15

Molecular and Cellular Biochemistry 228: 15–23, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

Bilirubin binding to normal and modified humanerythrocyte membranes: Effect of phospholipases,neuraminidase, trypsin and CaCl

2

Huma Rashid, Mohammad Owais and Saad TayyabInterdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh, India

Received 20 April 2001; accepted 31 July 2001

Abstract

Binding of bilirubin to human erythrocyte membranes was studied after various enzymatic treatments as well as calcium load-ing. Whereas phospholipase D treatment of erythrocyte membranes resulted in 23% increase in bilirubin binding, phospholi-pase C-treated membranes showed remarkable enhancement in bilirubin binding. Polar head groups in general and negativelycharged phosphate moieties, in particular, of phospholipids of the membrane appear to inhibit a large amount of bilirubin frombinding to the membranes. Neuraminidase treatment of the membranes also led to a slight increase in bilirubin binding ascompared to untreated membranes. Membrane proteins and carbohydrates seem to play significant regulatory role in bilirubinbinding. However, no direct correlation was found between the increase in bilirubin binding and the amount of carbohydratereleased upon tryptic digestion of membranes. Increase in bilirubin binding to trypsin-treated membranes can be ascribed tothe increase in free bilirubin concentration in the incubation mixture as a result of tryptic hydrolysis of albumin by the mem-brane-bound tryptic activity. Calcium-loaded erythrocyte membranes also showed remarkable increase in bilirubin binding asa result of negative charge shielding and calcium-induced hydrophobic aggregation. Taken together, these results suggest theinhibitory role of polar head groups of phospholipids (phosphate in particular), carbohydrate and sialic acid in the binding ofbilirubin to erythrocyte membranes. (Mol Cell Biochem 228: 15–23, 2001)

Key words: bilirubin, CaCl2, erythrocyte membrane, neuraminidase, phospholipase C, phospholipase D, trypsin

of these binding sites on the erythrocyte membranes have notbeen fully worked out.

Bilirubin binds to both outer and inner layers of erythro-cyte membrane [18], being more specific to the outer layer[19]. Though, it is accepted that membrane phospholipids aredirectly involved in bilirubin binding, contradictory resultshave been reported in terms of the involvement of specificgroups of phospholipids, i.e. polar head groups or non-polarmoieties, as well as mode of interaction [20, 21]. Vazquez etal. [22] reported a rapid electrostatic interaction between ani-onic bilirubin and polar head groups on membrane surface,followed by inclusion of bilirubin into the hydrophobic coreof membrane leading to membrane-induced aggregation ofbilirubin acid. Nagaoka and Cowger [20] also showed the

Introduction

The interaction of bilirubin, a bile pigment, with cellularmembranes may have important physiological implicationsin the pathogenesis of kernicterus (bilirubin-induced enceph-alopathy) in newborns [1–3]. Despite extensive studies on bi-lirubin-membrane interaction, the toxicity of bilirubin, itsentry mechanism and localization in the bilayer have not yetbeen completely elucidated [4–9]. Both free diffusion of bi-lirubin through biological membranes as well as facilitatedmode of binding have been reported [10–12]. Though, thepresence of bilirubin binding sites on biological membraneshas been suggested [13–18], characterization of the individualmembrane component involved in binding and the location

Address for offprints: S. Tayyab, Interdisciplinary Biotechnology Unit, Aligarh Muslim University, Aligarh 202002, India (E-mail: btisamua@nde.vsnl.net.in)

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involvement of ionic interactions between a cationic headgroup of the lipid and anionic bilirubin. If these conclusionsare correct, then phospholipase D treatment of the membranesshould reduce the binding of bilirubin to the membrane. Con-trary to this, phospholipase D-treated membranes showed ahigher binding constant whereas phospholipase C treatmentresulted in a decrease in binding constant for bilirubin-mem-brane interaction [22]. Further, no change in bilirubin-bind-ing properties of the membrane was observed by Sato andhis group [18] after phospholipase D treatment whereas phos-pholipase C treatment greatly enhanced bilirubin binding.Recently, we have shown [23] that negatively charged phos-phate moieties of phospholipids inhibit a large amount ofbilirubin from binding to the membrane as their removal byphospholipase C greatly enhanced the binding. This seemsto be understandable in terms of the large increase in non-specific binding of bilirubin to the non-polar fatty acid moi-ety of diacylglycerols [18].

