Analysis of electrophoretic behavior of cytochrome c oxidase subunits. Retardation coefficient versus molecular weight in dodecyl sulfate urea gels.

1. Standard proteins were examined by electrophoresis in highly cross-linked polyacrylamide gels containing sodium dodecyl sulfate and urea. Their behavior was analyzed at a single gel concentration (by molecular weight vs. relative mobility) and at several gel concentrations (molecular weight vs. retardation coefficient). The validity of the latter method of analysis was established for this gel system. 2. Cytochrome c oxidase was subjected to analysis by this method. Compared to standards, subunits I and III showed increased free electrophoretic mobility, while that of subunit V was slightly decreased. The molecular weight values derived were: I, 44 600; II, 22 700; III, 23 500; IV, 16 900; V, 9400; VI, 7600; VII, 4300. The standard errors were all less than +/- 7%. 3. Isolated V and VI were analyzed by two dimensional dodecyl sulfate electrophoresis, in which the second dimension also contained urea. In contrast to their behavior when the holo-enzyme was examined, these isolated subunits V and VI no longer exchange migrating positions relative to each other during the two dimensional analysis. The molecular weight values of the isolated subunits agree with those of the holo-enzyme.     

Immunological studies on cytochrome c oxidase: arrangements of protein subunits in the solubilized and membrane-bound enzyme.

Seven protein subunits of cytochrome c oxidase from bovine heart were isolated by gel filtration in the presence of sodium dodecyl sulphate (subunits I, II and III) and guanidine hydrochloride (subunits V, VI and VII), and ion-exchange chromatography in 6 M urea (subunit IV) after the enzyme had been dissociated in 6 M guanidine hydrochloride. When analysed by highly cross-linked sodium dodecyl sulphate/polyacrylamide gel electrophoresis in the presence of urea, the apparent molecular weights were = I, 36700; II, 24300; III, 20400; IV, 17300; V, 12300; VI, 8700: and VII, 5100. Monospecific rabbit antisera were obtained against subunits I, IV, V, VI and VII and a mixture of subunits II and III. These subunit-specific antisera with the exception of anti-I serum all cross-reacted with the detergent-solubilized native oxidase. Enzymatic studies on purified oxidase indicated that immunoglobulins against subunits II + III, IV, V, VI and VII respectively caused 25, 65, 20, 30 and 25% inhibition while anti-I immunoglobulin did not inhibit the activity. The subunit-specific antisera were used to examine the arrangements of the subunits in the membrane. Enzymatic studies using bovine heart mitochondria and rat liver mitochondrial digitonin particles showed that anti-(II + III) serum, anti-V serum and anti-VII serum all inhibited the oxidase activity while the other antisera did not. On the other hand, results of using 125I-labelled immunoglobulins showed that anti-IV, anti-V and anti-VII sera were bound to the surface of inverted vesicles (matrix side) while all other antisera were not. These results indicate that cytochrome oxidase subunits II and III are situated on the outer surface, and subunit IV is exclusively on the matrix surface while subunits V and VII are exposed on both surfaces of the mitochondrial membrane. Subunits I and VI are buried within the membrane, not exposed on either side.     

Large scale isolation and properties of subunits from bovine heart cytochrome oxidase

The subunits of the cytochrome oxidase from bovine heart were isolated in large quantities suitable for amino acid sequence studies. The preparation of subunits III, IV, V, VI, and VII for sequence determination can be achieved without employing sodium dodecyl sulfate. The method presented essentially involves pyridine extraction, pH fractionation, ammonium sulfate fractionation, and various types of column chromatography. However, subunits I and II can be prepared only in the presence of sodium dodecyl sulfate by molecular sieve chromatography; subunit III can also be isolated in this manner. The separation of subunits is found to be hindered by phospholipids associated with the enzyme and therefore the phospholipid-depleted preparation is used as the starting material. The molecular weights of subunits I, II, III, IV, V, VI, and VII are 40,000, 21,000, 14,800, 13,500, 11,600, 9,500, and 7,600, respectively. These values are based on the results of the conventional Weber and Osborn method of gel electrophoresis in the presence of sodium dodecyl sulfate. The amino termini of subunits I and II have been determined as N-formylmethionine, and those of subunits III, IV, V, VI, and VII are alanine, alanine, serine, alanine, and an N-acetyl-blocked residue, respectively. The carboxyl termini for subunits I to VII are lysine, leucine, lysine, histidine, valine, isoleucine, and valine, respectively. The complete amino acid sequence of some subunits has been published and that of other subunits will be reported elsewhere in collaboration with the Amino Acid Sequence Group of Cytochrome Oxidase at the University of Hawaii.     

