- Email: [email protected]
[30] Purification of bovine rhodopsin over concanavalin A-sepharose
[30]
PURIFICATION
197
OF BOVINE RHODOPSIN
into the 11-cis form completely (> 98%). s In our laboratory, 11-c/s-retinal is prepared by this reaction when needed for experiments. If a mixture of various isomers of retinal is applied, the 11-c/s-retinal is successively produced first from all-trans, then from 13-cis, and finally from 9-cis-retinal. Such a catalytic function of retinochrome protein in visible light would be useful for changing a variety of retinal isomers to only one type in the 11cis form, which is required for rhodopsin formation in the retina.
[30] P u r i f i c a t i o n o f B o v i n e R h o d o p s i n Concanavalin A- Sepharose By WILLEM
J.
DE
over
GRIP
Introduction Isolated photoreceptor membranes constitute a fairly pure rhodopsin preparation (about 90% of the total membrane protein), the main "contaminant" being lipid. Nevertheless, for studies, for example, on functional parameters of rhodopsin or specific lipid-protein interactions, a rhodopsin preparation may be required that is free from original lipids and/or other photoreceptor-membrane proteins. For this purpose detergents are required in order to disintegrate the membrane suprastructure into small units and as exchange for lipids. A variety of procedures for purification of rhodopsin have been reported (see footnote 1 for review, or elsewhere this volume). One of these, chromatography over immobilized concanavalin A, makes use of the presence in rhodopsin of two carbohydrate moieties, both containing three mannose and three N-acetylglucosamine residues 2 located near the N-terminus. a This technique shows, as compared to others, the common advantages of affinity chromatography: (1) Relatively small bed-volumina can be used, considerably reducing the time factor; (2) relatively large-sample volumina can be applied; and (3) the desired material can be eluted in high concentration (i> 100/zM). Additional advantages for rhodopsin are (4) it can be well separated from its apoprotein, opsin, and (5) it is easily completely delipidated. Drawbacks of this specific method are slow release of concanavalin A from its matrix, causing varying degree of contamination, and the fact that all glycocompounds present in the photoreceptormembrane, which have affinity for Con A, will tend to copurify with rhodopsin. Complementary to earlier W. J. De Grip, F. J. M. D a e m e n , and S. L. Bonting, this series, Vol. 67,p. 301. 2 M. N. F u k u d a , D. S. Papermaster, and P. A. H ~ g r a v e , J. Biol. Chem. 254, 8201 (1979). a p. A. Hargrave, Biochim. Biophys. Acta 492, 83 (1977). 1
METHODS IN ENZYMOLOGY, VOL. 81
Copyright© 1982by AcademicPress, Inc. All rights of reproduction in any form reserved. 1SBN 0-12-181981-7
198
CHARACTERIZATION OF VISUAL PIGMENTS
[30]
reports on the purification of rhodopsin by means of Con A 1"4-6 we will show here that under proper conditions both drawbacks of this technique can be appropriately dealt with. Principle Concanavalin A, isolated from jack beans (Canavalia ensiformis), is a lectin with preference for a-D-derivatives of mannose and, to a lesser extent, glucose.r Below pH 5.6 the protein exists as a dimer with subunits of MW 26 K, above the pH tetramers form. For activity Con A requires two metal ions (Ca R+ or Zn 2÷ and Mn 2+ or Ni~+), which at pH higher than 6.0 are bound sufficiently strongly so that in the absence of chelators these ions may be absent in the medium during chromatography. Hence, although Con A can be used in the pH range 4.0-9.0, the range 6.0-7.0 is most convenient in view of the high thermal stability of both Con A and rhodopsin under these conditions. In addition, at least 100 mM NaC1 or KC1 should be present for maximal stability of Con A. Con A can be immobilized onto various supports (Sepharose, cellulose, polyacrylamide, glass) by well-established procedures, s,9 In addition, some ready-to-use matrices are commercially available (Con A-Sepharose, Pharmacia, Sweden; Con A-Agarose, Sigma, USA; Con A-Ultrogel, Rractifs IBF, France). Of the latter, Con A-Sepharose has the highest Con A load per ml settled gel (approximately 10 mg vs about 6 and 4 mg) and is used for the following procedures. The use of detergents, required first of all to dissolve rhodopsin, has the additional advantage that the hydrophobic interaction, which Con A shows with several compounds, 7 is minimized. The choice of detergent is of some importance, as the thermal stability and leakage rate of Con A is somewhat detergent dependent9-11 and the thermal stability of rhodopsin is strongly detergent dependent. 12 In view of the latter, the procedure is 4 R. Renthal, J. S. Pober, A. Steinemann, and L. Stryer, in "Concanavalin A as a Tool" (H. Bittiger and H. P. Schnebli, eds.), p. 429. Wiley, New York, 1976. 5 j. j. Platner and E. L. Kean, J. Biol. Chem. 251, 1548 (1976). 6 Abbreviations used: Con A, concanavalin A; CTAB, cetyltrimethylammonium bromide; DTAB, dodecyltrimethylammonium bromide; DTE, dithioerythritol; EDTA, ethylenediaminotetraacetic acid; MW, molecular weight; PAGE, polyacrylamide gel electrophoresis; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); SDS, sodium dodecyl sulfate; Tris, tris(hydroxymethyl)aminemethane; TTAB, tridecyltrimethylammonium bromide. 7 I. J. Goldstein and C. E. Hayes, Adv. Carbohydr, Chem. Biochem. 35, 127 (1978). s This series, Vol. 34, entire volume. a Various articles in "Concanavalin A as a Tool" (cf. footnote 4). 10 R. Lotan, G. Beattie, W. Hubbell, and G. L. Nicolson, Biochemistry 16, 1787 (1977). 11 W. J. De Grip, unpublished. 12 See elsewhere this volume.