It has been suggested that membrane proteins function aseffective barriers to bilirubin binding based on the results ofincreased bilirubin binding with trypsinized membranes [13].These results seem to be questionable in view of the mem-brane-bound tryptic activity [24], which may digest the al-bumin present in the incubation mixture thereby increase thefree bilirubin concentration. Moreover, membrane proteinsmight have some type of non-specific effect on the proper-ties of lipids in bilayers, thus enhancing the capacity of thebilayer to sequester bilirubin as compared with protein-freebilayers [25]. All these contradictory results call for reinvesti-gation of bilirubin binding properties of membrane. There-fore, we investigated in detail the effect of phospholipases Cand D, trypsin, neuraminidase and Ca2+ on the mode of bind-ing of bilirubin to erythrocyte membranes. Here we presentour data on the bilirubin binding properties of normal andmodified human erythrocyte membranes.

Materials and methods

Materials

Phospholipase C, type XIV from Clostridium welchii (Lot55H6334), phospholipase D, type I from cabbage (Lot 57H0373), phenylmethylsulfonylfluoride (PMSF) (Lot 67H1645),neuraminidase, type V from Clostridium perfringens (Lot31H82302), trypsin, TPCK-treated from bovine pancreas(Lot 117H7261) and bovine serum albumin, fraction V werepurchased from Sigma Chemical Co., USA. Bilirubin wasprocured from Sisco Research Laboratories, India. CaCl

2 was

obtained from Qualigens Fine Chemicals, India. Human se-rum albumin was isolated by the method of Tayyab and Qasim[26]. Other reagents used were of analytical grade.

Human blood (in 1.32% sodium citrate and 1.47% dex-trose) was obtained from the blood bank of J.N. MedicalCollege, Aligarh Muslim University, Aligarh, India.

Preparation of erythrocyte membrane suspension

Blood was centrifuged at 1000 × g for 20 min at 4°C. Plasmaand buffy coat were removed by careful aspiration and cellswere then resuspended in 50 mM Tris/HCl buffer, pH 7.4containing 150 mM NaCl followed by triple washing withthe same buffer. After each step of centrifugation, the surfaceof the pellet was thoroughly aspirated. The packed, washedcells were diluted with an equal volume of the same bufferto obtain 50% hematocrit value.

Erythrocyte membranes were prepared in the same way asdescribed by Palfrey and Waseem [27]. Erythrocyte suspen-sion of 50% hematocrit value was hemolyzed with 10 vol.of cold 10 mM Tris/HCl buffer, pH 7.4 containing 0.01 mMEDTA and 0.01 mM PMSF followed by gentle swirling andcentrifugation at 16,000 × g for 20 min at 4°C. The dark redsupernatant was removed carefully by gentle aspiration. Eachtube was tilted and rotated to allow the loose ghost pellet toslide off to another tube, leaving the tightly packed ‘buttons’at the bottom of the tube. This minimized the contaminationof the ghost with proteinases as suggested by Fairbanks etal. [28]. The ghost pellet, thus obtained, was washed severaltimes with the same buffer followed by centrifugation at16,000 × g for 20 min at 4°C until membranes were free fromhemoglobin. After final washing, the erythrocyte membranepellet was resuspended in the same hemolyzing buffer suchthat the total volume of membrane suspension was equal tothe volume of erythrocyte suspension of 50% hematocritvalue initially taken for hemolysis. This suspension wasstored at 10°C and washed once with 50 mM Tris/HCl buffer,pH 7.4 prior to use and the volume was restored to the initialvolume taken with the same buffer. However, erythrocytemembrane suspension stored in 10 mM Tris/HCl buffer, pH7.4 could be used within 5 days when kept at 10°C.