Catalytic activity and arrangement of subunit polypeptides in rat liver cytochrome c oxidase as studied by proteolysis

Cytochrome c oxidase from rat liver was incubated with various proteinases of different specificities and the enzymic activity was measured after various incubation times. A loss of catalytic activity was found after digestion with proteinase K, aminopeptidase M and a mitochondrial proteinase from rat liver. In each case the decrease in enzymic activity was compared with the changes in intensities of the polypeptide pattern obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibilities of the subunit polypeptides of the soluble cytochrome c oxidase to proteinases were very different. Whereas subunit I was most susceptible, subunits V--VII were rather resistant to degradation. From the relative inaccessibility of subunits V--VII to proteinases it is likely that these polypeptides are buried in the interior of the enzyme complex.     

Further analysis of the polypeptide subunits of yeast cytochrome c oxidase. Isolation and characterization of subunits III, V, and VII

By using a modified purification procedure in which we have substituted detergent exchange gel filtration for DEAE-cellulose or hydroxylapatite chromatography (Mason, T. L., Poyton, R. O., Wharton, D. C., and Schatz, G. (1973) J. Biol. Chem. 248, 1346-1354), we have isolated yeast cytochrome c oxidase preparations which are low in contaminating polypeptides and which have been successfully used for the large scale purification of subunits. Subunits have been purified from this preparation by a simple two-step procedure which involves: 1) the release of subunits IV and VI from an "insoluble" core composed of subunits I, II, III, V, and VII; and 2) gel filtration of the "core" subunits in the presence of sodium dodecyl sulfate. Molecular weights of the isolated subunits, obtained from sodium dodecyl sulfate gel retardation coefficients (KR) derived from Ferguson plots, were: I, 54,000; II, 31,000; III, 29,500; IV, 14,500; V, 12,500; VI, 9,500; VII, 4,500. In their purified state all subunits, except for subunit V, exhibited electrophoretic behavior similar to that exhibited by unpurified subunits in sodium dodecyl sulfate-dissociated holoenzyme preparations. As purified, subunit V exhibits a slightly smaller apparent molecular weight than its counterpart in the holoenzyme. Amino acid analysis of the isolated subunits revealed that subunit III, a mitochondrial translation product, contained 41.9% polar amino acids, whereas subunits V and VII, cytoplasmic translation products, each contained 47.7% polar amino acids. These results extend and support our previous finding that the mitochondrially translated subunits of yeast cytochrome c oxidase are more hydrophobic than the cytoplasmically translated subunits.     

Specific labeling and partial inactivation of cytochrome oxidase by fluorescein mercuric acetate

Addition of 1 eq of fluorescein mercuric acetate (FMA) to beef heart cytochrome oxidase was found to inhibit the steady-state electron transfer activity by 50%, but further additions up to 10 eq had no additional effect on activity. The partial inhibition caused by FMA is thus similar to that observed with other mercury compounds (Mann, A. J., and Auer, H. E. (1980) J. Biol. Chem. 255, 454-458). The fluorescence of FMA was quenched by a factor of 10 upon binding to cytochrome oxidase, consistent with the involvement of a sulfhydryl group. However, addition of mercuric chloride to FMA-cytochrome oxidase resulted in an increase in fluorescence, suggesting that FMA was displaced from the high affinity binding site. Cytochrome c binding to FMA-cytochrome oxidase resulted in a 10% decrease in the fluorescence, possibly caused by Forster energy transfer from FMA to the cytochrome c heme. The binding site for FMA in cytochrome oxidase was investigated by carrying out sodium dodecyl sulfate gel electrophoresis under progressively milder dissociation conditions. When FMA-cytochrome oxidase was dissociated with 3% sodium dodecyl sulfate and 6 M urea, FMA was predominantly bound to subunit II following electrophoresis. However, when the dissociation was carried out at 4 degrees C in the absence of urea with progressively smaller amounts of lithium dodecyl sulfate, the labeling of subunit II decreased and that of subunit I increased. These experiments demonstrate that mercury compounds bind to a high affinity site on cytochrome oxidase, possibly located in subunit I, but then migrate to subunit II under the normal sodium dodecyl sulfate gel electrophoresis conditions. A definitive assignment of the high affinity binding site in the native enzyme cannot be made, however, because it is possible that mercury compounds can migrate from one sulfhydryl to another under even the mildest electrophoresis conditions.     

