Influence of heat treatment on rabbit liver ferritin

202

Bioehimica etBiophvsicaActa, 745 (1983) 202-208 Elsevier

BBA 31627

INFLUENCE OF HEAT TREATMENT O N RABBIT LIVER FERRITIN ERIK J. FRENKEL *, BEP VAN DEN BELD, BERNARD A. VAN OOST ** and JOANNES J.M. MARX

Department of Haematology, University Hospital Utreeht, Catharijnesingel 101, 35ll GV Utrecht (The Netherlands) (Received November 17th, 1982)

Key words: Heat treatment; Ferritin; (Rabbit liver)

Ferritin was purified from rabbit livers either by heat treatment and immunoaffinity chromatography, or by immunoaffinity chromatography alone. The immunoreactivity of ferritin with antibodies raised against heat-treated ferritin was significantly higher for heat-treated preparations than for non-heated preparations. The amount of ferritin protein could be estimated with equal reliability by the assay according to Lowry et al. and by nitrogen determination. Heat treatment favoured the L-subunit-rich ferritin fraction, as measured by densitometric scanning of S D S gradient-pore polyacrylamide gels. Amino acid analysis showed small changes in the amounts of valine, isoleucine and histidine in the heat-treated ferritin, possibly due to selective partial degradation of H-subunit-rich forms of ferritin. These results illustrate that heat treatment, which is a commonly used step in most purification procedures, induces partial denaturation of the ferritin molecules.

Introduction Ferritin is an almost-spherical, water-soluble protein with an apparent M r of 450000, and an external diameter of approx 12 nm, which is composed of 24 rather similar subunits [1,2]. These subunits, each with an apparent M r of about 19 000, surround a central cavity which can accommodate up to 4000 iron atoms per molecule of ferritin [3]. Ferritins extracted from different organs show distinct electrophoretic components on isoelectrofocussing with a p I between 4.5 and 5.5. The differences are mainly a result of the subunit composition. Two types of subunit can be discriminated: the heavier H-type subunits predominating in the more acidic ferritins extracted from heart tissue, and the lighter L-type subunits

* To whom correspondence should be addressed. ** Present address: Thrombosis Research Center, Temple University, Philadelphia, PA 19140, U.S.A. Abbreviations: L- and H-type subunits, light- and heavy-type subunits. 0167-4838/83/$03.00 © 1983 Elsevier Science Publishers B.V.

predominating in the more basic ferritins from liver tissue [4]. The iron content in the central cavity of the protein, which is highest in most basic ferritins, affects only slightly the electrofocussing profiles [5,6]. The highly ordered structure of ferritin results in an extraordinary stability with respect to heat and other denaturating conditions. The classical isolation procedure therefore includes heat treatment of a tissue homogenate at 75°C for 10 min [7-9], serving a two-fold purpose:(i) most contaminating proteins coagulate and can be removed easily by centrifugation;(ii) the sample can be freed from proteolytic action, because most proteolytic enzymes do not resist this treatment. Another common isolation procedure includes repeated ultracentrifugation, taking advantage of the greater density of iron-loaded ferritin molecules [10,11]. Due to the greater stability with respect to temperature of iron-rich ferritins, both procedures tend to favour iron-loaded forms. Most investigations on ferritin have used ferritin purified by methods which included heat treatment [12,13]. The results of the present paper

203

show that heat treatment has specific effects on the composition and properties of ferritin, and is therefore less suitable for purification purposes. Materials and Methods

All chemicals were of analytical grade and purchased either from Merck, Darmstadt, F.R.G., or from BDH, Poole, U.K. Fresh rabbit livers were obtained from the National Institute of Public Health RIV, Bilthoven, The Netherlands. Isoelectrofocussing agarose, Isogel Agarose EF and Ampholines (pH 4.0-6.5) were obtained from LKB, Bromma, Sweden).