Enzymatic and other treatments of erythrocyte membranes

Phospholipase C treatment of erythrocyte membranes wasperformed according to the method of Sato et al. [18]. To 0.5ml of membrane suspension (~4 mg protein/ml) in 50 mM Tris/HCl buffer, pH 7.4 was added 4.25 ml of 0.1 M Tris/HCl buffer,pH 7.4, 50 µl of 1.0 M CaCl

2 and 200 µl of the enzyme solu-

tion (0.15 mg/ml) and the mixture was incubated for differenttime periods at 37°C. After a desired period of incubation, allthe contents were centrifuged at 16,000 × g for 20 min at 4°Cand the released phosphate (Pi) in the supernatant was meas-ured by the method of Chen et al. [29]. The treated membrane

17

pellet was washed once with cold 50 mM Tris/HCl buffer, pH7.4 containing 10 mM EDTA followed by centrifugation at16,000 × g for 20 min at 4°C. This reaction mixture for thebinding assay became turbid during the incubation with phos-pholipase C and turbidity increased with time.

Phospholipase D digestion of erythrocyte membranes wascarried out following the method of Sato et al. [18]. To 0.5ml of erythrocyte membrane suspension was added 1.4 mlof 0.1 M Tris/HCl buffer, pH 7.4 and 100 µl of enzyme solu-tion (9.3 units). The mixture was incubated for different timeperiods at 37°C. After the desired period of incubation, thecontents were centrifuged at 16,000 × g for 20 min at 4°C.The supernatant was assayed for released choline by themethod of Kates and Sastry [30].

Ghost suspension (0.5 ml) was digested with neuraminidase(2 units) for different time periods at 37°C in 1.0 ml of 0.1 MTris/HCl buffer, pH 7.4. The mixture was centrifuged at 16,000× g for 20 min at 4°C after desired incubation and the super-natant containing released sialic acid was collected and sub-jected to sialic acid estimation as described by Warren [31].

Method of Steck et al. [32] was used for tryptic digestionof erythrocyte membrane which was performed by incubat-ing 0.5 ml of membrane suspension with 1.0 ml of 2.5 mMTris/HCl buffer, pH 8.1 containing 5 mg of solid trypsin at37°C and the mixture was shaken gently to dissolve the en-zyme completely. After the desired time of incubation, themixtures were centrifuged at 16,000 × g for 20 min at 4°C andthe released glycopeptides in the supernatant were measuredfor carbohydrate content according to Svennerholm [33].

Membrane suspension (0.5 ml) was incubated with differ-ent volumes of 1.0 M CaCl

2 in 50 mM Tris/HCl buffer, pH

7.4 making the total volume of 1.5 ml for about 30 min at37°C. Unbound metal ions were removed by centrifugationof the membranes at 16,000 × g for 20 min at 4°C.

Treated membranes stated above were washed with cold50 mM Tris/HCl buffer, pH 7.4 twice followed by centrifuga-tion at 16,000 × g for 20 min at 4°C and the final pellet, thusobtained, was resuspended in the same buffer after making upthe volume to 1.0 ml. These preparations were directly usedfor bilirubin binding experiments.

Extraction and analysis of phospholipids

Untreated and phospholipase C-treated membranes were sub-jected to phospholipid extraction by the method of Gier andDeenen [34]. To 1.0 ml of membrane suspension was added5.0 ml of isopropanol with intermittent shaking. After 2 h,2.0 ml of chloroform was added and the suspension was keptovernight at 10°C. The supernatant, obtained by centrifuga-tion at 16,000 × g for 20 min, was dried under reduced pres-sure in a rotary evaporator, washed thrice with benzene andfinally dissolved in 500 µl of chloroform.