Catalytic activity and arrangement of subunit polypeptides in rat liver cytochrome c oxidase as studied by proteolysis

Cytochrome c oxidase from rat liver was incubated with various proteinases of different specificities and the enzymic activity was measured after various incubation times. A loss of catalytic activity was found after digestion with proteinase K, aminopeptidase M and a mitochondrial proteinase from rat liver. In each case the decrease in enzymic activity was compared with the changes in intensities of the polypeptide pattern obtained after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The susceptibilities of the subunit polypeptides of the soluble cytochrome c oxidase to proteinases were very different. Whereas subunit I was most susceptible, subunits V–VII were rather resistant to degradation. From the relative inaccessibility of subunits V–VII to proteinases it is likely that these polypeptides are buried in the interior of the enzyme complex.     

Characterization of a seventh different subunit of beef heart cytochrome c oxidase. Similarities between the beef heart enzyme and that from other species.

Beef heart cytochrome c oxidase has been resolved into seven subunits by electrophoresis in highly cross-linked gels containing urea and sodium dodecyl sulfate. The molecular weights of the polypeptides are estimated to be I, 35 400; II, 24 100; III, 21 000; IV, 16 800; V, 12 400; VI, 8200; and VII, 4400. It has been shown that subunits II and III can coelectrophorese on standard sodium dodecyl sulfate-polyacrylamide gels and appear as a single component with an apparent molecular weight of 22 500. This accounts for previous reports that the beef heart enzyme contains only six subunits. Amino acid analysis of the isolated subunits I, II, and III revealed that they have polarities of 35.5, 44.7, and 39.9%, respectively. All three subunits have an extremely high leucine content and a low percentage of basic amino acids relative to subunits IV-VII. The size, number, and properties of subunits in the beef heart cytochrome c oxidase complex suggest that it has essentially the same subunit structure as the complexes isolated from Saccharomyces cerevisiae and Neurospora crassa.     

Cytochrome c oxidase from bakers' yeast. III. Physical characterization of isolated subunits and chemical evidence for two different classes of polypeptides.

Earlier studies have shown that cytochrome c oxidase from bakers' yeast is an oligomeric enzyme which contains three polypeptides (I to III) synthesized on mitochondrial ribosomes and four polypeptides (IV to VII) synthesized on cytoplasmic ribosomes. These polypeptide subunits have now been isolated by a simple protocol which utilizes differences in polypeptide charge, solubility, and size. Their molecular weights determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, gel filtration in the presence of guanidine hydrochloride, and amino acid analysis were: I, 40,000; II, 33,000; III, 22,000; IV, 14,500; V, 12,700; VI, 12,700; and VII, 4,600. All seven polypeptide subunits exhibited acidic isoelectric points; cytoplasmically made subunits were more acidic than mitochondrially made ones. The amino acid composition of two mitochondrially made subunits and two cytoplasmically made subunits was determined. The two mitochondrial translation products, I and II, contained only 34.7% and 42.1% polar amino acids, respectively, whereas the two cytoplasmic translation products, IV and VI, contained 48.3% and 49.3%, respectively. This agreed with the observation that Subunits I and II are very insoluble, requiring detergents for solubility, whereas Subunits IV and VI are water-soluble in the absence of any added detergent. These results indicate that the cytochrome c oxidase subunits synthesized on mitochondrial and cytoplasmic ribosomes are fundamentally different in size, isoelectric properties, and hydrophobicity. They also suggest the possibility that at least some of the mitochondrially made subunits are buried in the lipid phase of the mitochondrial inner membrane.     

