Bioehimica et Biophysica Acta, 495 (1977) 232-247 © Elsevier/North-Holland Biomedical Press

BBA 37807

SUBUNIT STRUCTURE OF THE RECONSTITUTIVELY ACTIVE CYTO- CHROME b-c1 COMPLEX

DETERMINATION OF AMINO ACIDS AND MOLAR DISTRIBUTION OF SUBUNIT FRACTIONS FROM GEL ELECTROPHORESIS

LINDA YU, CHANG-AN YU and TSOO E. KING* Laboratory of Bioenergeties and Department of Chemistry, State University of New York at Albany, 1400 Washington Avenue, Albany, N.Y. 12222 (U.S.A.)

(Received February 22nd, 1977)

SUMMARY

A quantitative method has been developed to analyze the amino acid com- position of protein subunits directly from the Coomassie Blue-stained band of poly- acrylamide gel columns after electrophoresis. It is an improved method originally reported by Houston (Houston, L. L. (1971) Anal. Biochem. 44, 81-88). The results obtained can be thus used for the calculation of the molar ratios ofsubunit components of protein. The manipulation of the method and computation of the results are illustrated by a very complicated lipoprotein complex.

The subunit molar ratios of the reconstitutively active cytochrome b-c1 com- plex were determined to be 2, 2, 2, 3, 2, 2, and 5 among the seven bands of the corre- sponding molecular weights of 53 000, 50 000, 37 000, 30 000, 28 000, 17 000, and 15 000, from gel electrophoretic columns.

The amino acid composition of each subunit fraction determined directly from hydrolysis of gel was comparable with that obtained by actual isolation of each subunit.

INTRODUCTION

The soluble cytochrome b-c1 complex [1, 2] is a "segment" of the mitochon- drial "chain" which mediates the electron (hydrogen) transfer from reduced ubi- quinone (Q) to cytochrome c. Unlike Complex III [3], the cytochrome b-el complex is characterized as being able to reconstitute with soluble succinate dehydrogenase [4] to form succinate-cytochrome c reductase, sensitive to antimycin A. The failure of Complex III to react directly with succinate dehydrogenase has been recently attri- buted to the lack of an apo-Q-protein in such complex. Such apo-Q-protein is essential in transferring electrons from succinate through succinate dehydrogenase to the cytochrome b-cl complex.

* To whom correspondence should be addressed.

233

As described previously, when the cytochrome b-q complex was subjected to sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, seven protein bands were observed. The number of the protein bands did not account for all the functional components and many reported factors in this region of the chain. This suggests that a rather complicated stoichiometry among subunits must exist. When the amino acid composition (unpublished results) of the pure subunits from purified cytochrome c1 was determined, the picture has then become very clear. The amino acid composition from the pure cytochrome c1 subunits is completely different from that of the bands in the cytochrome b-q complex corresponding to the positions of these cytochrome c1 subunits. These bands are thus obviously composed of more than one type of polypeptides, and cytochrome cl is only one of them. Moreover, the molar ratios or the protein distribution of these bands determined directly from the densitometric method by scanning the stained gel are not reliable, as we have found that the different proteins have different binding affinity toward dye*. Recently, we have improved a reported method [5] to quantify the amount of protein present in each band of gel electrophoretic column by directly hydrolyzing the stained gel slices followed by amino acid composition determination. With this method the molar ratios may thus be computed. However, the computation of molar ratios cannot be only just directly rounded off to the smallest integers, but also needs to consider at the same time results from experiments of different designs, such as fractionation and isolation, and direct chemical analysis ofthe essential components of the reductase.

In this communication we wish to report the improved method for the deter- mination of amino acid composition and molar ratio of subunits directly from gel electrophoresis and the results of the application of this method to the cytochrome b-c, complex. To substantiate the results obtained by the mentioned method, amino acid composition of actual isolated subunits of the cytochrome b-c, complex was also presented together with their isolation procedure. A partial assignment of functional subunits of the cytochrome b-c, complex has also been attempted.

EXPERIMENTAL PROCEDURE

Materials. The “soluble” cytochrome b-c, complex [l] and succinate-cyto- chrome c reductase [6] were prepared from the Keilin-Hartree preparation [7, 81 of bovine heart by the methods described previously.

QZ was synthesized from the reaction of Q0 (2,3-dimethoxy-5-methylbenzo- quinone) with geraniol by a reported method [9] with modifications. Q0 was syn- thesized from creasol (see references cited in ref. 9). Reduction of Q2 was effected by NaBH, in 50 mM phosphate buffer (pH 7.4). The QzHz was extracted with cyclo- hexane, evaporated under a stream of argon, and dissolved in 95 % ethanol up to a concentration of 5 mg/ml. The actual ubiquinone concentration was determined spectrophotometrically with a millimolar coefficient of 12.25 for A”;;irzt [lo].

Horse cytochrome c, type III, crystalline bovine serum albumin, trypsin and

* The blue color and its relative mobility of a band at the position equivalent to a molecular weight of 37 000 depend very much on the amount of dodecyl sulfate present in the incubation before electrophoresis of the system. We found that between 2 and 3 mg of the detergent per mg protein would give satisfactory results. Excess detergent slows the mobility and causes the inseparability of this band from the band of 50 000 daltons.

234

bovine pancreatic trypsin inhibitor were purchased from Sigma. Sodium dodecyl sulfate (Metheson, Coleman and Bell), creasol (Eastman Kodak), and geraniol (Aldrich) were commercial products. Reagents for polyacrylamide gel electrophoresis were obtained from Canalco, and acrylamide, from Eastman Kodak, was recrystall- ized from water. Reagents for amino acid analysis were procured from Beckman and the Pierce Company. All the other chemicals in the highest available purity were obtained commercially.

Enzyme assays. Succinate-cytochrome c reductase and Q2Hz-cyt0chrome c reductase activities were assayed spectrophotometrically in a 1 ml assay mixture containing 20 mM succinate or 50#M of QzH2, 0.1 mM cytochrome c, 0.3 mM EDTA, and 0.1 M phosphate buffer (pH 7.4) by measuring the increase of absorbance at 550 nm at approx. 23 °C after addition of an appropriate quantity of the enzyme.