[30]
PURIFICATION OF BOVINE RHODOPSIN
199
preferentially carried out at 4 °. Suitable detergents are, in order of increasing mildness, TTAB, CTAB, Ammonyx LO, Emulfogene BC-720, Triton X-100, and the alkylglucosides. (Con A has very little affinity for fl-alkylglucosides. 7) The nonionic detergents have the advantage that opsin is strongly retarded on the column (probably also due to aggregation phenomena and nonspecific interaction11), which facilitates separation from rhodopsin. If detergent-exchange or reconstitution studies with lipids are to follow purification, the alkylglucosides octyl- or nonylglucose are the best choice for detergent, since they are very easily removed by dialysis or dilution. 1"12In this respect the second best choices are dodecyldimethylaminoxide and TTAB. A mild and rather easily removable alternative is cholate, but this detergent should not be used below pH 7.0, and the commercial products are better purified first by recrystallization. We routinely employ nonylglucose. 1 The procedure is, however, independent of the detergent used, which therefore is not further specified. Finally, the procedure is also applicable to purification of opsin. Then, however, either very strong (DTAB, TTAB) or very mild detergents (digitonin, dodecylmaltose) have to be used. In the first case, opsin is irreversibly denatured; in the latter case complete delipidation is not easily achieved. H The slow leakage of Con A from the matrix usually does not interfere with subsequent experimentation. Generally, contamination of rhodopsin with Con A is less than 0.5%, often less then 0.1% (determined by rocket immunoelectrophoresis against Con A directed antibodies). Some batches of Con A-Sepharose, however, show uncontrollable high leakage rates, which cannot easily be suppressed by repeated washings with, e.g., media of high or low pH or BSA solutions. Under those conditions contamination of rhodopsin with Con A may increase to 5-10%, and the latter shows up on P A G E - S D S gels. Such high contamination levels are of course generally intolerable, but the low contamination levels previously mentioned may also be undesirable under specific conditions (e.g., certain immunological techniques). If Con A contamination does constitute a problem, two alternatives are offered to deal with it: (1) incubation of the Con A-Sepharose with low concentrations of glutaraldehyde, as described for other systems23 This treatment considerably stabilizes the affinity matrix, but at the same time decreases the capacity to a variable extent (at least 10, sometimes over 30%); and (2) passing of the column eluate through a Sephadex G-50 column. This simultaneously removes the small sugar competitor used in fairly high concentration (0.2 M) to elute rhodopsin from the affinity matrix. It is a rapid procedure, which does not add much to the time required for purification. Alter13 R. Kowal and R. G. Parsons, Anal. Biochem. 102, 72 (1980).
200
CHARACTERIZATION OF VISUAL PIGMENTS
[30]
natively, more lengthy procedures, like hydroxyapatite or ion-exchange chromatography, may be applied. Finally, the question of copurification of other glycoproteins present has to be addressed. It has recently been shown that, next to rhodopsin, one other Con A-binding glycoprotein is present in the photoreceptormembrane 14 with a MW of approximately 230,000 in cattle and about 270,000 in frog. In our experience, using nonylglucose as a detergent, this protein does not copurify with rhodopsin. It is not clear whether the large protein is very strongly bound to Con A-Sepharose and not eluted under the conditions suitable to elute rhodopsin or whether it is only weakly bound and elutes during washing. The small amount present in the photoreceptormembrane and its low solubility in most detergents make it difficult to trace. Reagents A. Con A-Sepharose (supplied by Pharmacia as a slurry in buffer solution) B. Photoreceptor membrane preparation C. Glutaraldehyde solution (70%; purified by distillation) D. 0.25 M sodium bicarbonate (pH 8.8) E. 1 M Tris-HCl (pH 7.8) F. 40 mM PIPES, containing 2 mM MgCl2, 2 mM CaCl~, 2 mM MnCl~, 0.2 mM EDTA, and 300 mM NaC1, pH 6.5 G. Same as F, but without MnCl~ H. 3 M NaCI I. Detergent solution (40-80 mM: concentration at least 4 x CMC) J. DTE, 100 mM K. Sephadex G-50 (medium grade) suspended in G/I/J(50:50: 1) All solutions are cleared by filtration through 0.2-t~m membrane filters and preferentially stored frozen in small batches. Reagent J is freshly prepared. Procedures For glutaraldehyde treatment, la the required volume of settled Con A Sepharose gel is equilibrated with 4 vol reagent D for 0.5 hr at 4 °. The beads are then allowed to settle, the supernatant is discarded and 4 vol of a freshly prepared mixture of C and D (1:2000; i.e., - 0 . 0 4 % glutaraldehyde) added. The suspension is incubated for 1 hr at 20° on a rotator. Dur14R. S. Moldayand L. L. Molday,J.
Biol. Chem.
254, 4653 (1979).
[30]
PURIFICATION OF BOVINE RHODOPSIN
201
ing incubation the beads turn orange-brown. Subsequently they are again allowed to settle and incubated with 4 vol E (1 hr, 20°) to block remaining reactive groups. Finally the beads are washed three times with 3 vol F / H / I (1 : 1 : 1) and stored in the cold in the same mixture or in F / H (2: 1). (The presence of detergent minimizes bacterial growth, but strong detergents may enhance leakage or thermal denaturation of Con A. 9-11) The required volume of Con A-Sepharose is poured into a column (per milliliter of settled gel 60-130 nmol of rhodopsin are bound), and washed with 3 vol of D / H (2 : 1) and 7 vol F / H (2 : 1) to remove residual carbohydrate and nonimmobilized material (flow rate: 5-10 ml/hr). Subsequent washings with 3 vol of F / H 2 0 (1 : 1) and 3 vol of G / I / J (50:50: 1)l~ make the column ready for sample application. All following operations are carried out in dim red light (~ > 650 nm) at 4 °. Preparation B is washed twice with water (under N2 or argon), once with G / H 2 0 / J (50:50: 1)1~; each time it is collected by centrifugation (30 rain; 100,000g; 4°C), and it is finally dissolved under nitrogen in I/J (100: 1) to a concentration of about 100/zM, by brief mixing on a Vortex mixer and incubation for about 0.5 hr in ice under occasional shaking. Then one volume of G is added and any remaining turbidity is removed by centrifugation (15 min, 100,000 g, 4°). An aliquot of the clear supernatant is withdrawn for analytical purposes, and the remainder is applied to the Con A column. For highest efficiency, the column can be loaded to its maximal capacity by applying a slight excess of rhodopsin. Flow rates depend on column size (about 2 ml/hr for columns ~< 2 ml, up to 10 ml/hr for columns > 10 ml). With higher flow rates part of the rhodopsin may pass through without binding and the eluate has to be reapplied for maximal loading. (Since Con A has higher affinity for opsin, high opsin content will seriously decrease the capacity of the column for rhodopsin.) Subsequently, nonbound material is washed out with at least 8 vol of G / I / J (50:50: 1) until the eluate is free of lipid (Fig. 1A). The bound rhodopsin is then eluted with G / I / J (50:50: 1) to which solid a-methylglucose or amethylmannose (Sigma, grade III) is added to a final concentration of 200 raM. 16 Rhodopsin-containing fractions with a A28o/Asoo ratio of 15 Mn2* binds very strongly to phospholipids (e.g., used for reconstitution studies) and may also interfere with subsequent ESR or NMR studies. It is therefore left out during chromatography. When its presence does not present a problem, everywhere in the procedure G can be replaced by F. 16 Alternatively, rhodopsin can be eluted, if so required, by lower concentrations of sugar (e.g., 50 mM) or a sugar gradient (e.g., 0-200 mM). Both conditions, however, retard elution of rhodopsin and yield eluates with a much lower rhodopsin concentration. This is probably due to heterogeneity in the affinity constants of the immobilized Con A as a result of chemical and steric factors inherent to immobilization.
