PhotorhPrnisrr-y and Phorohiology. Vol. 31. pp. 597 to 601 0 Pergamon Press Ltd 1980 Printed in Great Britain

RESEARCH NOTE

003 I -8655/80/0601-0S97S02.00/0

CROSS-LINKING OF MEMBRANE PROTEINS AND PROTOPORPHYRIN-SENSITIZED

PHOTOHEMOLY SIS* A. A. LAMOLA and F. H. DOLEIDEN

Bell Laboratories, Murray Hill, NJ 07974, U.S.A.

(Received 10 October 1979; accepted 28 November 1979)

Abstract-Irradiation of protoporphyrin-sensitized red cells with blue light in the presence of oxygen alters many components of their membranes and eventually leads to hemolysis. Extensive cross-linking of membrane proteins can be observed before hemolysis occurs (Girotti, 1976).

Facile oxidative hemolysis can be achieved without observable cross-linking of membrane proteins upon incubation (37°C) of red cells containing membrane-bound 3/l-hydroxy-5a-hydroperoxy-A6- cholcstene. Thus, protein cross-linking is not obligatory for oxidative lysis. Deoxygenation by Ar bub- bling strongly retards the light-induced increase in osmotic fragility and strongly inhibits eventual hemolysis of protoporphyrin-sensitized erythrocytes. However, similar reduction in oxygen concen- tration only partially inhibits cross-linking of membrane proteins. These results suggest that membrane protein cross-linking and photohemolysis are not coupled processes.

INTRODUCTION

The erythrocytes from patients with erythropoietic protoporphyria (EPP) undergo rapid hemolysis in the presence of light in the visible range due to sensitiza- tion by the protoporphyrin present in them. Soon after its discovery, (Harber et al., 1964; Fleischer et al., 1966) this photohemolysis phenomenon was shown to be a ‘photodynamic’ process, that is, it requires molecular oxygen (Schothorst et al., 1970; Hsu et a!., 1971). Normal red cells incubated with solutions of protoporphyrin undergo similar photody- namic hemolysis (Schothorst et al., 1970; Goldstein and Harber, 1972). These pseudo-EPP red cells have become a prototype for the study of photodynamic hemolysis and of photodynamic membrane damage, and, as such, have received much attention. However, many fundamental questions remain unanswered. Among these are: What are the relative contributions to cell lysis of the light-induced alterations of various membrane components? What are the important photochemical primary processes which lead to these lytic alterations?

It has been shown that both lipid and protein com- ponents of the membrane are oxidized early during the course of irradiation. Amino acid side chains of proteins, unsaturated fatty acid side chains of phos- pholipids, and cholesterol all undergo photooxidation

*Presented in part at the Baekeland Award Symposium, So. Orange, NJ, Oct. 24, 1977.

?Abbreviations used: Cholesterol bydroperoxide for 3/?- hydroxy-5a-hydroperoxy-A6-cholestene; PBS for phos- phate buffered saline (0.15 M NaCI, 0.01 M PO4, pH 7.4); SDS for sodium dodecyl sulfate.

(Schothorst el a!., 1970, 1972, 1974; Goldstein and Harber, 1972; Lamola et al., 1973). Lysis, which has been shown to be of the ‘colloid osmotic’ type (Fleischer et al., 1966), has been blamed on both lipid and protein damage. De Goeij, van Steveninck et al., have concluded that protein modification and not lipid oxidation is the determining factor in the protoporphyrin-sensitized hemolysis (De Goeij and van Steveninck, 1976; De Goeij et al., 1976).

One consequence of the irradiation of proto- porphyrin-sensitized erythrocytes is the aggregation and cross-linking of membrane proteins as visualized by electron microscopy and by SDS-gel electro- phoresis (Girotti, 1976; De Goeij et al., 1975, 1976). It has been suggested that this cross-linking of mem- brane proteins plays a crucial role in cell lysis (Girotti, 1976; De Goeij and van Steveninck, 1976; De Goeij et al., 1976; Dubbleman et al., 1978).

In this report data is presented which shows that oxidative damage to the membrane can lead to increased osmotic fragility and hemolysis in the absence of cross-linked membrane proteins. That pro- tein cross-linking and lysis are not necessarily coupled is also suggested by the differential effects of reduced oxygen concentration on these two processes.