The phospholipids, thus obtained, were separated by two-dimensional thin-layer chromatography following the meth-od of Dittmer and Wells [35] on silica impregnated aluminiumsheets (20 × 20 cm, silica gel 60F254 from Merck, Germany)using a pair of solvents introduced by Rouser [36]. Quanti-tative analysis of individual spots on the sheet was done af-ter scrapping off each spot and subjecting it to inorganicphosphate estimation according to Bartlett [37].

Other methods

Phospholipid content of erythrocyte membranes was deter-mined by the method of Chen et al. [29] after extraction ofphospholipids according to Gier and Deenen [34]. Proteinconcentration was determined by the method of Lowry et al.[38] after solubilization of membranes in 1% SDS. Total sialicacid was estimated after digesting the membranes in 0.1 NH

2SO

4 for 1 h at 80°C following the method of Warren [31].

Orcinol-H2SO

4 method was used for the determination of

carbohydrate content [33].

Bilirubin binding experiments

Bilirubin solution was prepared by dissolving a few crystalsof bilirubin in 38 mM sodium carbonate solution containing5 mM EDTA, pH 11.0. The concentration of bilirubin solu-tion was determined by Fog’s method [39]. However, forbilirubin binding study with CaCl

2-treated membranes, EDTA

was excluded from the solubilizing medium.Binding of bilirubin to erythrocyte membrane (untreated/

treated) was studied by incubating 1.0 ml of the membranepreparation in a final volume of 1.5 ml containing 250 µl ofbilirubin (150 nmoles for experiments with phospholipase C-and CaCl

2-treated membranes and 225 nmoles for rest of the

membranes) and 250 µl of albumin solution in 50 mM Tris/HCl buffer, pH 7.4 to achieve a bilirubin/albumin molar ra-tio (B/A) of 2.0. After 30 min of incubation at 37°C, the mix-ture was centrifuged at 16,000 × g for 20 min at 4°C andthe supernatant containing unbound bilirubin was discarded.Membranes were rinsed several times with the same bufferuntil the last supernatant was devoid of yellow color and themembrane-bound bilirubin was determined by modified Fog’smethod as described by Tayyab and Ali [40].

Results and discussion

The effect of phospholipase C (C. welchii) treatment of hu-man erythrocyte membranes can be seen from Fig. 1 wherethe amount of membrane Pi released is plotted against time

18

of incubation of the membranes with the enzyme. A signifi-cant increase in the amount of released Pi was observed upto 40 min of incubation, which became constant thereafter.Using the value of total membrane Pi content (Table 1), theenzyme treatment resulted in the release of about 62.3% ofthe membrane Pi. Similar enzymatic treatment of bilirubin-bound membranes (previously incubated with 450 nmoles ofbilirubin at B/A of 2.0 and washed subsequently) showed asignificant reduction in the amount of Pi released for the ini-tial time periods up to 30 min. However, increase in the timeof incubation up to 60 min was found to produce the similarrelease of membrane Pi as found with unbound membranes.Recalling that phospholipase C treatment of erythrocytemembranes results in the release of polar head groups from

phosphatidylcholine (PC) and sphingomyelin (Sph) fromthe outer layer of erythrocyte membranes, the lesser releaseof membrane Pi in the early periods observed with bilirubin-bound membranes can be ascribed to the protective effectof bound bilirubin. It appears that removal of a few polarhead groups of phospholipids from the outer layer facili-tates the release of more Pi over a period of time, whichseems to be hindered in the initial phase by the membrane-bound bilirubin. Extensive enzymatic treatment of the mem-branes led to enhanced packing of the ghosts, however, thecellular morphology is retained as suggested by Lenard andSinger [41].