Concentrations of cytochrome cl [11] and cytochrome b [12] were estimated as previously reported. Essentially, cytochrome c~ was determined from difference spectra of the ascorbate-reduced minus the ferricyanide-oxidized samples using the extinction coefficient of 17.5 for A552. 5 n m - - A540 n m ' Total cytochrome b was deter- mined from difference spectra of the dithionite-reduced minus the ascorbate-reduced samples using the extinction coefficient of 28.5 for A562 nm - - A575 nm.

Concentration of dilute solutions was made by placing the solution in a dialysis tubing which was immersed in powdered Aquacide (CalBiochem). Electro- phoresis on polyacrylamide gel and the staining with Coomassie Brilliant Blue were generally performed as reported [13]. Amino acid analysis was conducted on a Beckman amino acid analyzer, Model 120C [14], cysteine and cystine [15], and tryptophan [16] were determined by published methods.

Method for direct amino acid analysis of gel slices without elution. (1) Electro- phoresis: "l-he method is illustrated here by a very complicated lipoprotein complex, the cytochrome b-c1 complex from beef heart mitochondria. The complex was first incubated with sodium dodecyl sulfate at 3 mg per mg of protein in 1 ~o fl-mercapto- ethanol and 0.1 M phosphate buffer (pH 7.0) at 37 °C for 3 h. The electrophoresis was then d6ne on a 1070 polyacrylamide gel column [13] with a cross-linkage equi- valent to 0.3 70 methylene bisacrylamide in a medium containing 0.1 700 dodecyl sulfate and 0.1 M phosphate buffer (pH 7.0) at room temperature for approx. 5 h with 8 mA per column. Generally, 14 gel columns of 0.6 × 6 cm loaded with approx. 0.1 mg protein each were used. After electrophoresis, the column was stained by Coomassie Brilliant Blue and destained with acetic acid/methanol mixture [13]. The blue bands were sliced and pooled separately. The total length of the sliced gels of each protein band used in hydrolysis was not more than 4 cm. Single known proteins, cytochrome c or bovine serum albumin was first incubated with dodecyl sulfate at 3 mg per mg of protein in 1 ~o fl-mercaptoethanol and 0.1 M phosphate buffer (pH 7.0) at 37 °C for 3 h. The electrophoresis was then done on a polyacrylamide gel column also with a cross-linkage equivalent to 0.3 70 methylene bisacrylamide in a medium containing 0.1 ~o dodecyl sulfate and 0.1 M phosphate buffer (pH 7.0) at room temperature for approx. 1 h with 8 mA per column. 100/~g of protein were loaded to a gel column of 0.6 × 6 cm. After electrophoresis, the column was stained and destained with acetic acid/methanol mixture as the above.

(2) Hydrolysis: The pooled gel slices were mixed with 5 ml of 6 M HC1 plus 0.470 thioglycolic acid. The oxygen in the mixture must be removed as much as

235

possible by repeated evacuation and filled with argon, usually six cycles were required. The tube filled with argon was then sealed and the hydrolysis was conducted at 110 °C for 24 h. After hydrolysis, the mixture was dried in a rotary evaporator in vacuo under a slow stream of argon. The residue was redissolved in water and evaporated again; the process was repeated once more. Finally, it was dissolved in I0 ml water. The solution was divided into two portions.

(3) Removal of ammonia and amino acid analysis: One portion of 5 ml aliquot was dried again in vacuo and dissolved up to 0.8 ml with 0.2 M citrate buffer (pH 2.2). This solution was used for the determination of acidic and neutral amino acids in an amino acid analyzer. Another portion of the hydrolyzed mixture was brought to pH 9.8 with 1 M NaOH and incubated at room temperature for 1 h. After incubation, the solution was dried in a rotary evaporator in vacuo and the residue dissolved in about 5 ml water. The pH of the solution was readjusted to 9.8 and the solution was dried again. The residue was finally dissolved in 0.8 ml of 0.2 M citrate buffer (pH 2.2) and the solution was used for basic amino acid determination in a usual "short column" of an amino acid analyzer.

(4) Blank: A blank of a known length of polyacrylamide gel was carried out under the same conditions for corrections of certain amino acids.

RESULTS

The method The primary purpose for using stained gel slices for amino acid analysis is to

obtain a quantitative amount of protein present in each stained protein band of polyacrylamide gel column after the electrophoresis in order to elucidate the protein distribution among the protein bands. Under the conditions described, the method gives satisfactory results and produces no artifacts. "[he recovery of the protein, as calculated from the summarization of amino acid, is found to be between 95 and 100 ~ . Table I compares the amino acid composition of cytochrome c, bovine serum albumin and the cytochrome b-c1 complex obtained in the presence and absence of stained gel during hydrolysis. Similar satisfactory recoveries were also obtained with a "standard" amino acid mixture, as shown in the table. Moreover, the amino acid elution patterns of the cytochrome b-c1 complex in the presence and absence of gel, as depicted in Fig. 1, are practically the same with respect to the general behavior and the time of the appearance of the amino acids, q'he higher initial peaks were likely due to a higher thioglycolic acid concentration used in the case of Fig. 1B (cf. ref. 17).

It must be cautioned that small amounts of glycine, serine, and aspartic acid (cf. Fig. 2) were found in the blank, i.e. the gel in the absence of protein under the experimental conditions; therefore, a correction should be made, especially for those proteins low in these amino acids. Gel slices amounting to 5 cm produced a total of about 9 nmol of glycine, 7 nmol of serine, and lesser amounts of aspartic acid, gluta- mic acid, and alanine; no significant amount of other amino acids was found. The presence of a large amount of gel during the hydrolysis decreased the recovery of amino acids, as shown in Fig. 3. In actuality, the bands used in hydrolysis were less than 4 cm.