1.41.0-
Rh ,
~-MeMann
Rh
hv
A
Q8 ~ O.D.0.60402 + O-
B
1./*1.2-
-Me 10-
Mann
O.D. 0.8fl6 0./*0.20
C ,,-Me
-
Mann ÷
o.1+
Rh
h~.
1 M NaCt
+~-Me-
0.4 OD.
0.2 0
0.6 0D'0410.2
h,.
Rh
0
D
,~- M e -
NH20H
_
+~-Me-
Mann+ I M NoCI
,
I
I
I
I
i
L
I
5
I
I
I
I
I
10
i
I
I
i
L
I
15
I
I
I
I
20
I
I
~
t
I
25
I
I
i
I
I
30
FRACTION
FIG. 1. Con A-Sepharose chromatography of rhodopsin (A) and opsin (B, C, D) under various conditions. The column was not loaded to full capacity so as not to complicate the elution pattern with excess rhodopsin. To 1.6 ml of settled gel are applied, respectively, 84 (A), 95 (B), 96 (C), and 110 (D) nmol of rhodopsin, dissolved in 4 - 5 ml of 20 mM
[30]
PURIFICATION OF BOVINE RHODOPSIN
203
1.6-1.8 (corrected for contribution of the buffer and detergent to the A2s0) are combined. The column is then regenerated with 3 vol F / H / I (1 : 1 : 1), followed by 3 vol D / H (2 ; 1) and 5 vol F / H (2 : 1) and finally stored in F / H (2 : 1) or F / H / I (2: 2: 1) in the cold. The beads can be used repeatedly until the capacity has decreased too much. The entire purification takes 5 - 2 0 hr, depending on column size. Recovery of rhodopsin with an A2s0/Asoo ratio usually between 1.6 and 1.7 is over 90%. The A4oo/A~ooratio lies between 0.16 and 0.17. The lipid content is less than 0.5 tool/tool of rhodopsin. Spectra before and after purification are shown elsewhere. 1 S D S - P A G E gels are presented in Fig. 2. If any Con A present does not have to be removed, salts and sugar can be dialyzed out and the solution stored at - 70 ° in dark containers for at least half a year. In the case of dialyzable detergents, both lipid- and detergentfree rhodopsin can be obtained via dialysis and subsequent centrifugation (30 min, 100,000 g, 4 °) or the detergent used can be exchanged for another
PIPES buffer, pH 6.5, containing 130 mM NaCI, 3 mM MgCI2, 2 mM CaCI2, 1 mM MnCIz, 0.5 mM DTE, 0.1 mM EDTA and 20 mM nonylglucose. The column is washed with the same buffer, and (rhod)opsin eluted with the same buffer containing 0.2 M c~-l-methylmannoside, both at a flow rate of 10 ml/hr. Arrows indicate change of condition. Fractions of 1.7 ml are collected. Clear bar: A~0; solid bar: lipid; cross-hatched bar: As0o (A), Aa,t5 (B,C), Aa6~(D). (A) Following sample application, the column is washed until the eluate is free of protein (Az80) and lipid (assayed as phosphate). Then rhodopsin is eluted with a-1-methylmannoside. Recovery of rhodopsin (both on 280 and on 500-base) is 96%, 85% of which is recovered in the first four rhodopsin-containing fractions. This purified rhodopsin regenerates for over 70%. (B) Same as (A) until washing is completed. Then the column is illuminated at 4° with white light until no rhodopsin is left (several minutes: visual inspection). Immediate elution with sugar follows. Recovery of original chromophore as 375-nm absorbance is about 90%, recovery ofopsin protein is close to 100%, 80% of which is recovered in the first four fractions. Regeneration is 60-70% of a control sample. (C) Following sample application, the column is illuminated and washed until eluate is free of lipid and retinal (375 nm). This fraction comprises only 10-15% of the amount of chromophore originally applied. Subsequent elution with sugar releases only 40-50% of the opsin applied, part of which still contains 375-nm absorbance and shows variable regeneratiorr (30-60% of a control sample). The remainder of the opsin can be slowly eluted by including 1 M NaCl in the buffer. This fraction shows no measurable 375-nm absorbance and does not regenerate. Total recovery of original chromophore as 375-nm absorbance is only 30-35%. The first four opsin fractions comprise about 33% of the total applied. (D) Same as (C), but to the wash-buffer 20 mM hydroxylamine is added in order to convert all free and bound retinal into the oxime (hmax = 365 nm), and washing is continued until the eluate is also free of oxime. The recovery of original chromophore as the oxime is 95%. Subsequent elution with sugar only slowly releases the opsin in a nonregenerable form. The first four opsin fractions contain less than 20% of the total applied. Addition of l M NaCl somewhat enhances the rate at which opsin elutes, but more rapid release is only achieved under more extreme conditions such as using stronger detergents (not shown).