MATERIALS AND METHODS

Protoporphyrin-IX (>95%) obtained from Porphyrin Products, Inc. (Logan, UT) was used without further purifi- cation. 3/l-Hydroxy-5a-hydroperoxy-A6-cholestene (choles- terol hydroperoxide)? was prepared according to Schenck et al. (1957). All other chemicals were reagent grade. Red cells were isolated from hepar inized blood specimens obtained from two healthy volunteers. The cells were

597

598 A. A. LAMOLA and F. H. DOLEIDEN

washed twice in PBS before use which was always within 4 h after collection of the blood specimen and were kept at 4°C when not in use. Erythrocyte membranes were pre- pared according to the methods previously described (Fair- banks et al., 1971; Girotti, 1975). Membrane protein was determined according to Lowry e t al. (19.51).

Red cell suspensions were irradiated with blue light (Corning filter CS No. 5-57 and a dichroic mirror to remove infrared radiation) isolated from a tungsten iodide lamp operated at 90 W. The light beam was a 3 cm diam- eter circle at the point where it impinged the red cell sus- pension which was contained in a 50 mP round-bottomed flask capped with a rubber septum. The intensity was ap- proximately 350 W/m2, comparable to that employed by Girotti (1976). The cell suspension was stirred gently dur- ing irradiation with a magnetic stirrer and in some experi- ments was bubbled with Ar. Irradiations were carried out at room temperature.

Protoporphyrin-treated red cells were prepared as fol- lows: Packed red cells (1 mP) were suspended in 19 mP of 5 p M protoporphyrin in PBS and gently mixed at 4°C for 1 h in the dark. The erythrocytes were collected by centri- fugation and resuspended in 19 m/ of PBS. Treated cells were used immediately.

Hemolysis was determined in dense suspensions of erythrocytes by spinning down the cells and measuring hemoglobin in the supernatant solution by its absorbance at 540nm. The extent of hemolysis in dilute suspensions was also determined by light scattering measurements made with a Klett colorimeter equipped with a 630nm cut-off filter.

SDS-gel electrophoresis was carried out as described by Steck (1972) and Girotti (1976). Composite gels of 3.5% polyacrylamide-0.4% agarose were used. Membrane prep- arations were solubilized in 1% SDS and, unless otherwise noted, samples were reduced with 0.2 M dithiothreitol before electrophoresis. Coomassie blue was used to stain protein in the gels. The stained gels were photographed and scanned for OD at 5.50 nm.

Osmotic fragility data were obtained as follows: An ali- quot of a red cell suspension in PBS at a hematocrit of 5% which had been irradiated was diluted 20-fold into a saline solution of some concentration between 0.9% and 0.2%. The diluted suspension, contained in an appropriate test tube, was gently mixed for exactly 60s and then read for light transmission in the Klett colorimeter. Non-hemolyzed and totally hemolyzed specimens were read as references and the percent hemolysis was calculated. Solutions and cell suspensions were maintained at 25°C in a water bath.

RESULTS

As reported by Girotti (1976) and by De Goeij et al. (1975, 1976), irradiation of protoporphyrin-sensitized red cell with blue light in the presence of air causes rapid changes in the gel electrophoretic pattern of the SDS-denatured membrane proteins. In consonance with the findings of Girotti, we observed a progressive loss of some protein bands with irradiation and the appearance of high mol wt cross-linked polypeptides. The differences between the gel pattern obtained from an irradiated control (Fig. la) and that from protoporphyrin-sensitized cells irradiated for 20 min (Fig. lb) in the presence of air are chiefly the loss of spectrin, represented by the bands of relative mobility between 3 and 4, and the appearance of protein near the gel origin and three resolved bands of relative mobility near 0.5. These observed alterations are pro- gressive with the radiation dose but do not further

proceed in the dark after cessation of irradiation. In accordance with the previous reports these membrane protein alterations were observed to occur before sig- nificant hemolysis. For example, at the end of 20 min of irradiation there was no more hemolysis than that observed in the unirradiated control (1-273 The specimen irradiated for 20 rnin did undergo complete hemolysis upon standing for 8 h at room temperature in the dark. The control showed only 1-2% additional hemolysis. At the end of 40min of irradiation speci- mens of protoporphyrin-sensitized cells exhibited about 30% hemolysis on the average. Complete hemolysis was apparent after standing an additional 2 h in the dark.