The abundant production of ammonia is objectionable, which precludes accurate determination of amide. The removal of most ammonia, as described, is

236

TABLE I

Comparison of amino acid composition of bovine serum albumin, cytochrome c, the cytochrome b-c1 complex, and the "standard" amino acid mixture (Beckman No. A0620) obtained in the presence and absence of stained polyacrylamide gel in acid hydrolysis. The results for albumin, cytochrome c and the cytochrome b-c~ complex are expressed in tool percent, whereas those for the "standard" amino acid mixture contained 50 nmol each of the amino acids shown before hydrolysis. The columns marked " ÷ gel" signify for the results obtained by incorporation, in the hydrolysis, of 3 cm of polyacrylamide gel which was subjected to staining and destaining process. The percentage given for the "standard" mixture was based on the unhydrolyzed sample and uncorrected, tnd that for other proteins was corrected by "gel blanks". n.d., not determined.

Albumin Cytochrome c The cytochrome b-ct "Standard" amino complex acid mixture

- g e l ÷ g e l - gel -- gel - - g e l ÷ g e l - - g e l ÷ g e l

Lys 9.8 9.7 18.8 16.7 5.0 4.9 96 98 His 2.8 2.7 2.9 2.6 2.7 2.7 95 93 Arg 3.9 4.2 2.1 2.5 5.0 4.9 97 93 Asp 9.1 8.8 7.5 7.8 8.9 9.9 101 104 Thr 6.4 5.8 7.0 7.0 4.8 4.8 96 98 Ser 4.0 4.4 0.9 1.5 6.4 6.5 86 93 Glu 14.2 13.9 12.4 12.2 9.6 9.7 103 106 Pro 5.3 6.1 3.5 4.6 4.9 5.0 99 99 Gly 2.8 3.4 13.8 13.9 7.8 8.3 100 113 Ala 7.3 7.8 5.7 5.5 9.5 9.4 98 99 1/2 Cys n.d. n.d. n.d. n.d. 1.7 n.d. None Val 6.4 5.2 3.0 2.7 6.9 6.9 101 102 Met 0.7 0.6 2.0 1.2 2.5 2.4 90 90 lie 2.1 2.1 5.4 5.1 4.5 4.3 96 98 Leu 9.5 9.9 5.8 5.4 10.3 10.2 96 96 Tyr 3.2 3.1 3.5 3.6 3.9 3.9 97 87 Phe 4.3 4.2 3.4 4.1 4.2 4.4 95 92 Trp 0.3 n.d. 0.9 n.d. n.d. n.d. None

almost imperative, which Hous ton [5] did not do, for the es t imat ion o f basic amino acids. On the other hand, when traces o f oxygen remained during hydrolysis, lower

values have been found for methionine and tyrosine. Therefore, the importance o f

extensive evacuat ion to remove oxygen as much as possible must be emphas ized;

fortunately, the incorpora t ion o f a higher concentra t ion o f thioglycolic acid could, to a great extent, overcome the complicat ions because o f residual oxygen. A final concentra t ion o f 1 ~othioglycolic acid in the acid hydrolysis mixture has been found successful in preventing destruct ion o f the aforement ioned amino acids.

Succinate-cytochrome c reductase and the cytochrome b-c1 complex The active components as well as enzymic activities o f the samples used with

respect to succinate to cy tochrome c, and Q2H2 to cy tochrome c o f succinate-cyto- chrome c reductase and ub iqu inone-cy tochrome c reductase, i.e. the cy tochrome b-cx complex, were found the same as those previously reported [1]. They are summarized in Table II.

Without gel I.C

O.E N o-4

Asp (561 G)u,

°

~"LJ.-A.../3, 33 40 60

A

L6u (651

/ AI° Ilel Lys ~'I (321 Vol Met (28)jl ~ His (40) Tyr P~ II (18)

, , A . . . . . . . t-tA 80 I00 120 140 160 180"" 20- 40

TIME IN MINUTE

NH3 250

60 L

8O

237

't 402

0.1

With gel B

Leu (5.A.) Set Glu (64) Lys

i ~ IITh ~ (61) Gly AIo Ile~ (51~i

60 80 I:~0 140 160 80/' 40 TIME IN MINUTE

0 3o 4o ,oo 26

NHz

A 6o 8o

Fig. 1. Eluting patterns of amino acids from the cytochrome b-c1 complex hydrolyzed in the presence and absence of polyacrylamide gel. The cytochrome b-c1 complex, 6.85 mg/ml in 50 mM phosphate buffer (pH 7.4) was incubated with sodium dodecyl sulfate, 3 mg/mg protein and 1 ~ fl-mercapto- ethanol, at 38 °C for 3 h. The amino acid analysis was conducted as usual. (A) 20-ffl aliquots of the above-treated sample were hydrolyzed with 5 ml of 6 M HCI containing 0.05 ~ of thioglycolic acid in a sealed tube under argon at 110 °C for 24 h. (B) 20-#1 aliquots of the above-treated sample were subjected to polyacrylamide gel electrophoresis in the presence of 0.1 ~ dodecyl sulfate for about 1 h and the gel was removed from the tube, stained with Coomassie Brilliant Blue and destained with acetic acid and methanol mixture. The stained part of the gel was sliced off (about 2 cm) and hydro- lyzed with 6 M HCI containing 0.4 ~ thioglycolic acid under the same conditions as in A. The solid line was obtained by scanning at 570 nm for the calculation of all amino acids except proline which was done according to the dashed tracing at 440 nm.

Isolation of component fractions of the soluble cytochrome b-c1 complex F r o m the cy tochrome b-c1 complex, seven prote in bands were revealed f rom

analytical polyacrylamide gel ( 1 0 ~ gel with 0 . 3 ~ cross-linker) electrophoresis. These bands appeared at the posi t ions o f the molecular weights of, in thousands, 53,

238

30

o -6

E 20 -g

.~ 10 E <

Apparent production of amino acids by SDS-gel

Set . ._~ Asp, GIu, AIa

- Others I 3 5 7

Gel length (am)

120

~,IO(D L ®

u

so

6 0

~ Gly Ser

Asp, Glu,Ala.