A reversal in relative mobility of the two large subunits of brine shrimp (Na+ + K+)-adenosinetriphosphatase

The two large subunits of brine shrimp Na,K-ATPase can be resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis at neutral pH and at acidic pH. These subunits appear to reverse their positions on the gel relative to each other when resolved at acidic pH relative to neutral pH. The migration of both subunits is apparently affected by charge, even in the presence of 2.5% sodium dodecyl sulfate.     

Structure of cytochromec oxidase from baker's yeast—a progress report. Preparation of four subunits for amino acid sequence determination and attempts to localize the cytochromec binding site

Summary Cytochromec oxidase from the inner membrane of yeast mitochondria consists of seven nonidentical protein subunits, three being synthesized on mitochondrial ribosomes (molecular weights I: 43 K, II: 34 K, and III: 24 K) and four being made on cytoplasmic ribosomes (molecular weights IV: 14 K, V: 12 K, VI: 12 K, and VII: 4.5 K).In the present study all four cytoplasmically synthesized subunits of the enzyme were isolated on a large scale using ion exchange chromatography and gel filtartion. Their amino acid composition as well as their amino- and carboxy-terminal amino acid residues have been determined. Sequence determinations of sub-units IV and VI are already in an advanced state. The sequence of subunit VI is characterized by a large amino-terminal stretch dominated by charged amino acid residues followed by a cluster of hydrophobic amino acids.The binding site of yeast cytochrome oxidase for cytochromec was studied by chemical crosslinking experiments. The formation of a disulfide bridge between the two proteins was observed by using cytochromec from yeast modified with 5-thionitrobenzoate at the cysteinyl residue in position 107. Alternatively, a disulfide between yeast cytochromec and the oxidase could be formed directly by oxidation with copper phenanthroline. Gel electrophoresis of the crosslinked complexes in sodium dodecyl sulfate revealed a new protein band with an apparent molecular weight of 38 K. This new band appears to be derived from cytochromec and from subunit III of cytochrome oxidase.Recipient of a fellowship from the Swiss National Science Foundation. Present address: Department of Biology, University of California at San Diego, La Jolla, Calif. 92037 (USA).     

Human cytochrome c oxidase isoenzymes from heart and skeletal muscle; purification and properties

Human cytochrome c oxidase was isolated in an active form from heart and from skeletal muscle by a fast, small-scale isolation method. The procedure involves differential solubilisation of the oxidase from mitochondrial fragments by laurylmaltoside and KCl, followed by size-exclusion high-performance liquid chromatography. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate showed differences between the subunit VI region of cytochrome c oxidases from human heart and skeletal muscle, suggesting different isoenzyme forms in the two organs. This finding might be of importance in explaining mitochondrial myopathy which shows a deficiency of cytochrome c oxidase in skeletal muscle only. In SDS polyacrylamide gel electrophoresis most human cytochrome c oxidase subunits migrated differently from their bovine counterparts. However, the position of subunits III and IV was the same in the human and in the bovine enzymes. The much higher mobility of human cytochrome c oxidase subunit II is explained by a greater hydrophobicity of this polypeptide than of that of the subunit II of the bovine enzyme.     

The subunit structure of the cytochrome c oxidase complex.

The subunit structure of the cytochrome c oxidase complex has been obtained for three preparations each isolated by a different detergent procedure. Six polypeptides were present in all samples with the following molecular weights: subunits I, 36000; II, 22500, III, 17100; IV, 12500; V, 9700; and VI, 5300. These subunits have been purified by gel filtration in sodium dodecyl sulfate or in 6 M guanidine hydrochloride and their amino acid compositions have been determined. Subunit I is hydrophobic in character with a polarity of 35.7%. Subunits II through VI are more hydrophilic with polarities of 45.5, 48.6, 47.8, 49.7, and 53.7%, respectively.     