204
CHARACTERIZATION OF VISUAL PIGMENTS
[30]
Mw
x 10 -3
--TETRAMER
220 93 69
"TRIMER
5O 45 36 30 25
18 14 9
Ca
ROS
Rh
FIG. 2. SDS-polyacrylamide gels of rod outer segment membranes (ROS) and purified rhodopsin (Rh), together with a calibration gel (Ca). The tendency of opsin to aggregate is aggravated by purification as clearly expressed by the appearance of higher oligomers. Proof that these bands indeed are oligomers of opsin is obtained from the observations that these bands (1) all fluoresce after reductive fixation of the chromophore to opsin and (2) all react with antibodies against opsin, as determined by crossed immunoelectrophoresis (Margry and De Grip, unpublished). The arrow points at the 230,000 band in ROS representing the large glycoprotein, which is completely absent in the purified preparation. In order to separate a wide range of molecular weights, a linear polyacrylamide gradient is used (8.5-29% w/v; 4.5% cross-linker). The buffer system is adapted from U. K. Laemlli [Nature (London) 227, 680 (1970]. Staining was performed with Coomassie Blue R - 250 [G. Fairbanks, T. L. Steck, and D. F. M. WaUach, Biochemistry 10, 2606 (1971].
one (e.g., digitonin). Nondialyzable detergents are best exchanged for other types while rhodopsin is still bound to the column. If opsin is to be purified, the elution pattern depends on the detergent used. In strong detergents (DTAB, TTAB, CTAB) opsin is somewhat retarded with respect to rhodopsin but is easily eluted completely delipidated, albeit irreversibly denatured. The same chromatographic behavior is observed in very mild detergents (digitonin, dodecylmaltose), but here opsin remains fairly intact (regenerates for over 80%), while only partial delipidation is achieved, u In detergents of intermediate mildness, like nonylglucose, the behavior of opsin depends on its "illumination history" (Fig. 1B-D). When opsin is generated on the column and immediately desorbed with sugar (Fig. 1B), all protein and retinal elute with a constant
[30]
P U R I F I C A T I OOF N BOVINE RHODOPSIN
205
Azso/Aar~ ratio
o f about 1.9 and the material regenerates for o v e r 70% o f the control. H o w e v e r , when opsin is generated before the buffer washing (Fig. 1C), retinal is slowly eluted, and, following addition of sugar, that part o f the protein that still contains retinal is rapidly eluted and shows regenerability. The remainder of the protein is strongly retarded, does not contain retinal, and does not regenerate. Apparently at 4 ° illumination generates a fairly stable photoproduct with spectral properties similar to metarhodopsin II. This product behaves like rhodopsin in some ways. Loss of retinal seems to be followed by secondary conformational changes (partial or complete denaturation), resulting in higher affinity for Con A as well as nonspecific interaction a n d / o r aggregation phenomena. This is very clear in Fig. 1D, where, following illumination, retinal is first removed as the oxime derivative by including 20 mM hydroxylamine in the wash buffer. All opsin is then strongly retarded and can only be eluted completely under more extreme conditions, like including strong detergents a n d / o r ethyleneglycol or urea (not shown). Even more extreme results are obtained when opsin is generated by illumination at 20 ° before it is applied to the column. These observations explain why in this type of detergent rhodopsin is easily purified from opsin. If Con A present has to be removed, the volume of the combined (rhod)opsin fractions is first reduced in order to attain a rhodopsin concentration of at least 100/zM (in the cold, e.g., by pressure dialysis or vacuum filtration). The sample thus obtained is applied to a column o f K with a volume at least five times the sample volume. This guarantees that enough G-50 surface remains between the rapidly migrating rhodopsinmicelles and the small molecules lagging behind to retard the Con A. Rhodopsin is eluted with G / I / J (50: 50:1; flow rate: anywhere between 10 and 60 ml/hr) and elutes two to five times diluted with an elution volume equivalent to the void volume of the column. Except when contamination with Con A is very serious, the spectral properties of the combined rhodopsin fractions are practically unchanged. 17 The combined samples can be stored frozen or used for further treatments, as described previously after Con A chromatography. We have performed some careful amino acids analysis of rhodopsin purified in this way (see table). The results agree well with earlier reports 17Recently, we have worked out a further improved alternative to remove Con A from rhodopsin solutions (unpublished) by passage through a column of immobilized antibodies against Con A. With this procedure, denatured Con A is removed as well, while only small bed volumina are required (up to 100 nmol of Con A may be bound per milliliter of gel), thereby introducing only a small dilution factor. The antibody matrix can be freed from Con A with, e.g., 0.2 M glycin-HCl(pH 2.0) and reused after washing with the appropriate buffer.
206
CHARACTERIZATION OF VISUAL PIGMENTS
[30]
AMINO ACID COMPOSITION OF BOVINE RHODOPSIN IN RESIDUE PER MOLE OF RHODOPSIN Residue Asx Thr Ser Glx Pro Gly Ala Cys ½Cyn Val Met Ile Leu Tyr Phe Lys His Trp Arg Total
Results 23~ 28 15 35 21 26 28 6 4 27 13 17 29 17 31 13 7 8 8 356
24b 26 15 33 22 26 31 }5 30 10 20 30 16 33 14 6 nd e 8 357
26c 34 21 34 22 28 34 }8 21 11 13 27 14 30 11 5 nd 8
23a 26 18 32 20 24 30 }8 30 12 20 30 17 30 15 6 8 9
a Our results, expressed per mole of chromophore-bound retinal (¢s0o = 4050 m2/mol). Figures extrapolated from hydrolysis times up to 96 hr. Hydrolysis was performed at 110° with 6 N HCI in the presence of 0.1% phenol and 0.1% mercaptoacetic acid, which minimizes decomposition of methionine and tyrosine. Total Cys + Cyn was determined both spectrophotometrically and as cysteic acid (see elsewhere this volume). Cys and Trp were determined spectrophotometrically. b From J. J. Plantner and E. L. Kean, J. Biol. Chem. 251, 1548 (1976). Expressed per mole of rhodopsin ( ~ = 4060 m2/mol), purified by hydroxyapatite chromatography followed by Con A - S e p h a r o s e chromatography. Total includes 8 Trp. c From H. Shichi, M. S. Lewis, F. Irreverre, and A. L. Stone, J. Biol. Chem. 244, 529 (1969). Rhodopsin was purified by hydroxyapatite chromatography followed by ion-exchange chromatography. Figures were recalculated to obtain a MW of - 4 0 K. d From J. Heller, Biochemistry 7, 2906 (1968). Rhodopsin was purified by gel filtration. Figures were recalculated to obtain a MW of - 4 0 K. e Not determined.
[30]
PURIFICATION OF BOVINE RHODOPSIN
207
and from our data we calculate a MW for the protein part of rhodopsin of 39.8 K ---0.8 K. Together with carbohydrate and chromophore this adds up to a total molecular weight of about 42.3 K. The lower molecular weight obtained in various SDS-PAGE systems (32 K - 3 8 K) might indicate a relatively high level of SDS binding, a phenomenon observed also with other membrane proteins, TM including bacteriorhodopsin.