OtherTyr AA

~ M e t i i i i 1 3 5 7

Gel length (am)

Fig. 2. Production of amino acid by polyacrylamide gel. The gel electrophoresis on polyacrylamide column was conducted in 0.1 ~ dodecyl sulfate and 0.1 M phosphate buffer (pH 7.0) as usual except no protein was added to the column. The gel column was sliced, hydrolyzed, and treated as described in the text. The amino acid content was analyzed.

Fig. 3. Recovery of amino acids from the cytochrome b-cx complex as a function of the amount of the gel present during the hydrolysis. The cytochrome b-cl complex was hydrolyzed in the presence of the amount of the gel shown. The percentage was based on the system without gel.

TABLE II

COMPOSITION AND ENZYMIC ACTIVITY OF SUCCINATE CYTOCHROME c REDUC- TASE AND THE CYTOCHROME b-cl COMPLEX

Components are in the units of nmol or natoms per mg of protein except phospholipid which is in weight percent. Activities are in the unit of/~mol of the substrate (2 electron equivalent) oxidized per min per mg of protein at about 23 ° C.

Components and activity Reductase The cytochrome b-c1 complex

Flavin 0,9 0.0 Non-heine iron 12 6.0 Cytochrome b 4.0 6.5 Cytochrome cl 2.3 4.1 Ubiquinone 2.3 3.7 Phospholipids ~ 20 ~ 20

Succinate ~ cytochrome c activity 4 QzH2 ~ cytochrome c activity 25

0 39 +

+ In spite of the high activity of Complex III given in the literature [20], we found it has only comparable, if not less, activity to the cytochrome b-cl complex under our assay conditions.

50, 37*, 30, 28, 17, and 15"*. For convenience, the bands were numbered from III to IX according to the order of the decrease of the molecular weights. Bands I and II, which are not present in the cytochrome b-c1 complex are used for the subunits known as the flavoiron protein (Fp) and iron protein (Ip) of succinate dehydrogenase, respectively [21, 22].

* See footnote p. 233. ** Occasionally, we observed a protein band with a molecular weight lower than 10 000 present in

the cytochrome b-c1 complex. This band occurred in the cytochrome b-containing fraction after the complex was cleaved by ammonium sulfate and sodium cholate. However, the presence of such a band gave no correlation with the enzymic activity of the sample before electrophoresis. Therefore, we excluded the possibility of its being a component of the cytochrome b-c1 complex.

cytochrome b-c I complex

(1) succ~nylatlon [(2) SDS, #-ME

(3) 2x G-150

(~IZ + I]Z)

(1) 2°/. cholot% 20*/, said, (N H4)2504, 25"C, 3 h

(2) centrifugotlon

(1) SDS, J~-ME (2) 2 x G-150

239

precipitate rFr, TV', "~, 3Z~Ip, supernatant (Cyt. bs NHI) 13~ (Cyt. ci)

(1) SDS~ /3-ME (2) 2 x G-150

]Zip ~ 3Zl s

m, IE, 3ZII, 3Us,

( I) (NH4)2 SO 4 (2) dialysis (3) SDS, ~-ME (4) 2× G-150

1

Fig. 4. Cleavage of the cytochrome b-c1 complex and separation of fractions for subunit studies. The Roman numerals stand for the fraction numbers, as also shown in Table II. The Arabic numerals in parentheses are for the successive steps. Abbreviations here are: SDS, sodium dodecyl sulfate; fl-ME, fl-mercaptoethanol; and 2 x G-150, Sephadex chromatography on G-150, "superfine" twice, see the text for the details. It should be emphasized that both the supernatant and the precipitate were com- taminated with Fractions III, IV, and VII which were separated by fractionation and not used.

Fraction VII The isolation of fraction VII from the b-ct complex was made by the following

method. The dialyzed cytochrome b-ct complex, usually 18 mg in a total volume of 4 ml, was incubated with sodium dodecyl sulfate at the concentration of 3 mg deter- gent per mg of protein in the presence of 1% ~-mercaptoethanol at 37 °C for 3 h. The mixture was then applied to a Sephadex G-150 (super-fine) column of 2.5 x 100 cm, which had been equilibrated with 0.1 M phosphate buffer (pH 7.0) containing 1% dodecyl sulfate and 1% fl-mercaptoethanol at room temperature. The elution was made by the same solvent at a flow rate of 0.2 ml/min. 3-ml fractions were collected. The protein components present in the fractions were analyzed by analytical gel electrophoresis. The fractions, which were rich in Subunit VII (Fig. 4), were pooled, concentrated and rechromatographed on another column under the same conditions. The protein fractions at the peak, which contained only Subunit VII, were collected. Excess dodecyl sulfate was removed by precipitation in cold, before concentrating with Aquacide. The concentrated solution was stored at --20 °C until use.

Fractions I I I and I V Since the difference in molecular size between Fractions I I I and IV was rather

small, direct isolation from the cytochrome b-ca complex by Sephadex gel chromato- graphy was not easy. When the cytochrome b-c1 complex was succinylated, these two fractions became inseparable by analytical gel electrophoresis and travelled as one protein component with a higher molecular weight. The succinylation was conducted similar to that employed by Rieske [20]. The dialyzed cytochrome b-c1 complex in

240

50 mM phosphate buffer (pH 7.5) at the protein concentration of 20 mg per ml, was treated with succinic anhydride at about 2 mg of succinic anhydride per mg of protein. The addition was made with 5 mg increment and the pH was maintained between 7 and 8 during the reaction by addition of 1 M NaOH. The solution was allowed to stand until the excess of reagent was hydrolyzed as indicated by no further change in pH. This increase in molecular weight facilitated the separation of Fractions III and IV from Fraction V and others. Therefore, for the isolation of Fractions III together with IV, the cytochrome b-c~ complex was first reacted with succinic anhydride before the treatment with dodecyl sulfate and mercaptoethanol. The dissociation and the column chromatography were undertaken as described for the isolation of Fraction VII. No attempt was made, however, to further separate Fractions III and IV into single fractions by column chromatography.