Ferritin purification for the production of antibodies Fresh rabbit liver (100 g) was homogenized for 4 min at 4°C in 150 ml 0.01 M phosphate buffer, pH 7.4, containing 0.15 M sodium chloride, 10 -3 M benzamidine hydrochloride, 5 • 10-5 M phenylmethylsulphonyl fluoride and 3.10 -3 M sodium azide, using a blender (Braun MX 32). The homogenate was heated for 10 min at 75°C in portions of 100 ml under continuous stirring, followed by rapid cooling to 4°C. Heat coagulate was removed by centrifugation (10 min, 3500 x g, 4°C) and the supernatant was brought to 60% ammonium sulphate saturation. The sample was stored overnight at 4°C. After centrifugation (10 min, 3500 x g, 4°C, Sorval RC 3B) the pellet was redissolved in 0.15 M sodium chloride, concentrated by ultrafiltration (Amicon XM-300 membranes) and subsequently diluted five times and once more concentrated. During this step most of the remnant contaminating small proteins were removed, as judged by polyacrylamide gel electrophoresis. The iron-loaded fraction was purified by repeating the following scheme nine times: centrifugation for 30 min at 10000 × g and discarding the pelleted material; ultracentrifugation for 3 h at 4°C and 100000 x g and discarding the supernatant; resuspension of the pelleted material in Tris-HC1 buffer, pH 7.4. The purity of the preparation was confirmed by electrophoresis on 7% polyacrylamide gels [14] and staining for protein with Coomassie brilliant blue [15] and for iron with potassium ferrocyanide,

which both showed the same bands for ferritin and for its dimer.

Preparation of goat ant#rabbit liver ferritin antibody A goat was immunized by subcutaneous injection in multiple sites using 200 ttg heat-treated purified rabbit liver ferritin. The immunization procedure consisted of four successive subcutaneous injections every 10 days. The initial injection was given in Freunds' complete adjuvant, the subsequent booster injections in incomplete adjuvant. Antibody response was tested by the Ouchterlony technique [16] against rabbit liver ferritin and total rabbit serum. The latter did not show any precipitation line, the first only a single one. The immunoglobulin fraction was partially purified by ammonium sulphate precipitation followed by ion-exchange chromatography on DEAE-cellulose (Whatman DE 52).

Purification of rabbit liver ferritin All ferritin samples which were analyzed in this study were purified as summarized in Fig. 1, from either fresh rabbit liver or rabbit liver which was stored for not more than 14 days at -60°C. After

PURIFICATION PROCEDURE OF RABBIT LIVER FERRITIN

OMOGENIZATION:blender, 5 rnin. PBS with proteolysis inhbitors HEAT-TREATMENT:l~-imin,75°C, J I rapd heating, c o o l i n ~ CENTRIFUGATION:20 rain,3500 x g, If trot on w th Whotmon GF/D

I

AMMONIUM SULPHATEPRECIPITATION I in 60 % ornmoniumsulphQte,pH 6.6 | IMMUNO-AFFINITY CHROMATOGRAPHY goat IgG; CNBr-Sephorose-/. B

IGELFILTRATION 1 SepharoseCL6B; 50mMTRISpH7./., Fig. 1. Flow diagram for the two methods used for isolating ferritin from rabbit liver. PBS, phosphate-buffered saline.

204

thorough cleaning, the organs were homogenized at 0°C using an Ystral type X 1020 homogenizer, equipped with a 16-mm generator for tough tissue, during 5 min with 1-min intervals to prevent heating the sample. Homogenization was performed in 0.01 M phosphate buffer, pH 7.4, containing 0.15 M sodium chloride, 10 -3 M benzamidine hydrochloride, 5 . 1 0 -5 M phenylmethylsulphonyl fluoride and 3. 10 - 3 M sodium azide. The tissue homogenate was centrifuged (20 min, 3500 x g, 4°C) and the supernatant was filtered through Whatman G F / D filters. The filtrate was brought to 60% ammonium sulphate saturation and centrifuged (10 min, 3500 x g, 4°C, Sorval RC 3B). The pellet was redissolved in 0.01 M phosphate buffer, pH 7.4, containing 0.15 M sodium chloride, and transferred to a plastic tube, containing the immunoaffinity column matrix, prepared by coupling 150 mg goat immunoglobulin to 5 g activated CNBr-Sepharose 4B, obtained from Pharmacia, Uppsala, Sweden, essentially as described by Marcus and Zinberg [17]. The tube was slowly rotated overnight on an 'orbital shaker' at 4°C to couple the ferritin to the matrix. The content was subsequently poured into a column and washed with 0.01 M phosphate buffer, containing 0.15 M sodium chloride, pH 7.4, until no more protein was detectable at 280 nm. The bound ferritin was eluted with 3 M potassium thiocyanate, immediately followed by dialysis against ten changes of 0.01 M phosphate buffer, pH 7.4, containing 0.15 M sodium chloride. As a final step gel filtration using a Sepharose CL-6B column was performed, eluting the ferritin with 50 mM Tris, pH 7.4. The samples were concentrated using Amicon XM-300 membranes. After the initial homogenization step part of the samples was subjected to the classical heat treatment, as also applied for ferritin used for antibody production, to detect differences introduced by this treatment (Fig. 1). For purity control, samples were applied on a 4-30% (w/v) gradient-pore polyacrylamide slab gel (Precast gel PAA 4/30, Pharmacia, Uppsala, Sweden) and electrophoresed in 0.09 M Tris/0.08 M boric acid at pH 8.4 (4°C) for 20 min at 70 V constant voltage. After the samples had entered the gel, electrophoresis was continued for 16 h at 150 V constant voltage. Bromophenol blue was

used as tracking dye. Immediately after the run the gels were stained for protein with Coomassie brilliant blue R 250 or for iron with potassium ferrocyanide.