18 A. Helenius and K. Simons, Biochim. Biophys. Acta 41S, 29 (1975).
PURIFICATION
197
OF BOVINE RHODOPSIN
into the 11-cis form completely (> 98%). s In our laboratory, 11-c/s-retinal is prepared by this reaction when needed for experiments. If a mixture of various isomers of retinal is applied, the 11-c/s-retinal is successively produced first from all-trans, then from 13-cis, and finally from 9-cis-retinal. Such a catalytic function of retinochrome protein in visible light would be useful for changing a variety of retinal isomers to only one type in the 11cis form, which is required for rhodopsin formation in the retina.
[30] P u r i f i c a t i o n o f B o v i n e R h o d o p s i n Concanavalin A- Sepharose By WILLEM
J.
DE
over
GRIP
Introduction Isolated photoreceptor membranes constitute a fairly pure rhodopsin preparation (about 90% of the total membrane protein), the main "contaminant" being lipid. Nevertheless, for studies, for example, on functional parameters of rhodopsin or specific lipid-protein interactions, a rhodopsin preparation may be required that is free from original lipids and/or other photoreceptor-membrane proteins. For this purpose detergents are required in order to disintegrate the membrane suprastructure into small units and as exchange for lipids. A variety of procedures for purification of rhodopsin have been reported (see footnote 1 for review, or elsewhere this volume). One of these, chromatography over immobilized concanavalin A, makes use of the presence in rhodopsin of two carbohydrate moieties, both containing three mannose and three N-acetylglucosamine residues 2 located near the N-terminus. a This technique shows, as compared to others, the common advantages of affinity chromatography: (1) Relatively small bed-volumina can be used, considerably reducing the time factor; (2) relatively large-sample volumina can be applied; and (3) the desired material can be eluted in high concentration (i> 100/zM). Additional advantages for rhodopsin are (4) it can be well separated from its apoprotein, opsin, and (5) it is easily completely delipidated. Drawbacks of this specific method are slow release of concanavalin A from its matrix, causing varying degree of contamination, and the fact that all glycocompounds present in the photoreceptormembrane, which have affinity for Con A, will tend to copurify with rhodopsin. Complementary to earlier W. J. De Grip, F. J. M. D a e m e n , and S. L. Bonting, this series, Vol. 67,p. 301. 2 M. N. F u k u d a , D. S. Papermaster, and P. A. H ~ g r a v e , J. Biol. Chem. 254, 8201 (1979). a p. A. Hargrave, Biochim. Biophys. Acta 492, 83 (1977). 1
METHODS IN ENZYMOLOGY, VOL. 81
Copyright© 1982by AcademicPress, Inc. All rights of reproduction in any form reserved. 1SBN 0-12-181981-7
198
CHARACTERIZATION OF VISUAL PIGMENTS
[30]
reports on the purification of rhodopsin by means of Con A 1"4-6 we will show here that under proper conditions both drawbacks of this technique can be appropriately dealt with. Principle Concanavalin A, isolated from jack beans (Canavalia ensiformis), is a lectin with preference for a-D-derivatives of mannose and, to a lesser extent, glucose.r Below pH 5.6 the protein exists as a dimer with subunits of MW 26 K, above the pH tetramers form. For activity Con A requires two metal ions (Ca R+ or Zn 2÷ and Mn 2+ or Ni~+), which at pH higher than 6.0 are bound sufficiently strongly so that in the absence of chelators these ions may be absent in the medium during chromatography. Hence, although Con A can be used in the pH range 4.0-9.0, the range 6.0-7.0 is most convenient in view of the high thermal stability of both Con A and rhodopsin under these conditions. In addition, at least 100 mM NaC1 or KC1 should be present for maximal stability of Con A. Con A can be immobilized onto various supports (Sepharose, cellulose, polyacrylamide, glass) by well-established procedures, s,9 In addition, some ready-to-use matrices are commercially available (Con A-Sepharose, Pharmacia, Sweden; Con A-Agarose, Sigma, USA; Con A-Ultrogel, Rractifs IBF, France). Of the latter, Con A-Sepharose has the highest Con A load per ml settled gel (approximately 10 mg vs about 6 and 4 mg) and is used for the following procedures. The use of detergents, required first of all to dissolve rhodopsin, has the additional advantage that the hydrophobic interaction, which Con A shows with several compounds, 7 is minimized. The choice of detergent is of some importance, as the thermal stability and leakage rate of Con A is somewhat detergent dependent9-11 and the thermal stability of rhodopsin is strongly detergent dependent. 12 In view of the latter, the procedure is 4 R. Renthal, J. S. Pober, A. Steinemann, and L. Stryer, in "Concanavalin A as a Tool" (H. Bittiger and H. P. Schnebli, eds.), p. 429. Wiley, New York, 1976. 5 j. j. Platner and E. L. Kean, J. Biol. Chem. 251, 1548 (1976). 6 Abbreviations used: Con A, concanavalin A; CTAB, cetyltrimethylammonium bromide; DTAB, dodecyltrimethylammonium bromide; DTE, dithioerythritol; EDTA, ethylenediaminotetraacetic acid; MW, molecular weight; PAGE, polyacrylamide gel electrophoresis; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid); SDS, sodium dodecyl sulfate; Tris, tris(hydroxymethyl)aminemethane; TTAB, tridecyltrimethylammonium bromide. 7 I. J. Goldstein and C. E. Hayes, Adv. Carbohydr, Chem. Biochem. 35, 127 (1978). s This series, Vol. 34, entire volume. a Various articles in "Concanavalin A as a Tool" (cf. footnote 4). 10 R. Lotan, G. Beattie, W. Hubbell, and G. L. Nicolson, Biochemistry 16, 1787 (1977). 11 W. J. De Grip, unpublished. 12 See elsewhere this volume.
