

Formation of dityrosine cross-links in proteins by t reatment with peroxi- dase and hydrogen peroxide and quantification of dityrosine were done as described by Aeschbach et al. [24].

Illumination of solutions and cell suspensions in the presence of protopor- phyrin was performed as described previously [4]. Reactions between amino acids, sensitive to photooxidat ion, and other amino acids were studied by illuminating mixtures of two amino acids in the presence of protoporphyrin. In these experiments one of the amino acids in the mixture was labeled with

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~4C. Analysis of the reaction products was done via thin layer chromatography on silica gel with the solvent systems: n-butanol/acetic acid/water (4 : 1 : 1, v/v/v) and phenol/water (3 : 1, w/v) and subsequent autoradiography of the dried chromatogram. Radioactivity was assayed in a liquid scintillation counter. Photodynamic coupling of compounds to ghosts was measured by illumination of ghosts in the presence of these ~4C-labeled compounds and protoporphyrin. Subsequently the ghosts were washed five times with buffer and analyzed for radioactivity.

Succinylation of membrane protein amino groups was done by the procedure described by Habeeb et al. [25]. Oxygen consumption during photooxidation was measured with an YSI oxygen monitor equipped with a Clark-type electrode and a time-base recorder.

Results

Cross-linking of membrane proteins during illumination in the presence of protoporphyrin is shown in Fig. 1. With increasing illumination time a pro- gressive accumulation of high molecular weight protein complexes on top of the gel can be observed, with a concomitant disappearance of the normal proteins from the gels. As discussed in detail elsewhere, spectrin bands 4.1 and 6 are more susceptible to cross-linking than the other membrane proteins, with the formation of covalent cross-linked protein complexes with apparent molecular weights above 106 [14]. These cross-links are not acid-labile; incuba- tion of the ghosts at pH 1.9 for 20 min subsequent to illumination did not abolish the cross-linking.

A series of experiments was conducted to investigate the possible role of dityrosine formation in this cross-linking reaction. Protein cross-linking, caused by intermolecular dityrosine formation via enzymic oxidation has been described by several authors [24,26,27]. Enzymic oxidation of tyrosine, insulin and ribonuclease with peroxidase and H202 led to dityrosine formation. Dityrosine was detected by its characteristic fluorescence spectrum [24]. These experiments thus confirmed the results described in recent literature. Enzymatic oxidation of purified spectrin on the other hand, did not cause dityrosine formation. Moreover, protoporphyrin-induced photodynamic oxidation did not cause any detectable dityrosine formation with tyrosine, spectrin, insulin and ribonuclease. These results contradict a possible involve- ment of dityrosine bridges in photodynamic cross-linking.

The possible interaction of reaction products of photooxidizable amino acids with other amino acids was studied in model experiments. During illumi- nation of photooxidizable amino acids in the presence of protoporphyrin a gradual decrease of the specific chemical reactions for these amino acids took place. With thin layer chromatography and autoradiography a propor- tional decrease of the amino acid with the concomitant formation of one or more oxidation products was observed. In these experiments illumination was continued until the photooxidizable amino acid had completely disappeared. Fig. 2 shows the results of photooxidation of [14C]methionine. Identical autoradiograms were obtained with [~4C]methionine alone and with [14C]- methionine mixed with a n0n-photooxidizable amino acid. Samples of [14C]-

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Fig. 1. P r o t o p o r p h y r i n i n d u c e d cross-l inking of e r y t h r o c y t e m e m b r a n e p ro te ins as revea led by SDS poly- a c r y l a m i d e gel e lec t rophores i s . I l l u m i n a t i o n t imes: 0 - - 5 - - 1 5 rain. P r o t o p o r p h y r i n c o n c e n t r a t i o n : 0 .05 m M ; p H 7.5.

glycine, illuminated in the presence of p ro toporphyr in gave only one spot on autoradiograms with an Re value corresponding to free glycine. Addition of unlabeled methionine to the reaction mixture had no effect (Fig. 3c). Similar results were obtained with the other non-photooxidizable amino acids serine, lysine and leucine. These results thus contradict an interaction between non- photooxidizable amino acids and the photooxida t ion products of methionine.

In a similar series of experiments with cysteine, instead of methionine, the same results were obtained. Again there was no indication of interaction between non-photooxidizable amino acids and photooxida t ion products of cysteine.