Characterization of rabbit liver ferritin Amino acid analysis was carried out after hydrolysis of the purified ferritin for 18 h in 6 M HC1 at 110°C under nitrogen in a sealed ampulla on a Liquimat amino acid analyzer. The amount of tryptophan was not determined. Proline was detected separately at 400 nm, all other amino acids at 570 nm. Ferritin samples were dissolved in 3 ml distilled water and extensively dialyzed. The amount of protein was assayed according to the method of Lowry et al. [18] using bovine serum albumin as a standard. Ferritin concentrations were determined by rocket immunoelectrophoresis [ 19] using as a reference a single batch of highly purified rabbit liver ferritin whose purification procedure had included heat-treatment. The nitrogen determination was carried out by destruction of 50-/~1 samples with concentrated sulphuric acid at a temperature of 450°C. Ammonia was estimated with ninhydrin [20]. Isoelectrofocussing of liver ferritin samples was performed using an LKB Multiphore 2117 and an LKB 3371 E power supply, either in 0.5 mm thin-layer agarose gels (1% w / v ) or in LKB readyto-use PAG plates. As marker for the isoelectric points a pI-determination calibration kit from Pharmacia (Uppsala, Sweden) was used. The pH gradient was measured in a number of gels before fixation, using a surface electrode coupled to Metrohi]r equipment. Thereafter the gel was refocussed for approximately 10 min. The gel was immersed immediately after focussing in fixing solution for 60 min, dried with a stream of hot air, stained with Coomassie brilliant blue R 250 and destained overnight. Subunit analysis was performed on purified rabbit liver ferritin samples by 3-25% gradient-pore polyacrylamide slab gel electrophoresis in 0.1% SDS using low molecular weight markers (Pharmacia, Uppsala, Sweden) as internal molecular weight standards. Gels stained for protein with Coomassie brilliant blue R 250 were scanned with a Vitratron U F D 500 spectro-

205

photometer with an integrating densitometer attachment.

1.0 ¸

O t'-

Results

•

E

Ferritin from rabbit livers purified with and without tt3.

heat treatment

o

Omission of the heat treatment necessitates replacement by other purification procedures. Immunoaffinity chromatography was chosen for this purpose. The overall recovery of ferritin, using the immunoaffinity chromatography procedure alone, was 56%, compared to 35% when heat treatment was included in the purification of ferritin from the same liver homogenate, as shown in Table I. The immunoreactivity of both types of preparation was c o m p a r e d by rocket immunoelectrophoresis (Fig. 2). Two separate curves were obtained for the heat-treated and non-heated sampies. This difference in slope is indicative of a higher immunoreactivity of the heat-treated ferritin. To rule out the possibility that these differences were the results of a different reaction of the ferritins with the Lowry reagents, due to a distinct protein configuration, we compared both types of preparation in a micro ninhydrin nitrogen determination, performed on all samples. A good correlation (correlation coefficient 0.94 for nonheated and 0.97 for heat-treated preparations) was

as o E

E

0

o

0

!

o.5

,

mg protein mt~ Lowry method. Fig. 2. Comparison of the immunoreactivity of heat-treated and non-heated rabbit liver ferritin preparations. The amount of protein detected by the Lowry protein assay [ 18] was compared with the amount of ferritin measured with Laurell immunoelectrophoresis [19]. The standard used for the Lowry protein assay is bovine serum albumin. In the Laurell immunoassay the antigenicity of both types of preparation was tested against goat antibodies raised against highly purified, iron-loaded, heat-treated rabbit liver ferritin. The bars represent the range of at least three assays performed on the same sample. The assays were performed with either heat-treated (©) or nonheated (e) ferritin samples.