[30]
PURIFICATION OF BOVINE RHODOPSIN
199
preferentially carried out at 4 °. Suitable detergents are, in order of increasing mildness, TTAB, CTAB, Ammonyx LO, Emulfogene BC-720, Triton X-100, and the alkylglucosides. (Con A has very little affinity for fl-alkylglucosides. 7) The nonionic detergents have the advantage that opsin is strongly retarded on the column (probably also due to aggregation phenomena and nonspecific interaction11), which facilitates separation from rhodopsin. If detergent-exchange or reconstitution studies with lipids are to follow purification, the alkylglucosides octyl- or nonylglucose are the best choice for detergent, since they are very easily removed by dialysis or dilution. 1"12In this respect the second best choices are dodecyldimethylaminoxide and TTAB. A mild and rather easily removable alternative is cholate, but this detergent should not be used below pH 7.0, and the commercial products are better purified first by recrystallization. We routinely employ nonylglucose. 1 The procedure is, however, independent of the detergent used, which therefore is not further specified. Finally, the procedure is also applicable to purification of opsin. Then, however, either very strong (DTAB, TTAB) or very mild detergents (digitonin, dodecylmaltose) have to be used. In the first case, opsin is irreversibly denatured; in the latter case complete delipidation is not easily achieved. H The slow leakage of Con A from the matrix usually does not interfere with subsequent experimentation. Generally, contamination of rhodopsin with Con A is less than 0.5%, often less then 0.1% (determined by rocket immunoelectrophoresis against Con A directed antibodies). Some batches of Con A-Sepharose, however, show uncontrollable high leakage rates, which cannot easily be suppressed by repeated washings with, e.g., media of high or low pH or BSA solutions. Under those conditions contamination of rhodopsin with Con A may increase to 5-10%, and the latter shows up on P A G E - S D S gels. Such high contamination levels are of course generally intolerable, but the low contamination levels previously mentioned may also be undesirable under specific conditions (e.g., certain immunological techniques). If Con A contamination does constitute a problem, two alternatives are offered to deal with it: (1) incubation of the Con A-Sepharose with low concentrations of glutaraldehyde, as described for other systems23 This treatment considerably stabilizes the affinity matrix, but at the same time decreases the capacity to a variable extent (at least 10, sometimes over 30%); and (2) passing of the column eluate through a Sephadex G-50 column. This simultaneously removes the small sugar competitor used in fairly high concentration (0.2 M) to elute rhodopsin from the affinity matrix. It is a rapid procedure, which does not add much to the time required for purification. Alter13 R. Kowal and R. G. Parsons, Anal. Biochem. 102, 72 (1980).
200
CHARACTERIZATION OF VISUAL PIGMENTS
[30]
natively, more lengthy procedures, like hydroxyapatite or ion-exchange chromatography, may be applied. Finally, the question of copurification of other glycoproteins present has to be addressed. It has recently been shown that, next to rhodopsin, one other Con A-binding glycoprotein is present in the photoreceptormembrane 14 with a MW of approximately 230,000 in cattle and about 270,000 in frog. In our experience, using nonylglucose as a detergent, this protein does not copurify with rhodopsin. It is not clear whether the large protein is very strongly bound to Con A-Sepharose and not eluted under the conditions suitable to elute rhodopsin or whether it is only weakly bound and elutes during washing. The small amount present in the photoreceptormembrane and its low solubility in most detergents make it difficult to trace. Reagents A. Con A-Sepharose (supplied by Pharmacia as a slurry in buffer solution) B. Photoreceptor membrane preparation C. Glutaraldehyde solution (70%; purified by distillation) D. 0.25 M sodium bicarbonate (pH 8.8) E. 1 M Tris-HCl (pH 7.8) F. 40 mM PIPES, containing 2 mM MgCl2, 2 mM CaCl~, 2 mM MnCl~, 0.2 mM EDTA, and 300 mM NaC1, pH 6.5 G. Same as F, but without MnCl~ H. 3 M NaCI I. Detergent solution (40-80 mM: concentration at least 4 x CMC) J. DTE, 100 mM K. Sephadex G-50 (medium grade) suspended in G/I/J(50:50: 1) All solutions are cleared by filtration through 0.2-t~m membrane filters and preferentially stored frozen in small batches. Reagent J is freshly prepared. Procedures For glutaraldehyde treatment, la the required volume of settled Con A Sepharose gel is equilibrated with 4 vol reagent D for 0.5 hr at 4 °. The beads are then allowed to settle, the supernatant is discarded and 4 vol of a freshly prepared mixture of C and D (1:2000; i.e., - 0 . 0 4 % glutaraldehyde) added. The suspension is incubated for 1 hr at 20° on a rotator. Dur14R. S. Moldayand L. L. Molday,J.
Biol. Chem.
254, 4653 (1979).
[30]
PURIFICATION OF BOVINE RHODOPSIN
201
ing incubation the beads turn orange-brown. Subsequently they are again allowed to settle and incubated with 4 vol E (1 hr, 20°) to block remaining reactive groups. Finally the beads are washed three times with 3 vol F / H / I (1 : 1 : 1) and stored in the cold in the same mixture or in F / H (2: 1). (The presence of detergent minimizes bacterial growth, but strong detergents may enhance leakage or thermal denaturation of Con A. 9-11) The required volume of Con A-Sepharose is poured into a column (per milliliter of settled gel 60-130 nmol of rhodopsin are bound), and washed with 3 vol of D / H (2 : 1) and 7 vol F / H (2 : 1) to remove residual carbohydrate and nonimmobilized material (flow rate: 5-10 ml/hr). Subsequent washings with 3 vol of F / H 2 0 (1 : 1) and 3 vol of G / I / J (50:50: 1)l~ make the column ready for sample application. All following operations are carried out in dim red light (~ > 650 nm) at 4 °. Preparation B is washed twice with water (under N2 or argon), once with G / H 2 0 / J (50:50: 1)1~; each time it is collected by centrifugation (30 rain; 100,000g; 4°C), and it is finally dissolved under nitrogen in I/J (100: 1) to a concentration of about 100/zM, by brief mixing on a Vortex mixer and incubation for about 0.5 hr in ice under occasional shaking. Then one volume of G is added and any remaining turbidity is removed by centrifugation (15 min, 100,000 g, 4°). An aliquot of the clear supernatant is withdrawn for analytical purposes, and the remainder is applied to the Con A column. For highest efficiency, the column can be loaded to its maximal capacity by applying a slight excess of rhodopsin. Flow rates depend on column size (about 2 ml/hr for columns ~< 2 ml, up to 10 ml/hr for columns > 10 ml). With higher flow rates part of the rhodopsin may pass through without binding and the eluate has to be reapplied for maximal loading. (Since Con A has higher affinity for opsin, high opsin content will seriously decrease the capacity of the column for rhodopsin.) Subsequently, nonbound material is washed out with at least 8 vol of G / I / J (50:50: 1) until the eluate is free of lipid (Fig. 1A). The bound rhodopsin is then eluted with G / I / J (50:50: 1) to which solid a-methylglucose or amethylmannose (Sigma, grade III) is added to a final concentration of 200 raM. 16 Rhodopsin-containing fractions with a A28o/Asoo ratio of 15 Mn2* binds very strongly to phospholipids (e.g., used for reconstitution studies) and may also interfere with subsequent ESR or NMR studies. It is therefore left out during chromatography. When its presence does not present a problem, everywhere in the procedure G can be replaced by F. 16 Alternatively, rhodopsin can be eluted, if so required, by lower concentrations of sugar (e.g., 50 mM) or a sugar gradient (e.g., 0-200 mM). Both conditions, however, retard elution of rhodopsin and yield eluates with a much lower rhodopsin concentration. This is probably due to heterogeneity in the affinity constants of the immobilized Con A as a result of chemical and steric factors inherent to immobilization.