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ORIGI N -,,

a b c d e f Fig. 2. Autoradiography of a silica gel thin layer chromatogram of pho toox ida t ion products of [ 14C]- methionine, i l luminated in the presence of p ro toporphyr in (0.3 raM) in phosphate buffer (10 raM, pH 9.0). The react ion mixture contained: (a) rnethionine (2 raM), not i l luminated (reference); (b) methionine (2 raM); (c) meth ionine (2 raM) + glyeine (2 raM); (d) methionine (2 mM) + glutamic acid (2 raM); (e) meth ionine (2 mM) + glutamine (2 raM); (f) methionine (2 mM) + cysteine (2 raM). Solvent system: butanol /acet ic acid/water . With the solvent sys tem phenol /water similar results were obtained.

In further experiments [14C]glycine alone, or combined with one of the photooxidizable amino acids was illuminated in the presence of protopor- phyrin. The results of a typical experiment are depicted in Fig. 3. With glycine alone, or combined with cysteine or methionine, only free glycine was present. Combined with histidine or t ryptophan, and occasionally with tyrosine, addi- tional spots could be detected. This indicates a reaction between glycine and photooxida t ion products of histidine and t ryptophan and possibly tyrosine. It is clear from Fig. 3 that the reaction with histidine oxidation products is much more pronounced than with t ryptophan and tyrosine oxidation products. Quantitative measurements of radioactivity revealed that under these experi- mental conditions 24.8, 4.9 and 2.9% of the glycine reacted with photooxi- dation products o f respectively histidine, t ryptophan and tyrosine. With [14C]lysine, arginine, leucine, serine and ethylamine instead of [14C]glycine, similar results were obtained. In all cases a weak reaction with tyrosine and t ryptophan photooxida t ion products and a much stronger reaction with histidine-derived photooxida t ion products was observed (Table I). The fact that ethylamine was also reactive indicates that the NH2-group is essential for the reaction. Considering these results, the possible reaction between hydroxyl- amine and the photooxida t ion products was of interest. It appeared that this agent reacts readily with photooxidat ion products Of histidine, t ryptophan and tyrosine.

A similar reaction appeared to occur during photooxida t ion between mem- brane proteins and [14C]glycine, lysine and ethylamine, when ghosts were illuminated in the presence of protoporphyr in and varying concentrations of these agents. A Scatchard plot of the results showed that the total number of

146

Fig. 3. A u t o r a d i o g r a p h y of a silica gel th in l ayer c h r o m a t o g r a m of [ 1 4 C]g ly c in e , i l l umina ted in the p resence of a - - , b his t idine, c m e t h i o n i n e , d cys te ine , e t r y p t o p h a n , f tyros ine . Final c o n c e n t r a t i o n s o f a m i n o acids: 2 mM, of p r o t o p o r p h y r i n : 0 .3 m M and of p h o s p h a t e bu f fe r : 10 m M , p H 9.0. A: so lvent sys t em: b u t a n o l / a c e t i c a c id /wa t e r . B: so lvent s y s t e m: p h e n o l / w a t e r .

147

T A B L E I

R E A C T I O N OF N O N - P H O T O O X I D I Z A B L E A M I N O ACIDS A N D E T H Y L A M I N E W I T H P H O T O O X I - D A T I O N P R O D U C T S OF H I S T I D I N E , T R Y P T O P H A N , T Y R O S I N E , M E T H I O N I N E A N D C Y S T E I N E

Each o f the above- l i s ted c o m p o u n d s was p r e s e n t in a final c o n c e n t r a t i o n of 2 mM. I l l umina t i on was con- t i n u e d for 2 h in the p r e sence o f 0 .3 m M p r o t o p o r p h y r i n in 10 m M p h o s p h a t e bu f fe r , p H 9.0. Each value (pe rcen tage o f t he n o n - p h o t o o x i d i z a b l e c o m p o u n d t h a t has r eac t ed ) is the m e a n of 2 d e t e r m i n a t i o n s .

His t id ine T r y p t o p h a n Tyros ine Meth ion ine Cyste ine

Glyc ine 24.8 4.9 2.9 0 0 Lys ine 25.3 11.8 5.1 < I < I Ser ine 18 ,3 1.4 < 1 0 0 Leuc ine 28.1 5.1 5.7 0 0 E t h y l a m i n e 31.7 8.9 4.0 0.6 0.5

reactive groups created by the photodynamic process was identical with all three amino compounds.

If the photodynamic cross-linking of membrane proteins depends on a similar reaction as observed in the model systems, photooxida t ion products of histidine, t ryptophan and/or tyrosine residues in one polypept ide should be expected to react with free NH2-groups in other polypeptides. This hypothesis was tested with several experimental approaches.