TABLE I P U R I F I C A T I O N OF RABBIT LIVER F E R R I T I N W I T H A N D W I T H O U T H E A T T R E A T M E N T , F R O M T H E SAME LIVER HOMOGENATE Results are expressed as percentages. Protein content is based on the Lowry protein assay [18]. The 100% value is based on mg protein, using bovine serum albumin as a standard. Ferritin protein content is based on the Laurell immunoelectrophoretic assay [19]. The 100% value is based on the antigenic reaction of the fractions compared to that of highly purified iron-loaded, heat-treated ferritin. Fraction

Liver homogenate Heat supernatant Supernatant low-speed centrifugation A m m o n i u m sulphate precipitate Affinity-chromatography eluate Sepharose CL-6B eluate

Without heat treatment

With heat treatment

Protein

Ferritin protein

Protein

Ferritin protein

100.0

100.0

100.0

87.2 63.6 2.0 1.0

81.4 72.3 63.8 56.1

100.0 66.7 57.5 53.4 40.6 34.6

5.3 5.0 4.0 1.0 0.4

206

+ --4,55

-- 5 . 2 0 -

--5.501

M

2

--

3

4

5

M

Fig. 3. (left-hand figure) 3-25% gradient-pore polyacrylamide slab gel electrophoresis under reducing conditions in the presence of 0.1% SDS. This type of gel allows gel scanning to determine the subunit ratio and calculation of the M, of both types of subunit. The figure shows from left to right: a heat-treated preparation, an M, marker and a non-heated preparation. The subunit M, calculated from this gel is 21000 for the high-Mr subunit and 19 100 for the low-M, subunit. Fig. 4. Isoelectrofocussing patterns of rabbit liver ferritin preparations purified either with or without heat treatment. The pH range shown is 4.5-5.5. The range of the pl values of the analyzed ferritin samples is 4.75-5.35. The pattern of pl-marker proteins (M) is shown in the middle and on the right. Preparations 1 and 2 were prepared without heat treatment. For preparations 3-5 the purification included heat treatment. Protein was stained with Coomassie brilliant blue R 250.

f o u n d between the Lowry a n d the nitrogen assay, which was only slightly d e p e n d e n t on the isolation p r o c e d u r e used. T h e curves o b t a i n e d were y = 7.0x - 0.13 for the n o n - h e a t e d a n d y = 6.0x - 0.02 for the h e a t - t r e a t e d p r e p a r a t i o n . The slope o b t a i n e d is very similar to the factor (6.25) used in literature for conversion from nitrogen to p r o t e i n [21]. G r a d i e n t - p o r e p o l y a c r y l a m i d e slab gel electrophoresis u n d e r reducing c o n d i t i o n s in the presence of SDS revealed a c o m p l e t e s e p a r a t i o n of the Ha n d L - s u b u n i t s (Fig. 3) with a relative decrease of H - s u b u n i t s in h e a t - t r e a t e d ferritins (45 + 2%) as c o m p a r e d with the n o n - h e a t e d p r e p a r a t i o n s (56 _+ 4%). The a p p a r e n t 34, values c a l c u l a t e d from the gel (Fig. 3) were 21 000 a n d 19 100 for the high-M, subunit a n d the l o w - M r subunit, respectively. All samples were subjected to p o l y a c r y l a m i d e a n d agarose isoelectrofocussing. The range for the isoelectric p o i n t s of b o t h p r e p a r a t i o n s was

4.75-5.3~ (Fig. 4). The h e a t - t r e a t e d samples show less variability in their isoelectrofocussing p a t t e r n s t h a n the n o n - h e a t e d samples, p r o b a b l y due to p a r t i a l d e g r a d a t i o n of the ferritins. O u r results d e m o n s t r a t e d not only quantitative differences but also a slight shift in the p a t t e r n of multiple molecular forms o b t a i n e d on isoelectrofocussing of b o t h types of p r e p a r a t i o n . The a m i n o acid c o m p o s i t i o n of b o t h r a b b i t liver ferritin p r e p a r a t i o n s was d e t e r m i n e d a n d the results are p r e s e n t e d in T a b l e II. It was not possible to detect any cysteine in the hydrolysate. Statistical evaluation of the differences f o u n d in the a m i n o acid analysis revealed that the changes in the a m o u n t s of alanine and arginine ( P < 0.02) a n d of valine, isoleucine, tyrosine, p h e n y l a l a n i n e a n d histidine ( P < 0.001) were significant using t-statistics for two means.