1.41.0-
Rh ,
~-MeMann
Rh
hv
A
Q8 ~ O.D.0.60402 + O-
B
1./*1.2-
-Me 10-
Mann
O.D. 0.8fl6 0./*0.20
C ,,-Me
-
Mann ÷
o.1+
Rh
h~.
1 M NaCt
+~-Me-
0.4 OD.
0.2 0
0.6 0D'0410.2
h,.
Rh
0
D
,~- M e -
NH20H
_
+~-Me-
Mann+ I M NoCI
,
I
I
I
I
i
L
I
5
I
I
I
I
I
10
i
I
I
i
L
I
15
I
I
I
I
20
I
I
~
t
I
25
I
I
i
I
I
30
FRACTION
FIG. 1. Con A-Sepharose chromatography of rhodopsin (A) and opsin (B, C, D) under various conditions. The column was not loaded to full capacity so as not to complicate the elution pattern with excess rhodopsin. To 1.6 ml of settled gel are applied, respectively, 84 (A), 95 (B), 96 (C), and 110 (D) nmol of rhodopsin, dissolved in 4 - 5 ml of 20 mM
[30]
PURIFICATION OF BOVINE RHODOPSIN
203
1.6-1.8 (corrected for contribution of the buffer and detergent to the A2s0) are combined. The column is then regenerated with 3 vol F / H / I (1 : 1 : 1), followed by 3 vol D / H (2 ; 1) and 5 vol F / H (2 : 1) and finally stored in F / H (2 : 1) or F / H / I (2: 2: 1) in the cold. The beads can be used repeatedly until the capacity has decreased too much. The entire purification takes 5 - 2 0 hr, depending on column size. Recovery of rhodopsin with an A2s0/Asoo ratio usually between 1.6 and 1.7 is over 90%. The A4oo/A~ooratio lies between 0.16 and 0.17. The lipid content is less than 0.5 tool/tool of rhodopsin. Spectra before and after purification are shown elsewhere. 1 S D S - P A G E gels are presented in Fig. 2. If any Con A present does not have to be removed, salts and sugar can be dialyzed out and the solution stored at - 70 ° in dark containers for at least half a year. In the case of dialyzable detergents, both lipid- and detergentfree rhodopsin can be obtained via dialysis and subsequent centrifugation (30 min, 100,000 g, 4 °) or the detergent used can be exchanged for another
PIPES buffer, pH 6.5, containing 130 mM NaCI, 3 mM MgCI2, 2 mM CaCI2, 1 mM MnCIz, 0.5 mM DTE, 0.1 mM EDTA and 20 mM nonylglucose. The column is washed with the same buffer, and (rhod)opsin eluted with the same buffer containing 0.2 M c~-l-methylmannoside, both at a flow rate of 10 ml/hr. Arrows indicate change of condition. Fractions of 1.7 ml are collected. Clear bar: A~0; solid bar: lipid; cross-hatched bar: As0o (A), Aa,t5 (B,C), Aa6~(D). (A) Following sample application, the column is washed until the eluate is free of protein (Az80) and lipid (assayed as phosphate). Then rhodopsin is eluted with a-1-methylmannoside. Recovery of rhodopsin (both on 280 and on 500-base) is 96%, 85% of which is recovered in the first four rhodopsin-containing fractions. This purified rhodopsin regenerates for over 70%. (B) Same as (A) until washing is completed. Then the column is illuminated at 4° with white light until no rhodopsin is left (several minutes: visual inspection). Immediate elution with sugar follows. Recovery of original chromophore as 375-nm absorbance is about 90%, recovery ofopsin protein is close to 100%, 80% of which is recovered in the first four fractions. Regeneration is 60-70% of a control sample. (C) Following sample application, the column is illuminated and washed until eluate is free of lipid and retinal (375 nm). This fraction comprises only 10-15% of the amount of chromophore originally applied. Subsequent elution with sugar releases only 40-50% of the opsin applied, part of which still contains 375-nm absorbance and shows variable regeneratiorr (30-60% of a control sample). The remainder of the opsin can be slowly eluted by including 1 M NaCl in the buffer. This fraction shows no measurable 375-nm absorbance and does not regenerate. Total recovery of original chromophore as 375-nm absorbance is only 30-35%. The first four opsin fractions comprise about 33% of the total applied. (D) Same as (C), but to the wash-buffer 20 mM hydroxylamine is added in order to convert all free and bound retinal into the oxime (hmax = 365 nm), and washing is continued until the eluate is also free of oxime. The recovery of original chromophore as the oxime is 95%. Subsequent elution with sugar only slowly releases the opsin in a nonregenerable form. The first four opsin fractions contain less than 20% of the total applied. Addition of l M NaCl somewhat enhances the rate at which opsin elutes, but more rapid release is only achieved under more extreme conditions such as using stronger detergents (not shown).