Induction of rat hepatic alkaline phosphatase and its appearance in serum: electrophoretic characterization of liver-membranous and serum-soluble forms

Simultaneous bile duct ligation and colchicine injection (2 mg/kg body weight) in rats caused a remarkable induction of alkaline phosphatase in the liver. Concomitantly, a marked elevation of the enzyme activity occurred in the serum, and three activity peaks (peaks I, II, and III) were separated by Sephadex G-200 gel filtration. By several criteria for alkaline phosphatase isoenzymes it was determined that the liver-derived enzyme was distributed in peak I (30% of total serum activity) as a vesicle-bound form and in peak II (65%) as a soluble form, while the intestinal enzyme was contained in peak III (5%). The serum alkaline phosphatase in peaks I and II was compared with the liver enzyme extracted from plasma membrane with n-butanol. Under non-reducing conditions, the soluble form of peak II showed an electrophoretic mobility different from that of the liver enzyme; in the presence of sodium dodecyl sulfate the serum-soluble form migrated a little more slowly than the liver one, while in the presence of Triton X-100 the former migrated much faster than the latter. The sedimentable fraction of peak I was found to contain two forms corresponding to the serum-soluble and liver-membranous forms. Neuraminidase treatment of these two forms reduced their mobilities but did not abolish the relative difference in their mobilities on gel electrophoresis in the presence of either Triton X-100 or sodium dodecyl sulfate. Under reducing conditions, however, each form (which was dissociated into single subunits) migrated with an identical mobility on sodium dodecyl sulfate gel electrophoresis. These results suggest that the hepatic alkaline phosphatase exists as conformationally different forms in the serum and the liver membrane (even solubilized), but the difference is no longer preserved after their denaturation into subunits.     

The reactivity of thiol groups in bovine heart cytochrome c oxidase towards 5,5'-dithiobis(2-nitrobenzoic acid)

Bovine heart cytochrome c oxidase consists of 12 stoicheiometric polypeptide chains of at least 11 different types. The enzyme contains 14--16 cysteine residues; the distribution of nearly all cysteine residues over the subunits has been established. In native cytochrome c oxidase two thiol groups reacted rapidly and stoicheiometrically with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). These thiol groups are located in subunits I and III, respectively. This implies that subunit I is not fully buried in the hydrophobic core of the enzyme. After dissociation of the enzyme by sodium dodecyl sulphate more thiol groups became available to DTNB, in addition to those in subunits I and III, at least one in subunit II, two in fraction V/VI and one to two in the smallest subunit fraction. It is shown that separation of the subunits of cytochrome c oxidase by gel permeation chromatography in the presence of sodium dodecyl sulphate depends on the pH of the elution medium. The elution volume of subunits I, III and VII is dependent on pH, that of the others independent.     

Detection of iron-sulfur center-containing subunits of mitochondrial NADH dehydrogenase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by high-performance gel permeation chromatography

Soluble NADH dehydrogenase resolved from Complex I of the mitochondrial electron-transfer chain was subjected to gel electrophoresis in the presence of sodium dodecyl sulfate at 4 degrees C, and then the gel was stained for iron with bathophenanthroline disulfonate and thioglycolic acid. The 23,000-dalton subunit was markedly stained, and the 51,000-dalton subunit was also stained, but only slightly. High-performance gel permeation chromatography using an eluant containing 0.1% sodium dodecyl sulfate also demonstrated that these subunits contain an iron-sulfur center: the elution pattern recorded by light absorption at 400 nm gave two peaks corresponding to the positions of the subunits.     

Cytochrome c oxidase from bakers' yeast. Photolabeling of subunits exposed to the lipid bilayer.

Yeast mitochondria and purified yeast cytochrome c oxidase incorporated into micelles of the nonionic detergent Tween 80 were equilibrated with the hydrophobic aryl azides 5-[125I]iodonaphthyl-1-azide or S-(4-azido-2-nitrophenyl)-[35S]thiophenol. The azides were then converted to highly reactive nitrenes by flash photolysis or by illumination for 2 min and the derivatized cytochrome c oxidase subunits were identified by gel electrophoresis and radioactivity measurements. 5-[125I]Iodonaphthyl-1-azide labeled mainly the three mitochondrially made Subunits I to III and the cytoplasmically made Subunit VII. Subunits IV to VI or cytochrome c bound to the purified enzyme were labeled 9- to 90-fold less. Essentially the same result was obtained with S-(4-azido-2-nitrophenyl)-[35S]thiophenol except that Subunit V was labeled as well. In contrast, all seven subunits as well as cytochrome c were heavily labeled when the enzyme was dissociated with dodecyl sulfate prior to photolabeling with either of the two probes. These data indicate that all subunits of yeast cytochrome c oxidase except Subunits IV and VI are at least partly embedded in the lipid bilayer of the mitochondrial inner membrane.     