Incubation of untreated as well as phospholipase C-treatedhuman erythrocyte membranes with constant amount of bi-lirubin (150 nmoles at B/A of 2.0) led to an increase in themembrane-bound bilirubin. As much as 121% increase inmembrane-bound bilirubin was noticed with the modifiedmembranes treated with enzyme for 40 min (Fig. 1). This in-crease in the membrane-bound bilirubin upon phospholipaseC treatment correlated very well with the release of membranePi which suggests that polar head groups of phospholipids playinhibitory role in the bilirubin-binding phenomenon. Even avery short (5 min) treatment of the membranes with phos-pholipase C resulted in 18% increase in membrane-boundbilirubin. These results were similar to the results observedby Sato and his group [18]. As phospholipase C treatment ofbilirubin-bound membranes failed to release the bound bi-lirubin from these membranes, which otherwise resulted inthe release of Pi, it appears that bilirubin binding to the mem-branes involves hydrophobic interactions. This is supportedby an earlier finding in which it has been suggested that bi-lirubin is hydrophobically inserted into the apolar region ofthe bilayer [42].

Degradation of phospholipids of human erythrocyte mem-branes by phospholipase C as analysed by thin-layer chro-matography (Table 2) shows about 70% degradation of totalphospholipids, which comprised of total degradation of Sph,86% degradation of PC and 28% of phosphatidylethanol-amine (PE) after 20 min of enzyme treatment. This suggeststhat exposure of diacylglycerols or ceramides potentiated alarge increase in the membrane-bound bilirubin.

Fig. 1. Time course of phospholipase C (C. welchii) treatment of humanerythrocyte membranes as monitored by Pi release and its effect on thebinding of bilirubin. Open circles show the amount of Pi released uponenzyme treatment from normal membranes whereas closed circles repre-sent Pi released from bilirubin-bound membranes. Amount of bilirubinbound to phospholipase C-treated membranes is shown by closed triangles.Each point is the mean of two independent experiments.

Table 2. Quantitative analysis of phospholipid degradation of human eryth-rocyte membrane by phospholipase C under different time periods

Time % Phospholipid recovered after treatment(min)

Total PE PC Sph

0 88.1 ± 7.8 23.9 ± 1.8 30.1 ± 2.8 23.8 ± 1.220 26.4 ± 6.0 17.0 ± 2.8 4.3 ± 1.8 0.040 25.7 ± 4.6 15.3 ± 2.2 4.7 ± 1.1 0.060 26.5 ± 4.6 17.2 ± 2.1 4.2 ± 1.6 0.0

Each value represents the mean ± S.D. of six observations obtained withthree different experiments.

Table 1. Chemical composition of human erythrocyte membranes (equiva-lent to 1.0 ml of 50 % hematocrit value)

Component Amount

Protein (mg) 3.8 ± 0.2Total phosphorus (Pi, µg) 111.7 ± 4.2Organic phosphorus* (µg) 53.0 ± 1.7Phospholipid (mg) 1.3 ± 1.7Sialic acid (nmoles) 319.5 ± 8.7

*Pi as determined in isopropanol-chloroform extracted phospholipids.Each value is the mean ± S.D. of 12 observations obtained with six differ-ent membrane preparations.

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Phospholipase D treatment of erythrocyte membranes forvarying time periods was monitored by the estimation ofcholine released (Fig. 2) as the enzyme is known to removethe polar head groups from PC, PE and phosphatidylserine(PS) to produce phosphatidic acid. The enzymatic digestionof membranes for 60 min resulted in the release of 47.6 µg ofcholine/mg of protein, which corresponded to the degradationof about 45% of PC. Presence of bilirubin at the membranesurface, as observed with erythrocyte membranes pre-treatedwith 450 nmoles of bilirubin at B/A of 2.0, significantly in-hibited the release of choline as 38.5% degradation of PC wasnoticed after phospholipase D treatment for 60 min. The lowerextent of choline released from bilirubin-bound membranesobserved at prolonged enzyme treatment can be attributed tothe inaccessibility of enzyme cleavage sites due to boundbilirubin. Lack of difference in the amount of choline releasedfrom both normal and bilirubin-bound erythrocyte mem-branes at shorter incubation with the enzyme and the sig-nificant reduction in the amount of choline released frombilirubin-bound membranes compared to normal membranesat prolonged incubation of enzyme with these membranessuggest that release of few choline molecules from PC resultsin the clustering of bilirubin molecules around PC molecules,thereby, making some of the cleavage sites inaccessible to theenzyme.