The amino acid composi t ion of purified spectrin before and after illumina- tion in the presence of protoporphyrin is given in Table II. The decrease of photooxidizable amino acid residues is shown in Fig. 4. Besides cysteine, histidine and t ryptophan appeared to be photooxidized at an appreciable rate, whereas tyrosine did not decrease.

Based on the model experiments the inhibition of cross-linking by agents,

T A B L E II

A M I N O A C I D C O M P O S I T I O N OF I S O L A T E D S P E C T R I N B E F O R E AND A F T E R 30 MIN I L L U M I N A - T I O N

For cond i t i ons see Fig. 4.

A m i n o acid mol%

Before i l lumina t ion Af t e r i l l umina t ion

ASP 1 0 . 8 ± 0 . 8 1 1 . 2 ± 0 . 3 Th r 4 . 4 ± 0 . 6 4 . 6 ± 0 . 4 Set 7 . 2 ± 2 . 7 6 . 7 ± 0 . 7 Glu 1 7 . 2 ± 1 . 7 1 7 . 8 ± 0 . 5 Pro 2 . 8 ± 0 . 5 2 . 6 ± 0 . 2 Gly 7 . 5 ± 1 . 3 7 . 1 ± 0 . 6 Ala 8 . 9 ± 0 . 6 9 . 2 ± 0 . 2

Val 5 . 0 ± 0 . 4 5 . 0 ± 0 . 5 Met 2 . 1 ± 0 . 3 2 . 2 ± 0 . 4 Ile 4 . 2 ± 0 . 2 4 . 2 ± 0 . 3 Leu 1 0 . 0 ± 0 . 9 1 0 . 3 ± 0 . 5 T y r 1 . 7 ± 0 . 2 1 . 6 t 0 . 2 Phe 2 . 4 ± 0 . 3 2 . 6 ± 0 . 2 Lys 7 . 3 ± 0 . 8 7 . 5 ± 0 . 2 His 3 . 3 ± 0 . 5 2 . 1 ± 0 . 3 Arg 4 . 6 ± 0 . 5 4 . 5 ± 0 . 4 ~ p 2.1 1.6

148

m ~J

loo

!i .....

50

i ;

10 20 3; min Fig. 4. P h o t o o x i d a t i o n o f h i s t i d ine CA), t r y p t o p h a n (v) , t y r o s i n e (©) a n d S H - g r o u p s (o) in i so l a t ed spec t r i n . R e a c t i o n m i x t u r e : 0 .7 m g / m l s p e c t r i n , 0 . 0 5 m M p r o t o p o r p h y r i n , a n d 10 m M p h o s p h a t e b u f f e r , p H 7.6. E a c h p o i n t is t he average o f a t l eas t t h r e e d e t e r m i n a t i o n s .

Fig. 5. P r o t e c t i o n aga in s t p h o t o d y n a m i c c ro s s - l i nk ing o f e r y t h r o c y t e m e m b r a n e p r o t e i n s (A) a n d i so l a t ed s p e c t r i n (B) a f t e r t r e a t m e n t w i t h succ in i c a n h y d r i d e . G h o s t s u s p e n s i o n s (2 m g p r o t e i n / m l ) a n d s p e c t r i n s o l u t i o n s (1 m g / m l ) we re i l l u m i n a t e d fo r 0, 5 a n d 10 m i n in 10 m M p h o s p h a t e b u f f e r p H 8 .0 in t he p r e s e n c e o f 0 . 0 5 m M p r o t o p o r p h y r i n , a, c o n t r o l ; b , t r e a t e d w i th s u c c i n i c a n h y d r i d e (2 r a g / r a g p r o t e i n ; p H 8 .0 ) .

reacting with photooxidation products was studied. Both with ghosts and with isolated spectrin glycine, lysine and ethylamine yielded a slight degree of inhibition, whereas hydroxylamine gave a much stronger protection.

Finally, succinylation of membrane protein amino groups prior to photo-

149

oxidation yielded a very strong protect ion against photodynamic cross-linking in ghosts and in isolated spectrin (Fig. 5).

In control experiments it appeared that neither the addition of non-photo- oxidizable amino acids or amines nor succinylation of amino groups inhibited photooxida t ion of susceptible amino acid residues per se, as judged from mea- surements of oxygen consumption.