PROTOPOR PHY R IN AIR IRRADIATED

1.5

5 8 "O 4 as

Y w 1 *.:

CHOLESTEROL a HYDROPEROXIDE

1.5 2 4 d 1.0 K

0.5

" PROTOPORPHYRIN 1.5 ARGON BUBBLED -

r IRRADIATED

0 I I I I I o a2 0.4 0.6 0.8 1.0 RELATIVE MOBILTY

Figure 1. Gel electrophoresis of erythrocyte membrane protein. Membrane suspensions containing 40 pg protein were dissolved in Sop! 1% SDS with or without 0.2M dithiothreitol and electrophoresed in 0.2% SDS on 3.0% acrylamide/0.4% agarose composite gels (Girotti, 1976). Gels were stained and scanned. The scale for relative mobi- lity follows Steck (1972). Gel patterns shown are examples including those obtained from untreated control erythro- cytes, protoporphyrin-sensitized cells irradiated 20 min in the presence of air, cells lysed due to the action of choles- terol hydroperoxide, and protoporphyrin-sensitized cells

irradiated for 40 min under an A t purge.

Research Note 599

40

60

I- % NaCP

Figure 2. Osmotic fragility curves of 0.25% suspensions of erythrocytes. The cells were sensitized with protoporphyrin and then kept dark for 40 min (A); irradiated 40 min with an A r purge to remove air (W); irradiated 20min in the presence of air (0); irradiated 40 rnin in the presence of air (0). The dashed curve is the renormalized curve for the cells irradiated 40 min in air which were already partially

lysed at the end of the irradiation period.

Cholesterol hydroperoxide was incorporated into the membranes of intact erythrocyte to the level of about 1% of the total steroid by a method previously described (Lamola et a!., 1972). When these peroxide- containing cells were incubated at 37°C at a hemato- crit of 10% in PBS for 3 h, complete hemolysis resulted. However, the gel electrophoretic patterns of the membranes prepared from such hemolysates (Fig. I c) were within experimental variability un- changed from that of membranes from untreated controls. In particular, no cross-linked membrane proteins could be observed in the gels employed. Gel electrophoresis of specimens run without prior reduction with dithiothreitol also did not reveal cross- linked proteins.

Suspension (57; hematocrit) of protoporphyrin- sensitized erythrocytes contained in a septum-capped flask could be deaerated by gentle bubbling with Ar for 1/2 h in the dark. When such deaerated cells were irradiated under continuous Ar bubbling, membrane protein alterations visualized by the gel electro- phoretic pattern were similar to those obtained upon irradiation of aerated protoporphyric red cells (Fig. Id). The production of cross-linked proteins a t the top of the gels and the loss of spectrin bands were much less extensive for the deaerated cells compared to the cells irradiated in the presence of air. Up to the longest period of irradiation employed, 40 min, the extents of production of cross-linked protein and of spectrin loss in the Ar-bubbled specimens were observed to be from 25 to 35% of those for aerated specimens.

The osmotic fragility of irradiated protoporphyrin- sensitized red cells was assessed by measurement of the extent of hemolysis after 60s of suspension in

saline solutions of reduced osmolalities. Some fragility data are shown in Fig. 2. While cells irradiated in the presence of air exhibited a large increase in osmotic fragility with only 15 rnin of irradiation, deaerated (Ar-bubbled) cells irradiated for 40 rnin exhibited a fragility curve similar to that obtained for the un- irradiated control cells.

DISCUSSION

Our observations concerning the light-induced alterations of SDS-gel electrophoretic patterns of the membrane proteins of protoporphyrin-sensitized erythrocytes are in full agreement with those reported by Girotti (1976) and by De Goeij et al. (1975, 1976). The most conspicuous effects of the radiation on the gel pattern are the loss of intensity of the spectrin bands and the appearance of bands due to higher mol wt cross-linked polypeptides. These protein changes progress with the radiation dose and d o not further increase in the dark. They therefore appear to be a more-or-less direct effect of the irradiation. The protein alterations precede hemolysis which does pro- gress in the dark. It is therefore possible that mem- brane lesions associated with these protein changes are responsible for the eventual cell lysis. However, these observations d o not demand that this be so.