Bilirubin binding results obtained with phospholipase D-treated membranes at B/A of 2.0 (with 225 nmoles of bi-

lirubin) showed 23% increase in the bound bilirubin uponprolonged enzyme treatment (60 min) compared to untreatedmembranes (Fig. 2). Although these results suggested theinhibitory role of polar head groups in bilirubin-binding tothe membranes to some extent, bilirubin-binding results ob-tained with phospholipase C-treated membranes were foundto be more convincing as 121% increase in bilirubin-bind-ing was observed upon phospholipase C treatment against23% increase in phospholipase D-treated membranes. Fromthese results it appears that the presence of negative chargecontributed by Pi restricts the bilirubin entry inside the hy-drophobic milieu of membrane to a greater extent. In viewof the stimulation of endogenous phospholipases (C and D)by receptor tyrosine kinases [43], growth factors [44], tu-mour necrosis factor α [45] etc., it is likely that membranesof various cells may be acted upon by these phospholipasesand thus become more prone towards bilirubin toxicity.Therefore, activities of endogenous phospholipases shouldbe checked to prevent bilirubin toxicity under jaundicedconditions.

Incubation of human erythrocyte membranes with neu-raminidase for varying time periods resulted in the releaseof sialic acid. Time course of neuraminidase-treatment oferythrocyte membranes is shown in Fig. 3, which suggeststhat greater incubation of membranes with the enzyme ledto a greater release of sialic acid that became constant after60 min. A value of 84.1 nmoles of sialic acid/mg of protein

Fig. 2. Time course of phospholipase D (cabbage) treatment of humanerythrocyte membranes as monitored by choline release and its effect onthe binding of bilirubin. Open circles show the amount of choline releasedupon enzyme treatment from normal membranes whereas closed circles rep-resent choline released from bilirubin-bound membranes. Amount of bi-lirubin bound to phospholipase D-treated membranes is shown by closedtriangles. Each point is the mean of two independent experiments.

Fig. 3. Time course of neuraminidase (C. perfringens) treatment of humanerythrocyte membranes as monitored by sialic acid release and its effect onthe binding of bilirubin. Open circles show the amount of sialic acid releasedupon enzyme treatment from normal membranes whereas closed circlesrepresent sialic acid released from bilirubin-bound membranes. Amount ofbilirubin bound to neuraminidase-treated membranes is shown by closedtriangles. Each point is the mean of two independent experiments.

20

(Table 1) was determined for untreated membranes. Thisvalue was found to be higher than the value reported by Satoet al. (50.9 nmoles of sialic acid/mg of protein) [18] but wasin good agreement with the value reported by Choy [46].Neuraminidase treatment for 2 h resulted in the release ofabout 77% of sialic acid whereas Sato et al. [18] reported therelease of 88% of sialic acid upon neuraminidase treatment.The possible discrepancy between our results and those re-ported earlier can be ascribed to the lower values of sialic acidin untreated membranes obtained by Sato and his group [18].Bilirubin-bound membranes (preincubated with 450 nmolesof bilirubin at B/A of 2.0) produced the same results uponneuraminidase treatment as compared to those observed withcontrol membranes. The only difference was observed in theinitial phase (up to 20 min) of enzyme treatment where bi-lirubin-bound membranes showed some protection as judgedby the lesser release of sialic acid from these membranes. Itappears that the presence of bilirubin at the membrane sur-face offers some resistance to the enzymatic attack on someof the cleavage sites for the initial time period which alsobecome accessible to the enzyme during prolonged enzymetreatment.