Fractions V, VIp, and VIII For the isolation of Fractions V, VIp, VIII, VIs, and IX, the cytochrome b-c1

complex was first cleaved into two fractions by sodium cholate in the presence of ammonium sulfate [23]. One contained cytochrome b, non-heme iron protein plus perhaps other factors and another fraction contained mainly cytochrome cl. Both fractions were contaminated with Fractions III, IV, and VII. The dialyzed cytochrome b-el complex was dissolved in phosphate buffer containing 2 ~ cholate at a protein concentration of about 10 mg/ml. The undissolved residue, if any, was removed by centrifugation. The clear reddish solution was brought up to 20~ saturation of (NH4)2SO4 by adding neutralized saturated (NH4)2SO4. The solution was then in- cubated at room temperature for 3 h. At the end of incubation, the crude b cytochrome fraction was collected as precipitate by centrifugation and the crude cl cytochrome fraction remained in the supernatant. The supernatant was dialyzed extensively to remove cholate and ammonium sulfate and saved for the isolation of Fractions VIs and IX. The precipitates were washed once with 2 ~o cholate and used for the isolation of Fractions V, VIp and VIII.

The washed precipitates were dissolved in 50 mM phosphate buffer (pH 7.0) containing dodecyl sulfate and mercaptoethanol. The protein concentration was adjusted to 5 mg per ml and dodecyl sulfate was added up to 3 mg per mg protein and mercaptoethanol to 1 ~. The solution was then incubated at 37 °C for 3 h before chromatography. The latter was done on a Sephadex G-150, superfine, column of 2.5 × 100 cm, equilibrated with 0.1 M phosphate buffer containing 1 ~ dodecyl sulfate and 1 ~ fl-mercaptoethanol. The column was eluted with the same solvent at a flow rate of 0.2 ml/min. 3-ml fractions were collected and fractions, which were en- riched with Fractions V, VIp and VIII, were separately pooled. The pooled fractions were concentrated and rechromatographed once more as before. Only the fractions which showed a single band on analytical gel electrophoresis were pooled and used. The excess detergent was removed by precipitation in cold. After removing dodecyl sulfate, the pooled fraction was concentrated by Aquacide and stored at --20 °C for the use of amino acid analysis.

Fractions VIs and IX Fractions VIs and IX were obtained from the cytochrome cl-rich fraction by a

241

similar operat ion, as described in Fract ions V, VIp, and VIII. A summary of the scheme for the isolat ion of these fractions is given in Fig. 4.

It should be stated that in the isolat ion the emphasis was placed on the puri ty rather than the yield. In all cases, only a few fractions with absolute puri ty by the cri terion of analytical gel electrophoresis were used to avoid cross-contaminat ion.

Isolat ion of Fract ions VIs and IX was also achieved from purified cytochrome ct prepara t ion (unpubl ished results). However, ment ion may be made at the onset that Frac t ion IX isolated from purified cytochrome c~ was no t identical to Frac t ion IX isolated from cytochrome cl fract ion of the cytochrome b-c1 complex of Fig. 4. This fact is because Frac t ion IX from the cytochrome b-cl complex conta ined more than one polypeptide (see below), whereas the Subuni t IX from purified cytochrome c~ contains only one peptide (unpubl ished results). By now it is obvious that the rat ionale of the f ract ionat ion used here is mainly by the cleavage of the cytochrome b-c~ com- plex into the cytochrome b- and c-containing fractions. F rom these fractions the subuni ts of the cytochrome b-c~ complex with identical size can be separated by gel fil tration in the presence of SDS.

TABLE III

AMINO ACID COMPOSITION IN MOL PERCENT OF THE ISOLATED CYTOCRHOME b-c1 COMPLEX SUBUNITS

Amino acid Fractions III and IV V VIp VIs* VII VIII IX** IXcx**

Lys 4.9 3.7 4.9 5.3 5.6 5.0 8.5 6.8 His 2.5 3.0 3.7 3.0 2.4 2.8 2.4 3.3 Arg 4.9 2.9 6.4 5.5 3.9 7.2 4.1 6.7 Asp(Asn) 7.6 7.9 6.4 8.7 8.7 5.8 9.1 8.1 Thr 4.6 6.4 3.5 4.6 4.5 4.8 3.6 5.2 Ser 6.6 5.8 4.6 6.2 7.7 4.9 6.3 4.6 Glu(Gln) 8.8 5.6 9.9 9.8 9.1 8.8 14.9 19.8 Pro 5.2 6.0 8.3 6.7 4.5 3.4 4.8 3.9 Gly 7.3 7.9 7.6 7.5 7.5 6.3 6.5 3.7 Ala 9.6 7.9 7.5 8.5 8.0 6.6 7.8 7.0 1/2Cys 1.3 2.4 3.5 n.d. 1.1 2.7 n.d. 2.1 Val 8.4 5.1 6.3 6.0 7.4 5.6 5.2 6.2 Met 2.8 3.3 3.6 3.1 2.2 3.6 2.9 0.5 lie 5.5 7.6 2.1 4.1 4.6 3.4 4.7 2.5 ~ u 10.5 13.3 11.2 9.0 7.5 12.9 10.9 12.3 Tyr 3.4 3.6 6.0 4.1 3.1 4.7 2.5 2.2 Phe 3.7 5.5 3.6 3.3 3.4 6.6 2.6 4.1 Trp 2.1 2.6 1.0 n.d. 9.0 4.8 nd. 1.1 Hydro- phobiciW*** 49 54 47 41 ~ 50 39 42

* Calculation was made by assuming the same mol percent of half cysteine and tryptophon of the berne-containing subunit isolated from pure cutochrome Cl. See Fig. 1 for difference of VIp and VIs.

* * Calculation was made by assuming the same mol percent of half cysteine and tryptophon of IXcl which was the subunit without heme directly obtained from pure cytochrome cl.