Cholesterol hydroperoxide is sufficiently stable to be introduced without decomposition into the mem- branes of intact erythrocytes by steroid exchange with liposomes (Lamola et al., 1973). At a concentration of hydroperoxide corresponding to 1% of the total mem- brane steroid content, there is no change in the osmo- tic fragility of the erythrocytes due to the hydroperox- ide per se (Yamane and Lamola, 1973). After the cho- lesterol hydroperoxide is introduced at room tem- perature, the cells may be incubated at 37°C where a slow (7 2 1 h) catalyzed decomposition of the peroxide occurs. In the presence of oxygen this decomposition provides initiation events for radical chain autoxida- tion processes, the result of which is eventual cell lysis (Yamane and Lamola, 1973). The lysis induced by cholesterol hydroperoxide is inhibited by ct-toco- pherol and is accompanied by the loss of unsaturated fatty acids and loss of activity of several niembrane- associated enzymes (Yamane and Lamola, 1973; Kochevar and Lamola, 1979). Like the hemolysis which occurs upon irradiation of protoporphyric erythrocytes, the hemolysis induced with cholesterol hydroperoxide is clearly of the “colloid osmotic” type, i.e. most of the potassium ions are lost from the cells before hemolysis occurs (Yamane and Lamola, 1973).

When erythrocytes containing cholesterol hydro- peroxide at a level of the order of 1% of the mem- brane steroid level were incubated at 37°C in the pres- ence of air, complete hemolysis occurred after 2-3 h (cf. Lamola et al., 1973). The SDS-gel electrophoretic patterns of the stroma isolated from these lysates were not significantly different from those obtained from control non-hemolyzed specimens. Conspicuously

600 A. A. LAMOLA and F. H. DOLEIDEN

absent, both with and without reduction with dithio- threitol, was any evidence for cross-linked membrane proteins. Thus ‘colloid osmotic’ hemolysis due to oxi- dative damage to the erythrocyte membrane can occur by at least one pathway which does not require cross-linking of membrane proteins.

Protoporphyrin-sensitized photohemolysis appears to require molecular oxygen. No oxygen independent pathway has been demonstrated. Under the con- ditions of Ar bubbling used in the experiments de- scribed here, no hemolysis is observed during con- tinuous irradiation for 3 h, while in the presence of air complete hemolysis is achieved within 1 h of ir- radiation (cf. Schothorst et a/., 1970). An increase in the osmotic fragility of irradiated porphyric erythro- cytes is observed before hemolysis occurs. It appears, however, that this light-induced increase in fragility is not a linear function of the extent of irradiation (also, see Girotti, 1976). Therefore interpretation of data which compares fragility changes with the progress of some light-associated molecular event in the mem- brane must be made with caution. However, the observations reported here on the differential effect of a reduction in oxygen concentration upon osmotic fragility compared to membrane protein cross-linking certainly suggest that protein cross-linking is not coupled with hemolysis. Removal of most of the oxy- gen from a suspension of protoporphyric red cells apparently completely inhibits the great increase in fragility which was obtained after 40min of ir- radiation in the presence of air. However, similar re- moval of oxygen caused only about a three-fold reduction in the rate of formation of cross-linked pro- teins visualized by gel electrophoresis.

The patterns of cross-linked polypeptides for aer- ated and deaerated specimens were quite similar (cf. Figs Ib and d). Irradiation of deaerated sensitized cells for 40 min gave changes in the gel patterns com- parable to those obtained upon irradiation of aerated cells for 15 to 20 min. While little hemolysis of aerated

cells was obtained in 20min. a large increase in the osmotic fragility was observed.

It is possible that the cross-linking observed upon irradiation of Ar bubbled specimens of proto- porphyrin-sensitized cells was due to a small residual fraction of oxygen. The observation is also consistent with an oxygen independent pathway for protopor- phyrin-sensitized protein cross-linking. Whether or not the cross-linked products obtained under aerated and deaerated conditions are identical has not been tested.

