The isoelectric points of cytochromes in the reduced and oxidised forms

Comp. Biochem. PhysioL Vot. 72B, pp. 481 to 485, 1982 Printed in Great Britain.

0305-0491/82/030481-04S03.00/0 ©1982 PergamonPress Ltd

THE ISOELECTRIC POINTS OF CYTOCHROMES IN THE REDUCED AND OXIDISED FORMS BIRGIT A. HELM Department of Biochemistry, Bedford College, University of London, Regent's Park, London NW1 4NS, England (Received 12 November 1981)

Abstract--1. A number of acidic and basic cytochromes from eukaryotic organisms have different isoelectric points in the reduced and oxidised forms. 2. It appears that the direction of the redox-linked shift cannot be predicted.

INTRODUCTION Crystallographic studies have provided little evidence for a significant alteration in the tertiary structure following a change in the oxidation state of the prosthetic group of cytochrome c (Mandel et al., 1977). However, many indirect lines of evidence suggest that such a change occurs in the folding of the polypeptide chain as the haem group is reduced or oxidised. Such observations include different habits of crystallisation (Lemberg & Barrett, 1973), changes in affinity for biologically important anions including ATP and orthophosphate (Schejter & Margalit, 1970; Margoliash et al., 1970; Margalit & Schejter, 1973, 1974), different susceptibilities to proteolytic digestion and various denaturing conditions (Butt & Keilin, 1962; McLendon & Smith, 1978; Hantgan & Taniuchi, 1978; Drew & Dickerson, 1978). Differences have also been found in deuterium exchange experiments (Ulmer & K~igi, 1968; Nabedryk-Viala et al., 1976), calorimetric studies (Watt & Sturtevant, 1969) and measurements of the chemical reactivities of certain lysine residues (Bosshard & ZiJrrer, 1980). It is generally accepted that, in the reduced form, cytochromes of the c-type possess a more compact and stable conformation than in the oxidised state. Data for the isoelectric points of cytochrome c from a variety of sources give one figure only without stating whether this was obtained for the reduced or the oxidised form of the protein. The figure most commonly quoted for cytochrome c from horse or cow heart is pH 10.05. Evidence has now been obtained that cytochromes from a variety of sources have two isoelectric points, one corresponding to the reduced, the other to the oxidised state of the prosthetic group. The experiments described here were commenced during an investigation of a newly discovered cytochrome, provisionally named helicorubin P (Cheesman & Helm, 1979), which occurs in the egg jelly of the South American prosobranch mollusc Pomacea canaliculata. The study was subsequently extended to other cytochromes. METHODS

AND MATERIALS

Cytochrome c from horse heart, was obtained from British Drug Houses, while cytochrome c from tuna heart,

Saccharomyces cerevisiae and Candida krusei was supplied by the Sigma Chemical Company. After removal of ovorubin (Uhlenbruck et al., 1976) from a 25~oaqueous homogenate of the eggs of Pomacea canaliculata by affinity chromatography on Concanavalin A-Sepharose (Pharmacia AB, Uppsala, Sweden), helicorubin P was purified by isoelectric focussing in a 3 mm thick bed of granulated gel (Ultradex) containing equal quantities of Ampholine carrier ampholytes of pH-ranges 7-9 and 9 11 in the LKB 2117 Multiphor apparatus (all from LKBProdukter AB, Bromma, Sweden). Helicorubin and cytochrome h from the gut fluid of Helix pomatia were purified similarly in a gel containing equal quantities of Ampholine carrier ampholytes of pH-ranges 3.5-5 and 5-8 after removal of cellulase by affinity adsorption on Whatman No. 540 filter paper. All cytochromes were prepared in the reduced and oxidised state by reduction with dithionite or oxidation with ferricyanide followed by filtration through a Sephadex G-25 column. Electrophoresis on polyacrylamide gels (PAGE) was carried out in the Pharmacia GE 4 electrophoretic apparatus. For basic proteins, the method described by Gabriel (1971) was used. Polyacrylamide gel electrophoresis in sodium dodecyl sulphate gels (SDSPAGE) was performed according to the method of Weber & Osborn (1969). Absorption spectra were recorded with a Pye Unicam SP 8-100 spectrophotometer, pH-measurements were carried out with a Pye-Unicam (403.3) surface electrode and recorded on a Phillips (PW 9409) digital pH-meter.

RESULTS Helicorubin P is spectrophotometrically indistinguishable from the extracellular haemochromogen helicorubin of Helix pomatia, an acidic cytochrome with an isoelectric point reported to be in the region of pH4.3 (Keilin, 1968). However, helicorubin P, unlike the helicorubin investigated by Keilin, is a basic protein. The purified haemoprotein was shown to be electrophoretically homogeneous in SDS-PAGE (Fig. l(a)). In a fl-alanine acetic-acid buffer system, pH 4.5, two bands of closely similar mobility were observed (Fig. l(b)). Flat-bed electrofocussing of such preparafions~n a granulated gel (Ultradex) with LKB Multiphor showed two bands (Fig. l(c)). Owing to their native colour, the separated bands were easily collected, the protein was eluted from the gel with

481

482

BIRGIT A. HELM

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l

,I

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pH 5.1 lc

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250

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I

I

350

450

550

Wavelength

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Fig. 1. The spectra of ca. 75/~g ml- ~helicorubin P in water obtained after isoelectric focussing at pH 9,47 ( - - ) and pH 9.88 (..... t: (a) electrophoretogram of helicorubin P on SDS-PAGE in a Tris-glycine buffer, pH 8.3;(b)electrophoretogram of helicorubin P on PAGE in a fl-alanine-acetic acid buffer, pH 4.5: (c) helicorubin P focussed in a 3 mm thick bed of Ultradex gel with Ampholine carrier ampholytes, pH range 7--10 after 16 hr.