Rat kidney L-alpha-hydroxy acid oxidase: isolation of enzyme with one flavine coenzyme per two subunits.

L-alpha-Hydroxy acid oxidase (listed as EC 1.4.3.2, L-amino acid: O2 oxidoreductase) has been purified 100-fold from rat kidney to apparent homogeneity by gel electrophoresis. A subunit molecular weight of 47,500 was found by sodium dodecyl sulfate gel electrophoresis, but in contrast to previous reports, the enzyme has been found to have a molecular weight of ca. 200,000 by Sephadex gel filtration and by dodecyl sulfate gel electrophoresis of the enzyme cross-linked with dimethyl suberimidate. A somewhat higher value was found by sedimentation equilibrium, but a tetrameric structure for the active enzyme is definitely established. The enzyme was found to contain the FMN coenzyme at a concentration of one FMN/102,000 daltons or one flavine/two subunits, a highly unusual finding. This ratio was determined from spectroscopic analysis of the FMN in lyophilized samples of the enzyme and by titration of the coenzyme with the flavine specific enzyme inactivator 2-hydroxy-3-butynoate. The enzyme has the same specific activity as a crystalline sample of the enzyme reported to have twice as much flavine/milligram.     

Arrangement of the subunits in solubilized and membrane-bound cytochrome c oxidase from bovine heart.

The arrangement of the six cytochrome c oxidase subunits in the inner membrane of bovine heart mitochondria was investigated. The experiments were carried out in three steps. In the first step, exposed subunits were coupled to the membrane-impermeant reagent p-diazonium benzene [32S]sulfonate. In the second step, the membranes were lysed with cholate anc cytochrome c oxidase was isolated by immunoprecipitation. In the third step, the six cytochrome c oxidase subunits were separated from each other by dodecyl sulfate-acrylamide gel electrophoresis and scanned for radioactivity. Exposed subunits on the outer side of the mitochondrial inner membrane were identified by labeling intact mitochondria. Exposed subunits on the matrix side of the inner membrane were identified by labeling sonically prepared submitochondrial particles in which the matrix side of the inner membrane is exposed to the suspending medium. Since sonic irradiation leads to a rearrangement of cytochrome c oxidase in a large fraction of the resulting submitochondrial particles, an immunochemical procedure was developed for isolating particles with a low content of displaced cytochrome c oxidase. With mitochondria, subunits II, V, and VI were labeled, whereas in purified submitochondrial particles most of the label was in subunit III. The arrangement of cytochrome c oxidase in the mitochondrial inner membrane is thus transmembraneous and asymmetric; subunits II, V, and VI are situated on the outer side, subunit III is situated on the matrix side, and subunits I and IV are buried in the interior of the membrane. In a study of purified cytochrome c oxidase labeled with p-diazonium benzene [32S]sulfonate, the results were similar to those obtained with the membrane-bound enzyme. Subunits I and IV were inaccessible to the reagent, whereas the other four subunits were accessible. In contrast, all six subunits became labeled if the enzyme was dissociated with dodecyl sulfate before being exposed to the labeling reagent.     

The reactivity of thiol groups in bovine heart cytochrome c oxidase towards 5,5′-dithiobis(2-nitrobenzoic acid)

Bovine heart cytochrome c oxidase consists of 12 stoicheiometric polypeptide chains of at least 11 different types. The enzyme contains 14–16 cysteine residues; the distribution of nearly all cysteine residues over the subunits has been established. In native cytochrome c oxidase two thiol groups reacted rapidly and stoicheiometrically with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). These thiol groups are located in subunits I and III, respectively. This implies that subunit I is not fully buried in the hydrophobic core of the enzyme. After dissociation of the enzyme by sodium dodecyl sulphate more thiol groups became available to DTNB, in addition to those in subunits I and III, at least one in subunit II, two in fraction V/VI and one to two in the smallest subunit fraction. It is shown that separation of the subunits of cytochrome c oxidase by gel permeation chromatography in the presence of sodium dodecyl sulphate depends on the pH of the elution medium. The elution volume of subunits I, III and VII is dependent on pH, that of the others independent.