Discussion

The formation of covalent cross-links between membrane proteins in ghosts subsequent to protoporphyrin-sensit ized photooxida t ion has been demon- strated in preceding studies [4,6,7,14]. The chemical nature of the cross-link was still obscure, but cannot be ascribed to the formation of disulfide bridges [8]. As shown in this paper dityrosine formation, as observed during oxidative cross-linking of several proteins by peroxidase-H~O2 treatment [24,27], could also be excluded as the molecular background of photosensit ized cross- linking of ghost proteins.

The described experimental results strongly suggest that cross-linking is effectuated by a reaction between a photooxidat ion product of histidine residues and free amino groups in the involved polypept ide chains. This is based on the following observations.

In the model experiments with mixtures of amino acids no reaction was observed between non-photooxidizable amino acids or amines on the one hand, and cysteine or methionine on the other. Reactions did occur with tyrosine, t ryp tophan and much more pronounced histidine (Table I). This suggests a reaction between a photooxidat ion product of one of these amino acids and free amino groups as the molecular mechanism for cross-linking, with histidine as the most probable source of the involved photooxidat ion product . Direct measurements of photooxidat ion of amino acid residues in spectrin (Table II, Fig. 4) support this supposition. From the results given in Table II and the apparent molecular weight of spectrin (about 230 000) it can be calculated that a spectrin polypept ide chain contains about 34 tyrosine, 66 histidine and 42 t ryptophan residues. According to the results shown in Fig. 4 the number of photooxidized amino acid residues after an illumination period of 30 min (when cross-linking is virtually complete) is 0 for tyrosine, 26 for histidine and 10 for t ryptophan. Although these results make it very unlikely that tyrosine would be involved in the cross-linking reaction, they do not fully exclude this possibility. Theoretically the reaction between only one photooxidizable residue on each spectrin polypept ide with one free NH2-group on another chain would be sufficient for complete cross-linking. If tyrosine residues would be involved, this would require a minimal decrease of about 3%, which is within the experimental error.

The crucial role of free NH:-groups in the cross-linking reaction was con- firmed by the strong inhibition of cross-linking by succinylation of NHE-groups prior to photooxidat ion (Fig. 5).

Based on the proposed mechanism it could be expected that photooxidat ion of ghost membrane proteins in the presence of amines or amino acids would result in covalent binding of these agents and, via competi t ion, inhibition of

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protein cross-linking. The experimental results were in accordance with this anticipation. Analysis of the results of binding experiments showed that the total number of reactive groups amounts to about 64 nmol/mg membrane pro- tein. From the fact that spectrin comprises about 27% of the membrane protein [16,28] and the amino acid composi t ion given in Table II it can be calculated that the spectrin included in 1 mg membrane protein contains about 77 nmol histidine, 49 nmol t ryptophan and 40 nmol tyrosine. Although the amino acid composi t ion of not all membrane proteins is known it can be expected that the total number of histidine, t ryptophan and tyrosine residues per mg of membrane protein will be more or less proportional, thus roughly 3--4 times higher. On the other hand it should be expected that, due to sterical factors, many photooxidizable groups will not be reactive. Therefore, the value of 64 nmol/mg membrane protein may well be a reasonable reflection of the actual number of reactive groups.

Several studies on photosensit ized oxidation of tyrosine, histidine and t ryptophan appeared in recent literature [29--37]. Many photooxidat ion products of these amino acids have been identified. All three amino acids yield intermediates and products with aldehyde function. Reaction of free NH2-groups with these aldehydes might yield Schiff's bases. However, it seems unlikely that this type of reaction would be responsible for the observed cross-links in the membrane proteins, considering their acid stability. The for- mation of a stable covalent bond by a reaction between free NH2-groups and a particular intermediate of histidine photooxida t ion is more likely. This intermediate is very sensitive to nucleophilic addition at the C4 position [30]:

OH OH :X--H

R ]l ~ R I X H N - - - ~ N H N - - . ~ N H

0 0

During photooxida t ion of histidine nucleophilic at tack by the NH2-groups as well as by imidazole residues at the C4 position readily occurs, leading to different final photooxida t ion products [29]. A similar reaction with NH2- groups of other amino acid residues or amines seems very probable and could easily explain the experimental results described above. The very pronounced protect ion against cross-linking by the strong nucleophile hydroxylamine is in accordance with this interpretation.

Further experiments to clarify the exact mechanism of photodynamic cross- linking of membrane proteins are in progress.

Acknowledgments

The authors are much indebted to Miss Henny Vreeburg and Miss Karmi Christianse for their technical assistance and to Ing. W.J.M. Pluijms for carrying out the amino acid analysis. We thank dr. J.A. Maassen, dr. W. MSller and dr. R. Amons for helpful discussions.

1 5 1

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