distilled water and passed through a Sephadex G-25 column before spectrophotometric analysis (Fig. 1). It was found that the band focussing at pH 9.47 _+ 0.03 corresponded to the reduced cytochrome, while the second band with an isoelectric point of pH 9.88 + 0.05 showed the spectrum of the fully oxidised haemoprotein. When the reduced or the oxidised cytochrome was studied alone, only one band at pH 9.47 or 9.88 was observed. The reduced fraction could be converted into the oxidised form, and vice versa. Following such changes in oxidation state the isoelectric points of the products corresponded exactly to those previously observed for the appropriate forms of the haemoprotein. Tests for amino acid composition carried out by Dr M. W. McDonough revealed no difference between the oxidised and the reduced fractions, In view of the potential significance of this finding, cytochrome c from horse heart and tuna heart, Saccharomyces cerevisiae and Candida krusei together with helicorubin and cytochrome h from Helix pomatia were prepared in the Fe 2+ and Fe 3+ states by reduction with dithionite or oxidation with ferricya-

hide, followed by filtration through a Sephadex G-25 column. The isoelectric points of these cytochromes were investigated by preparative isoelectric focussing, which allowed easy separation and subsequent spectrophotometric analysis. The results obtained are shown in Table 1.

DISCUSSION

In most cases where the electrophoretic properties of cytochromes have been investigated, multiple components have been reported. Flatmark & Sletten (1968) suggest that various forms arise from the deamidation of labile asparagine and/or glutamine residues, while Margoliash & Lustgarten (1962) observe different polymeric forms. Barlow & Margoliash (1966) show that yet another variety of electrophoretic heterogeneity of mammalian ferricytochrome c occurs as a result of multiple anion binding in solution. The observations made in the present study which show that the isoelectric point for ferrocytochrome c

The isoelectric points of cytochromes A

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I 350

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Fig. 2. The spectra of approx. 100,ug ml-~ cytochrome c from horse heart obtained after isoelectric focussing at pH 10.12 ( ) and pH 9.81 (..... ): (a) equine cytochrome c focussed in a 3 mm thick bed of Ultradex gel with Ampholine, pH-range 7-10, after 16 hr.

from horse heart is higher than that of the oxidised form, while the reverse is true for helicorubin P, render it improbable that the alteration in charge on the Fe atom, or the loss of a proton observed with mammalian ferricytochrome c at alkaline pH (Davis et al., 1974) is the immediate reason for the observed differences in mobility between the reduced and the oxidised form of the cytochromes investigated. It appears, however, probable that Ampholine carrier ampholytes present in the gel support will bind to the oxidised form of the cytochrome. The observed differences in isoelectric point for the oxidised and reduced forms of cytochrome c from horse heart and tuna heart suggest anion binding to the ferric forms, since the isoelectric points observed in the present study are lower than the isoionic points of 10.05 and 10.04 reported for ferricytochrome c from these species (Barlow & Margoliash, 1966). The

finding that lysine residues 39, 53 and 55 of cytochrome c from horse heart undergo a small, but significant increase in pK upon reduction (Bosshart & ZiJrrer, 1980) is consistent with, and possibly accounts for, the observation made in the present study that ferrocytochrome c has the higher isoelectric point, since anions do not bind to reduced cytochrome c (Barlow & Margoliash, 1966) and in the presence of cations a reduction in electrophoretic mobility is observed (Margoliash et al., 1970). The results obtained suggest that the direction, if any, of the redox linked shift in isoelectric point, cannot be predicted. A comparison of isoelectric points for cytochromes of various species might hence provide interesting information concerning evolutionary and functional aspects, since differential ion binding might influence the electron transfer mechanism mediated by cytochromes in situ.

484

BIRGIT A. HELM Table 1. The isoelectric points of cytochromes in the reduced and oxidised forms Cytochrome Cytochrome e Cytochrome c Helicorubin Cytochrome h Helicorubin P Cytochrome c Cytochrome e

Isoelectric point Reduced Oxidised

Source Horse heart Tuna heart Helix pomatia Helix pomatia Pomacea canalieulata Candida krusei Saccharomyces cerevisiae

10.12 _ 0.05 9.81 + 0.03 10.09 9.76 4.8 5.12 6.23 5.94 9.47 _+ 0.03 9.88 + 0.05 9.42 9.76

9.42* 9.49

* Separation of the reduced and oxidised form of this cytochrome could not be achieved by this method. Careful inspection of the band suggested, however, that the leading edge (towards the higher isoelectric point) consisted of the reduced haemoprotein. The results shown represent the mean and the standard deviation of ten determinations for helicorubin P and equine cytochrome c focussed in a 3 mm thick bed of Ultradex gel with Ampholine carrier ampholytes, pHrange 7--11, (both from LKB-Produkter AB, Bromma, Sweden) after 16hr. All other results represent the mean values obtained from three determinations. The isoelectric points for helicorubin and cytochrome h were determined in a gel bed containing equal quantities of Ampholines of pH-ranges 3.5-5 and 5 8.

Acknowledgements--This study was supported by a grant from the Central Research Fund of the University of London. I am grateful to Dr M. W. McDonough for the determination of the amino acid compositions of reduced and oxidised helicorubin P and to Professor D. F. Cheesman for helpful discussions.

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