*** Calculated by consideration of Pro, Ala, Cys, Val, Met, Ile, Leu, Phe, and Trp as hydrophobic amino acids.

n.d., not determined.

242

Amino acid composition of the subunits of the cytochrome b-cx complex Table I I I summarizes the amino acid compos i t ion ob ta ined f rom the isolated

subunits . The da ta given are the average values o f three separate samples. The hydro- phobic i ty o f the subunits is calculated as the sum of mola r percent o f prol ine, alanine, cysteine, valine, methionine , isoleucine, phenyla lanine and t ryp tophan . Table IV shows the amino acid compos i t ions ob ta ined f rom direct hydrolysis o f s tained gel slice. The close resemblance between the da ta given in Table I I I and Table IV in- d ica ted tha t the me thod descr ibed in the previous sect ion d id indeed work.

TABLE IV

AMINO ACID COMPOSITION IN MOL PERCENT OF THE SEVEN BANDS OF THE CYTOCHROME b-c1 COMPLEX AFTER GEL ELECTROPHORESIS DETERMINED DI- RECTLY IN THE PRESENCE OF GEL SLICES

Amino acid** Bands*

III IV V VI VII ~'III IX

~ s 4.2 3.2 4.3 5.3 6.3 5.2 7.7 His 3.4 3.5 2.4 3.5 3.8 3.0 2.5 Arg 4.4 3.9 2.4 5.5 3.8 6.5 4.8 Asp(Asn) 8.7 8.5 8.4 7.7 8.0 6.3 9.0 Thr 4.9 4.3 6.1 3.2 4.1 5.1 3.7 Ser 6.2 7.1 5.5 5.4 7.4 6.2 6.8 Glu(Gln) 10.9 9.4 7.0 9.5 10.3 8.6 14.7 Pro 5.3 5.2 7.0 8.1 6.9 4.5 5.3 Gly 7.7 7.9 7.9 7.8 7.6 8.0 6.3 Ala 8.8 11.6 7.5 7.6 7.5 7.3 6.7 Val 7.4 7.6 4.9 6.1 7.2 5.4 4.7 Ile 4.5 4.2 6.7 3.4 4.1 3.0 4.3 Leu 10.6 10.1 12.1 10.2 6.6 8.2 10.7 ~ r 3.6 3.2 3.5 5.0 2.5 3.6 2.5 Phe 3.3 3.6 5.0 3.4 2.4 4.4 2.5 Trp 2.1 2.6 1.0 n.d. 9.0 4.8 n.d.

n.d., not determined. " For band numbers see Table III.

* * Methionine and half-cystine are not determined.

Determination of weight (prote&) distribution of components by amino acid analysis oj the slices directly from the gel electrophoretic columns

Direct analysis o f the amino acids after slicing each s tained band o f the gel co lumn after the usual s taining and desta ining processes, and assuming tha t the amino acid recoveries o f each subuni t are comparab le , then one can calculate the pro te in weight d i s t r ibu t ion o f each subuni t fractiOn. Dividing the weight d i s t r ibu t ion by its cor responding molecular weight, molar d is t r ibut ion among the subunits can be obtained. Table V summarizes all per t inent facts. Co lumn 1 gives the band numbers . The molecular weights were ob ta ined f rom, as usual , a s t andard curve o f known prote ins which consisted o f bovine serum albumin, t rypsin, cy tochrome c, and pan- creat ic t rypsin inhibi tor . The weight d i s t r ibu t ion was computed f rom the amino acid analysis o f each band. The weight d i s t r ibu t ion ob ta ined f rom the in tegra t ion o f densi tometr ic t rac ing is also included for compar i son , as shown in Co lumn 4 o f the

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TABLE V

PROTEIN DISTRIBUTION A N D PROBABLE MOLAR RATIO OF SUBUNITS OF THE CYTOCHROME b-c1 COMPLEX BY THE METHOD OF AMINO ACID ANALYSIS

The values in the parentheses were obtained through the calculation of the densitometric scanning of the blue color from Coomassie Blue-stained gel column.

Band number Molecular Weight Molar Smallest Probable we igh t distribution (%) distribution molar ratio molar ratio (X 10 -3)

(1) (2) (3) (4) (5) (6) (7)

III 53 22.4 (17) 0.42 2 2 IV 50 21.9 (17) 0.44 2 2 v 37 10.0 (5) 0.27 1 2 vI 30 15.1 (18) 0.50 2 3 vii 28 9.2 (14) 0.33 1 2 vii i 17 5.5 (9) 0.32 1 2 IX 15 15.8 (18) 1.05 3 5

table. Since the weight distribution is known, the molar distribution, as shown in Column 5, can be obtained. However, these numbers cannot be simply rounded off to obtain the smallest integers as molar ratios but must be reconciled with the results from other observations, such as the analysis of the components of the cytochrome b-ca complex (see Table II). One of the most probable molar ratios is shown in the last column. Thus the calculated nmol of cytochromes per mg of the b-cl complex are 3.7. for ca and 7.5 for b. These values are in fairly good agreement (in view of the complicated operations involved and especially the somewhat "arbitrary" extinction coefficients used) with the results of direct analysis, i.e. 4.1 and 6.5 for cytochromes cl and b, respectively.

Assignment of subunits The molar ratios given in Table V indicate probable subunit components in

these bands or fractions. In other words, each of Bands III, IV, V, VII, and VIII may contain two polypeptides where Bands VI and IX may possess three and five, respectively. Needless to say, the polypeptides in each fraction can be either identical or non-identical. However, not many useful facts are available for the assignment of the sgbunit components in contrast to the prevalent simplified manipulation of other workers [24-28].