Both membrane preparations (control as well as neurami-nidase-treated for various time periods) were used for bi-lirubin binding studies after incubation with 225 nmoles ofbilirubin at B/A of 2.0 and the results are shown in Fig. 3. Ascan be seen from the figure, 1 h enzyme treatment resultedin 41% increase in the bound bilirubin compared to untreatedmembranes. This increase in bound bilirubin upon neurami-nidase-treatment correlated to some extent with the releaseof sialic acid from membrane surface, which suggests someinhibitory role of sialic acid residues in the bilirubin-bind-ing phenomenon to the membranes. Although these resultsare contradictory in terms of the increase in bilirubin bind-ing upon neuraminidase-treatment with those of Sato and hisgroup [18] but agree very well in excluding the gangliosidesas the candidates for bilirubin binding.

Trypsin treatment of erythrocyte membranes resulted in therelease of glycopeptides, which was monitored by carbohy-drate measurement. Results of tryptic digestion of erythro-cyte membranes are shown in Fig. 4A, in which the amountof carbohydrate released is plotted against time of incubationof enzyme with the membrane. As is clear from the figure,there was significant increase in the amount of carbohydratereleased on increasing the time of incubation up to 60 minbeyond which it sloped off. As much as 74% of total mem-brane carbohydrate was released in 3 h. When tryptic diges-tion of bilirubin-bound membranes (previously treated with450 nmoles of bilirubin at B/A of 2.0) was performed undersimilar conditions, a slight decrease in the amount of carbo-hydrate released was noticed. This is understandable, as someof the tryptic cleavage sites of membrane proteins must havebeen protected by the bound bilirubin.

A significant increase in bilirubin binding to the mem-branes was noticed upon trypsin treatment when these treatedmembranes were incubated with 225 nmoles of bilirubin for30 min. An analysis of bilirubin-binding data of treated mem-branes and untreated membranes showed about 57% increasein the binding of bilirubin upon 3 h trypsin treatment of themembranes. These results were similar to those reported bySato and Kashiwamata [13] who suggested that the trypsintreatment of the membranes disclosed a great number of low-affinity bilirubin binding sites and that the bilirubin bindingsites are not composed of proteins. These conclusions seem tobe questionable in view of the retention of activity of mem-brane-bound trypsin [24] which may cleave the albuminpresent in the incubation medium, thereby, increase the freebilirubin concentration in the incubation mixture, being acces-sible to the membranes. To check this possibility, we estimatedthe protein content both in untreated and trypsin-treated mem-branes. As trypsin treatment of the membranes is supposed

Fig. 4. (A) Time course of trypsin treatment of human erythrocyte mem-branes as monitored by carbohydrate release and its effect on the bindingof bilirubin. Open circles show the amount of carbohydrate released uponenzyme treatment from normal membranes whereas closed circles repre-sent carbohydrate released from bilirubin-bound membranes. Amount ofbilirubin bound to trypsin-treated membranes is shown by closed triangles.Each point is the mean of two independent experiments. (B) Binding oftrypsin to human erythrocyte membranes as monitored by the measurementof protein content in the membrane suspension. Each point is the averageof two separate determinations.

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to degrade the membrane proteins, one should expect a de-creased protein content in trypsin-treated membranes com-pared to untreated membranes. Contrary to this, an increasein the amount of membrane protein was observed upon tryp-sin treatment (Fig. 4B), which suggested the binding of tryp-sin to the cell membranes. If this is really the case thenincubation of these trypsin-treated membranes (with boundenzyme) with bilirubin-albumin mixture at B/A of 2.0 for 30min should degrade the albumin present in the medium. Whenboth untreated as well as trypsin-treated membranes wereincubated with bilirubin-albumin mixture under the condi-tions of bilirubin binding experiment and the supernatant ob-tained after centrifugation was analysed by SDS-PAGE [28],