In summary, the irradiation of protoporphyrin- sensitized erythrocytes leads to alterations in mem- brane proteins. These alterations are characterized by SDS-gel electrophoresis predominantly as the forma- tion of high mol wt polypeptides due to protein cross- linking. Such cross-linking can occur before hemolysis is observed. The increase in osmotic fragility and sub- sequent hemolysis due to the combined action of pro- toporphyrin and light are very severely inhibited when most of the oxygen is removed from the cell suspension. However, similar deoxygenation appar- ently reduces the extent of membrane protein cross- linking by only a factor of about three. Finally, it is possible to observe osmotic hemolysis due to oxidative membrane damage induced with cholesterol hydroperoxide without the formation of such cross- linked membrane proteins. These observations strongly suggest that the cross-linking of membrane proteins is not pcr se the major cause of lysis of irradiated por- phyric red cells. This conclusion does not preclude the involvement of other kinds of protein alterations in cell lysis.

Similar conclusions have been drawn by Deziel and Girotti (1980) for the bilirubin-sensitized photodyna- mic lysis of red cells. These investigators found that certain antioxidants inhibited the lysis of resealed erythrocyte ghosts concomitant with inhibition of lipid oxidation. However, the same antioxidants did not inhibit protein cross-linking.

REFERENCES

De Goeij, A. F. P. M., P. H. J. Th. Ververgaert and J. van Steveninck (1975) Clin. Chirn. .4cta 62, 287-292.

De Goeij, A. F. P. M. and J. van Steveninck (1976) Clin. Chim. Acta 68 115-122. De Goeij, A. F. P. M., R. J. C. van Straalen and J. van Steveninck (1976) Clin. Chim. Acra 71,

Deziel, M. R. and A. W. Girotti Dubbleman, T. M. A. R., A. F. P. M. De Goeij and J. van Steveninck (1978a) Biochim. Biophgs. Acta

Dubbleman, T. M. A. R., A. F. P. M. De Goeij and J . van Steveninck (1978b) Photochm. Photobiol.

Fairbanks, G., T. C. Steck and D. F. H. Wallach (1971) Biochernisfry 10, 2606-2617, Fleisher, A. S., L. C. Harber, J. S. Cook and R. L. Baer (1966) J . Invest. Drrrnatol. 46, 505-509. Girotti, A. W. (1976) Biochem. Biophys. Res. Comniun. 72, 1367-1374. Girotti. A. W. (1978) J . Biol. Chern. 253, 71867193. Goldstein, B. D. and L. C . Harber (1972) J . Chi. Invest. 51, 892-902. Harber. L. C.. A. S. Fleischer and R. L. Baer Hsu, J., B. D. Goldstein and L. C . Harber (1971) P/iotoc/i~wi. Pliorohiol. 13, 67-77. Kochevar, I. E. and A. A. Lamola (1979) Photochem. Photobiol. 29, 791-796. Lamola, A. A., T. Yamane and A. M. Trozzolo (1973) Science 179, 1131-1133. Lowry, 0. H., N. J. Rosebrough. A. L. Farr and R. J. Randall (1972) J . B i d . Chern. 193, 265-275. Schenck. G. 0.. K. Gollnick and A. 0. Neumuller (1957)

485-494. (1980) Photochem. Phorobiol. 31, 593-596.

511, 141-151.

28, 197-204.

(1964) J . Am. Med. Assoc. 189, 191-194.

Ann. Chem 603, 46-54.

Research Note 60 I

Schothorst, A. A,, J. van Steveninck, L. N. Went and D. Suurmond (1970) Clin. Chim. Acta 28, 41-49. Schothorst, A. A., J. van Steveninck, L. N. Went and D. Suurmond (1972) Clin. Chim. Actn 31,

Schothorst, A. A,, J. van Steveninck, L. N. Went and D. Suurmond (1974) Clin. Chim. Acta 33,

Steck, T. L. (1972) J. Mol. B i d . 66, 295-305 Yamane, T. and A. A . Lamola (1973)

485494.

207-2 13.

American Societyfor Phqtobioloy)i-Abstracts 1, 66; and unpub- lished results.