Bands V and VIII are apparently derived from b cytochromes. The reasoning is based on that practically the same amino acid composition is obtained from the analysis of the bands sliced from the gel column and of purified samples directly obtained from repeated fractionation by a different method after cleavage of the cytochrome b-ca complex [23]. The difference of amino acid composition of Fractions VIp and VIs is because the former is almost free of cytochrome b, and VIs is almost free of cytochrome ca. The molecular weights for Bands VI and IX correspond to the subunits of cytochrome cl with and without berne, viz. 30.103 and 15- 103, respec- tively [11, 29]. Since the amino acid composition of Bands VI and IX is different from that of the subunits isolated from pure cytochrome ca, other components must exist in these bands.

244

Some evidence indicates at least one of the two subunits of Band IV as a b cytochrome protein. "[his subunit or polypeptide may associate with Band VIII of 17.10 a daltons but without an additional protoheme group to give the first protoheme- containing peak of Fig. 1 of Yu et al. [18] in the direct isolation of b cytochromes. Thus the cytochrome b-c~ complex may contain three b cytochrome apoproteins but only two protoheme groups. From the similarity of the molecular size, the remaining three subunits of Band IX may be the antimycin-binding factor reported by Gupta and Rieske [28]; this component does not contain any known prosthetic group or non-heme iron. It is possible that one subunit in each of Bands VI and VII may be non-heme iron proteins. But distribution of the iron atoms is completely unknown, although we have some evidence* to suggest at least two entities of non-berne iron proteins exist in the cytochrome b-c1 complex.

The nature of other subunits is far from known. These might be subunits associated with the aforementioned components of course, Slater's factor [30], Racker's oxidation factors [31], or likely a Q-carrying protein(s) (apoenzyme(s) for coenzyme Q)**, among other possibilities. One thing is certain that no evidence suggests the existence of the so-called structural proteins or core proteins [32, 33], which we have discussed previously [1]. According to the views of Schatz and Saltz- gaber [34], the "structural proteins" isolated are merely a mixture of various denatured components so that the "structural" or "core" proteins are rather insoluble.

In summary, the cytochrome b-c1 complex possesses a very complicated sub- unit composition. It could be composed of as many as 18 polypeptide chains (some of them could be identical) in a minimum complex weight of about 536.103. The molecular weight of the isolated cytochrome b-cl complex determined directly from analytical centrifugation is about 1.4. 106 with $20.2 = 40 S. This value suggests the isolated complex exists in a polymeric form in aqueous media.

DISCUSSION

Houston [5] has used direct analysis of amino acids of Amido Schwartz- stained gel slices after electrophoresis. A large amount of ammonia, formed during the operation, is not removed before analysis and greatly hinders the method. Am- monia can even induce precipitation on the column during the amino acid analysis; consequently, the author [5] must use a column longer than usual for the basic amino acids. Houston has also encountered some difficulties in recoveries of certain proteins, for example only about 76 ~o for bovine serum albumin comparable to control and about 7 0 ~ for ovalbumin [5]. Stein et al. [17] have ingeniously compensated the objection of ammonia by taking advantage of low fluorescence resulted from its reaction with fluorescanine. The method is applicable, however, only for fluorimetric technique as developed by Udenfriend and co-workers (e.g. ref. 35) for the extremely sensitive automatic determination of amino acid in a quantity of a few micrograms of proteins.

* We have found that only a fraction of the non-heine iron proteins is released upon treatment of the cytochrome b-ct complex with 2-fold excess ofp-hydroxymercuribenzoate. The other portion of the non-heine iron proteins was not affected even by prolonged reaction of the sulfhydryl reagent.

* * Evidence of the possible existence of a Q-carrying protein(s) will be reported elsewhere.

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In addition, both Houston [5] and Stein et al. [17] have worked on relatively simple proteins, i.e. bovine serum albumin, ovalbumin, and lactate dehydrogenase. The present communication describes the direct method which can be used for very complicated lipoprotein complexes from mitochondrial membranes.

We have thoroughly examined all the parameters which might affect the method. It has been found that the ammonia formed is very hazardous and causes interference in determination. Ammonia must be removed prior to the amino acid analysis. For- tunately, the method of the removal does not affect any amino acid subsequently determined. Consequently, the conventional automatic amino acid analyzer without modification may be routinely employed. However, the removal of oxygen or air in the course of hydrolysis cannot be overemphasized, although 1 ~ of thioglycolic acid may avoid, to a great extent, the destruction of some amino acids. Nonetheless, the removal of most air is very much advisable. In the presence of large amounts of gel during hydrolysis the recovery of tyrosine and methionine greatly decreased. On the other hand, glycine, serine, and, in a lesser degree, aspartic acid, glutamic acid, and alanine (Fig. 3) are increased to nearly 8 ~ of the recovery in the presence of 7 cm of polyacrylamide gel. All other amino acids are much less affected. In usual determina- tion, however, not more than 4 cm gel is included.

Comparison of amino acid analysis in the presence and absence of gel, bovine serum does not show much deviation (Table 1), except for valine. The literature value is 5.4 in the absence [36] and 6.0 in the presence of gel [17]. Serine in cytochrome c and glycine in the "standard" mixture show higher values in the presence of gel and may result from over-correction. At any rate, it is gratifying to see that the results of the cytochrome b-c1 complex for all amino acids are practically the same in the pre- sence and absence of gel. It is obvious that the direct method could avoid the densito- metric difficulty and satisfy complex samples.

Amino acid composition obtained from the isolated fractions gives only the qualitative nature of the fraction but obviously not the quantitative distribution of the fractions in the complex. This is because the yields of isolation of various fractions were quite different. Many investigators [24] have used the integration of the densito- metric tracings of stained electrophoretic gel to probe the distribution of fractions by assuming that each fraction shows the same color intensity proportional only to the protein content. This assumption is valid when the same protein or the same type of protein is involved. For the cytochrome b-cl complex, the nature of the fractions is is different; densitometric technique led to false information. We found that the color intensities, after staining with Coomassie Brilliant Blue, of different isolated fractions were not proportional to their protein content. For example, two cytochrome b pro- teins [18], which have been isolated and identified, showed very light color intensites compared to those of the other components; direct densitometric tracing would underestimate their content. Amino acid composition analyzed from the fractions after elution of protein from the gel suffered two disadvantages as far as the cytochrome b-c1 complex was concerned. Firstly, it was almost impossible to avoid cross-contami- nation of fractions which resulted from slicing the gel column without staining because several bands from the complex were very close together. It was almost impossible to elute stained protein from the gel column. Secondly, much difference in in eluting efficiency was found for different proteins in the different fractions.