significant degradation of albumin was observed as shownin Fig. 5A. Further, it should be noted that trypsin treatmentof the membrane for more than 1 h did not result in any in-crease in membrane-bound enzyme activity as similar al-bumin degradative pattern was observed with all the threetrypsin-treated membranes (see Fig. 5A). This is also under-standable from Fig. 4B in which no further increase in mem-brane protein was observed after 1 h enzyme treatment. Inorder to verify these results, 3 h trypsin-treated membraneswere incubated with increasing concentrations of albumin andthe supernatants obtained after centrifugation were analysedby SDS-PAGE. The results are shown in Fig. 5B in which theintensity of low molecular weight degradative products wasfound to increase with the increase in albumin concentrationin the incubate. Taken together, all these results (Figs 4B, 5Aand 5B) unequivocally suggest the degradation of albuminby the membrane-bound activity found in trypsin-treatedmembranes. Therefore, degradation of albumin in the in-cubation mixture will certainly increase the free bilirubinconcentration in the incubation medium, which may be avail-able to the trypsin-treated membranes. In view of this, in-creased bilirubin binding to erythrocyte membranes upontrypsin treatment cannot be ascribed solely to the degrada-tion of membrane proteins which have been suggested toact as a barrier in bilirubin binding to the membranes [18].From our results it appears that removal of carbohydratefrom membrane surface exposes more surface area of themembrane for bilirubin to interact.

The effect of Ca2+ ions on the binding of bilirubin to hu-man erythrocyte membranes was studied after incubatingthe membranes with different concentrations of CaCl

2 in the

range of 0–1.5 mM and then using these membranes after

A

B

Fig. 5. (A) SDS-PAGE analysis of the supernatant obtained by centrifu-gation of the incubation mixture containing untreated/trypsin-treated eryth-rocyte membranes and bilirubin-albumin mixture at B/A of 2.0. Lane 1shows the result when untreated membranes were taken whereas lanes 2, 3and 4 represent the results obtained with trypsin-treated (1, 2 and 3 h, re-spectively) membranes. Lanes 5, 6 and 7 show the results when trypsin-treated membranes were incubated with bilirubin in the absence of albumin.(B) SDS-PAGE analysis of the supernatant obtained by centrifugation ofthe incubation mixture containing 3 h trypsin-treated erythrocyte membranesand increasing concentrations of albumin. Lanes 1–7 show the results ob-tained when the concentration of albumin in the incubation mixture was 0,25, 50, 75, 100, 125, 150 µM, respectively.

Fig. 6. Effect of CaCl2 treatment of human erythrocyte membranes on thebinding of bilirubin. Each point is the average of two separate determinations.

22

washing, for bilirubin-binding experiments. Incubation ofCa2+-treated membranes with 150 nmoles of bilirubin at B/Aof 2.0 for 30 min resulted in an increase in membrane boundbilirubin as compared to the amount of bound-bilirubin ob-served with control membranes. This increase in membrane-bound bilirubin was dependent on CaCl

2 concentration as the

binding increased on increasing the CaCl2 concentration as

shown in Fig. 6. As much as 85% increase in the amount ofmembrane-bound bilirubin was observed with 1.0 mM CaCl

2

treatment beyond which it sloped off. The increase in bi-lirubin binding upon CaCl

2-treatment can be ascribed to the

shielding effect, redistribution of phospholipids as well as in-crease in surface hydrophobicity induced by calcium [47].It seems likely that this effect may also be mediated by en-dogenous phospholipases as activation of phospholipase Cin nonexcitable cells has been shown to cause the release ofCa2+ from intracellular stores [48].

In conclusion, these results suggest that polar head groups(phosphate in particular) of phospholipids along with carbo-hydrate and sialic acid produce hindrance in the binding ofbilirubin to erythrocyte membranes. Further, binding of bi-lirubin to the membrane seems to be hydrophobic in nature.

Acknowledgements

This work was financially supported by a research grant (SP/SO/D-52/95) from the Department of Science and Technol-ogy, New Delhi, India. Facilities provided by Aligarh Mus-lim University, Aligarh are gratefully acknowledged.

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