207 TABLE lI A M I N O ACID COMPOSITION OF RABBIT LIVER FERRITIN The figures are mean values of three separate analysis runs, using the same heat-treated and non-heated rabbit liver ferritin. All data are expressed as percentage of the total number of amino acids in the sample. Amino acid

Heattreated

Nonheated

Asx Thr Set Glx Pro Gly Ala Cys Val Met Ile Leu Tyr Phe Lys His Arg Trp

10.3 5.4 8.1 12.4 2.9 7.3 8.4 5.9 1.3 3.8 10.6 3.4 4.3 6.3 4.4 5.2

l 1.1 4.3 8.1 13.6 3.5 6.0 8.3 4,2 1,6 2.4 11,6 4.0 4.2 5.9 5.7 5.4

Non-polar Acidic Basic Uncharged

37.2 22.7 15.9 24.2

35.9 24.7 17.0 22.4

Discussion

Heat treatment of the tissue homogenate is generally used because it is an efficient step in the isolation of ferritin from various tissues. It is known that the subunit composition of ferritin reflects differences in iron-uptake and iron-release capacity. Ferritin that is mainly composed of H-type subunits appears to sequestrate less iron [22]. This phenomenon may be of great importance for physiological investigations on ferritin. The differences are important enough to affect the results from biochemical and physiological experiments with purified ferritin which has to be reinjected into animals. It is known from the literature [23] that prolonged heating is associated with a progressive loss of the more acidic ferritins,

suggesting that ferritin species rich in I-i-type subunits are less stable. Our data appear in line with this observation and show selective disappearance of H-type subunits during short heating periods as well. The frequently used cadmium sulphate crystallization was omitted from our purification procedure because the cadmium sulphate could not be removed from the rabbit ferritin by any means. Conformational differences might account for a selective crystallization of L-subunit-rich ferritins [16], thereby modulating the properties of the final preparation. The differences found in the immunoelectrophoretic analysis of heat-treated and non-heated rabbit liver-ferritin (Fig. 2) can be explained by quantitative variations in the antigenic determinants of both ferritin preparations. A different subunit composition accounts for a different packing of the final ferritin molecule and may therefore modulate the antigenic properties to a greater extent than may be judged by subunit analysis. Moreover, Arosio and Albertini [24] found that the H-subunit of horse ferritin seems to expose a higher number of histidine, leucine and arginine residues to the surface of the molecule than the L-subunit. This difference has to be kept in mind when performing serum ferritin assays because antibodies raised against heat-treated ferritins are used, while non-heated serum samples are assayed for ferritin concentration. The differences in antigenicity influence the comparison of the recovery by both methods (Table I). Because the recovery is calculated for heat-treated and non-heated samples from the same standard curve in the Laurell technique [ 19], the differences found become much greater when a correction for antigenicity is applied. In that case the recovery from heat-treated ferritin is as low as 19%. An alternative, preferentially functional assay for ferritin would be desirable. The difference in subunit composition of both ferritin preparations, as concluded from the subunit analysis, showed a decrease in the H / L ratio due to heat treatment. On analytical isoelectrofocussing gels we found a clear shift in the intensities of the various bands, accompanied by a shift to more acidic ferritin forms. This implies that ferritin in rabbit liver behaves more like horse liver ferritin than like ferritins from other animal origin.

208 Because the heat-treated samples show less variability in the b a n d i n g p a t t e r n from one sample to a n o t h e r it is possible that the heat treatment selects ferritin forms with a more stable s u b u n i t composition. Differences in the extent of m u l t i m e r i z a t i o n of the ferritin p r e p a r a t i o n s c a n n o t explain these differences because in both p r e p a r a t i o n s there was hardly a n y multimeric structure present. This could be seen from 3-25% gradient-pore polyacrylamide slab gels without SDS, r u n u n d e r n o n - r e d u c i n g conditions. The diversity in peptide structure, as seen from small differences in the a m i n o acid compositions of heat-treated a n d n o n - h e a t e d ferritin (for valine, isoleucine, tyrosine, p h e n y l a l a n i n e a n d histidine: P < 0.001), may also generate differences in biochemical behaviour, although the small n u m b e r of a m i n o acid analyses does not permit us to draw absolute conclusions with respect to these differences. We conclude that heat t r e a t m e n t should beavoided for p r e p a r a t i o n of ferritin to be used for biochemical and, p r o b a b l y even more so, for physiological experiments.

Acknowledgements We wish to t h a n k Dr. G.H. De Haas from the L a b o r a t o r y of Biochemistry from the State U n i versity of Utrecht for p e r f o r m i n g the a m i n o acid analyses. The N a t i o n a l Institute of Public Health RIV provided us with fresh r a b b i t livers. F o r valuable advice we are very grateful to Professor I. Listowsky, D e p a r t m e n t of Biochemistry, Albert Einstein College of Medicine, New York. The f o u n d a t i o n for Biological Research in the Netherlands, BION, which is supported by the Netherlands O r g a n i z a t i o n for the A d v a n c e m e n t of Pure Research (ZWO), is acknowledged for financial support (grant 14-96-009).

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