In order to avoid these difficulties, we determined the weight distribution of

246

the f ract ions o f the cy tochrome b-c1 complex by direct analysis o f amino acids f rom sta ined gel. This method has been adap ted for the subuni t o f cy tochrome oxidase [19] and N A D H dehydrogenase (unpubl i shed results).

I t should be ment ioned that amino acid compos i t ion o f subunits o f Complex I I I has been repor ted par t ia l ly [27]. Direc t compar i son between the repor ted da ta and the results ob ta ined in the present s tudy would yield imprope r in format ion , because o f the different na ture in the p r epa ra t i on and var ia t ion in ass ignment on the molecular weight o f subuni ts .

ACKNOWLEDGEMENTS

We are great ly indebted to Dr. Bernard Horecke r for cal l ing our a t ten t ion to Refs. 5 and 17 abou t the pr inciple o f direct de te rmina t ion o f amino acids f rom gel slices, which have been previously publ ished. This work was suppor ted by grants f rom the Na t iona l Science F o u n d a t i o n and the Uni ted States Publ ic Hea l th Service.

REFERENCES

1 Yu, C. A., Yu, L. and King, T. E. (1974) J. Biol. Chem. 249, 4905-4910 2 King, T. E. (1966) Adv. Enzymol. 28, 155-326 3 Green, D. E., Wharton, D. C., Tsagoloff, A., Rieske, J. S. and Brierley, G. P. (1965) in Oxidases

and Related Redox Systems (King, T. E., Mason, H. S. and Morrison, M., eds.), Vol. 2, pp. 1032- 1076, John Wiley and Sons, New York

4 King, T. E. and Takemori, S. (1964) J. Biol. Chem. 239, 3559-3569 5 Houston, L. L. (1971) Anal. Biochem. 44, 81-88 6 Yu, L., Yu, C. A. and King, T. E. (1973) Biochemistry 12, 540-546 7 King, T. E. (1961) J. Biol. Chem. 236, 2342-2346 8 King, T. E. (1967) Method Enzymol. 10, 202-208 9 Shunk, C. H., Linn, B. O., Wong, E. L., Wittreich, P. E., Robinson, F. M. and Folkors, K. (1958)

J. Am. Chem. Soc. 80, 4753 10 Crane, F. L. (1960) in Quinones in Electron Transport (Wolstenholme, G. E. W., ed.), pp. 36-78,

Little Brown and Co. 11 Yu, C. A., Yu, L. and King, T. E. (1972) J. Biol. Chem. 247, 1012-1019 12 Berden, J. A. and Slater, E. C. (1970) Biochim. Biophys. Acta 216, 237-249 13 Weber, K. and Osborn, M. (1969) J. Biol. Chem. 244, 4406-4412 14 Moore, S. and Stein, W. H. (1963) Methods Enzymol. 6, 819 15 Moore, S. (1962) J. Bioo. Chem. 238, 235-237 16 Matsubara, H. and Sasaki, R. M. (1969) Biochem. Biophys. Res. Commun. 35, 175-181 17 Stein, S., Chang, C. H., B6hlen, P., Imai, K. and Udenfriend, S. (1974) Anal. Biochem. 60, 272-

277 18 Yu, C. A., Yu, L. and King, T. E. (1975) Biochem. Biophys. Res. Commun. 66, 1194-1200 19 Yu, C. A. and Yu, L. (1977) Biochim. Biophys. Acta 495, 248-259 20 Rieske, J. S. (1967) Methods Enzyrnol. 10, 357-362 21 Davis, K. A. and Hatefi, Y. (1971) Biochemistry 10, 2509-2516 22 Felberg, N. T. and Hollocher, T. C. (1972) Biochem. Biophys. Res. Commun. 48,933-936 23 Yu, C. A., Chiang, Y. L., Yu, L. and King, T. E. (1975) J. Biol. Chem. 250, 6218-6221 24 Gellerfors, P. and Nelson, B. D. (1975) Eur. J. Biochem. 52, 433-443 25 Hare, J. F. and Crane, F. L. (1974) Sub-Cell. Biochem. 3, 1-25 26 Capaldi, R. A. (1974) Arch. Biochem. Biophys. 163, 99-105 27 Bell, R. L. and Capaldi, R. A. (1976) Biochemistry 15, 996-1001 28 Gupta, U. D. and Rieske, J. S. (1973) Biochem. Biophys. Res. Commun. 54, 1247-1253 29 Trumpower, B. L. and Katki, A. (1975) Biochemistry 14, 3635-3641 30 Slater, E. C. (1950) Biochem. J. 46, 484--499

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31 Nishibayashi-Yamashita, H., Cunningham, C. and Racker, E. (1972) J. Biol. Chem. 247, 698-704 32 Green, D. E., Tisdale, H. D., Criddle, R. S., Chen, P. Y. and Beck, R. M. (1961) Biochem.

Biophys. Res. Commun. 5, 109 33 Criddle, R. S., Beck, R. M., Green, D. E. and Tisdale, H. D. (1962) Biochemistry 1,827 34 Schatz, G. and Saltzgaber, J. (1971) in Probes of Structure and Function of Macromolecules and

Membranes (Chance, B., Yonetani, T. and Mildvan, A. S., eds.), Vol. 1, pp. 437444, Academic Press, New York

35 Stein, S., B6hlen, P., Stone, J., Dairman, W. and Udenfriend, S. (1973) Arch. Biochem. Biophys. 155, 202-212

36 Alexander, P. and Lundgren, H. P. (e~is.) (1966) A Laboratory Manual of Analytical Methods of Protein Chemistry, Pergamon Press, New York