204
CHARACTERIZATION OF VISUAL PIGMENTS
[30]
Mw
x 10 -3
--TETRAMER
220 93 69
"TRIMER
5O 45 36 30 25
18 14 9
Ca
ROS
Rh
FIG. 2. SDS-polyacrylamide gels of rod outer segment membranes (ROS) and purified rhodopsin (Rh), together with a calibration gel (Ca). The tendency of opsin to aggregate is aggravated by purification as clearly expressed by the appearance of higher oligomers. Proof that these bands indeed are oligomers of opsin is obtained from the observations that these bands (1) all fluoresce after reductive fixation of the chromophore to opsin and (2) all react with antibodies against opsin, as determined by crossed immunoelectrophoresis (Margry and De Grip, unpublished). The arrow points at the 230,000 band in ROS representing the large glycoprotein, which is completely absent in the purified preparation. In order to separate a wide range of molecular weights, a linear polyacrylamide gradient is used (8.5-29% w/v; 4.5% cross-linker). The buffer system is adapted from U. K. Laemlli [Nature (London) 227, 680 (1970]. Staining was performed with Coomassie Blue R - 250 [G. Fairbanks, T. L. Steck, and D. F. M. WaUach, Biochemistry 10, 2606 (1971].
one (e.g., digitonin). Nondialyzable detergents are best exchanged for other types while rhodopsin is still bound to the column. If opsin is to be purified, the elution pattern depends on the detergent used. In strong detergents (DTAB, TTAB, CTAB) opsin is somewhat retarded with respect to rhodopsin but is easily eluted completely delipidated, albeit irreversibly denatured. The same chromatographic behavior is observed in very mild detergents (digitonin, dodecylmaltose), but here opsin remains fairly intact (regenerates for over 80%), while only partial delipidation is achieved, u In detergents of intermediate mildness, like nonylglucose, the behavior of opsin depends on its "illumination history" (Fig. 1B-D). When opsin is generated on the column and immediately desorbed with sugar (Fig. 1B), all protein and retinal elute with a constant
[30]
P U R I F I C A T I OOF N BOVINE RHODOPSIN
205
Azso/Aar~ ratio
o f about 1.9 and the material regenerates for o v e r 70% o f the control. H o w e v e r , when opsin is generated before the buffer washing (Fig. 1C), retinal is slowly eluted, and, following addition of sugar, that part o f the protein that still contains retinal is rapidly eluted and shows regenerability. The remainder of the protein is strongly retarded, does not contain retinal, and does not regenerate. Apparently at 4 ° illumination generates a fairly stable photoproduct with spectral properties similar to metarhodopsin II. This product behaves like rhodopsin in some ways. Loss of retinal seems to be followed by secondary conformational changes (partial or complete denaturation), resulting in higher affinity for Con A as well as nonspecific interaction a n d / o r aggregation phenomena. This is very clear in Fig. 1D, where, following illumination, retinal is first removed as the oxime derivative by including 20 mM hydroxylamine in the wash buffer. All opsin is then strongly retarded and can only be eluted completely under more extreme conditions, like including strong detergents a n d / o r ethyleneglycol or urea (not shown). Even more extreme results are obtained when opsin is generated by illumination at 20 ° before it is applied to the column. These observations explain why in this type of detergent rhodopsin is easily purified from opsin. If Con A present has to be removed, the volume of the combined (rhod)opsin fractions is first reduced in order to attain a rhodopsin concentration of at least 100/zM (in the cold, e.g., by pressure dialysis or vacuum filtration). The sample thus obtained is applied to a column o f K with a volume at least five times the sample volume. This guarantees that enough G-50 surface remains between the rapidly migrating rhodopsinmicelles and the small molecules lagging behind to retard the Con A. Rhodopsin is eluted with G / I / J (50: 50:1; flow rate: anywhere between 10 and 60 ml/hr) and elutes two to five times diluted with an elution volume equivalent to the void volume of the column. Except when contamination with Con A is very serious, the spectral properties of the combined rhodopsin fractions are practically unchanged. 17 The combined samples can be stored frozen or used for further treatments, as described previously after Con A chromatography. We have performed some careful amino acids analysis of rhodopsin purified in this way (see table). The results agree well with earlier reports 17Recently, we have worked out a further improved alternative to remove Con A from rhodopsin solutions (unpublished) by passage through a column of immobilized antibodies against Con A. With this procedure, denatured Con A is removed as well, while only small bed volumina are required (up to 100 nmol of Con A may be bound per milliliter of gel), thereby introducing only a small dilution factor. The antibody matrix can be freed from Con A with, e.g., 0.2 M glycin-HCl(pH 2.0) and reused after washing with the appropriate buffer.
206
CHARACTERIZATION OF VISUAL PIGMENTS
[30]
AMINO ACID COMPOSITION OF BOVINE RHODOPSIN IN RESIDUE PER MOLE OF RHODOPSIN Residue Asx Thr Ser Glx Pro Gly Ala Cys ½Cyn Val Met Ile Leu Tyr Phe Lys His Trp Arg Total
Results 23~ 28 15 35 21 26 28 6 4 27 13 17 29 17 31 13 7 8 8 356
24b 26 15 33 22 26 31 }5 30 10 20 30 16 33 14 6 nd e 8 357
26c 34 21 34 22 28 34 }8 21 11 13 27 14 30 11 5 nd 8
23a 26 18 32 20 24 30 }8 30 12 20 30 17 30 15 6 8 9
a Our results, expressed per mole of chromophore-bound retinal (¢s0o = 4050 m2/mol). Figures extrapolated from hydrolysis times up to 96 hr. Hydrolysis was performed at 110° with 6 N HCI in the presence of 0.1% phenol and 0.1% mercaptoacetic acid, which minimizes decomposition of methionine and tyrosine. Total Cys + Cyn was determined both spectrophotometrically and as cysteic acid (see elsewhere this volume). Cys and Trp were determined spectrophotometrically. b From J. J. Plantner and E. L. Kean, J. Biol. Chem. 251, 1548 (1976). Expressed per mole of rhodopsin ( ~ = 4060 m2/mol), purified by hydroxyapatite chromatography followed by Con A - S e p h a r o s e chromatography. Total includes 8 Trp. c From H. Shichi, M. S. Lewis, F. Irreverre, and A. L. Stone, J. Biol. Chem. 244, 529 (1969). Rhodopsin was purified by hydroxyapatite chromatography followed by ion-exchange chromatography. Figures were recalculated to obtain a MW of - 4 0 K. d From J. Heller, Biochemistry 7, 2906 (1968). Rhodopsin was purified by gel filtration. Figures were recalculated to obtain a MW of - 4 0 K. e Not determined.
[30]
PURIFICATION OF BOVINE RHODOPSIN
207
and from our data we calculate a MW for the protein part of rhodopsin of 39.8 K ---0.8 K. Together with carbohydrate and chromophore this adds up to a total molecular weight of about 42.3 K. The lower molecular weight obtained in various SDS-PAGE systems (32 K - 3 8 K) might indicate a relatively high level of SDS binding, a phenomenon observed also with other membrane proteins, TM including bacteriorhodopsin.
18 A. Helenius and K. Simons, Biochim. Biophys. Acta 41S, 29 (1975).