In the present studies, respiratory chain pathogenic bacterium, Proteus mirabilis, was investigated. In the first phase, growth profile study was performed to optimize the P. mirabilis growth. Maximum bacterial growth could be obtained between 10 – 12 h of culturing time. Down-stream processing was performed by using sonication, ultracentrifugation and detergent solubilization techniques. Partially purified respiratory contents were analyzed spectrophotometrically. Pyridine-ferrochrome and redoxspectra showed the presence of heme-c

Ismail, T

Nazir, MS

Qureshi, MH

Zia, MA

English

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African Journal of Biotechnology Vol. 6 (9), pp. 1132-1135, 2 May 2007 Available online at http://www.academicjournals.org/AJB ISSN 1684–5315 © 2007 Academic Journals      Full Length Research Paper  Isolation and purification of membrane-bound cytochrome c from Proteus mirabilis  Muhammad Atif Zia1*, Muhammad Shahid Nazir, Tariq Ismail and Muhammad Hassan Qureshi   Biochemistry Laboratory, Department of Chemistry, Government College University, Lahore, Pakistan.  Accepted 4 May, 2007  In the present studies, respiratory chain pathogenic bacterium, Proteus mirabilis, was investigated. In the first phase, growth profile study was performed to optimize the P. mirabilis growth. Maximum bacterial growth could be obtained between 10 – 12 h of culturing time. Down-stream processing was performed by using sonication, ultracentrifugation and detergent solubilization techniques. Partially purified respiratory contents were analyzed spectrophotometrically. Pyridine-ferrochrome and redox spectra showed the presence of heme-c.   Key words: Cytochrome c, respiratory chain and Proteus mirabilis.   INTRODUCTION  Proteus mirabilis is facultative anaerobic, rod-shaped, gram negative bacterium. It belongs to family Enterobac-teriaceae and displays swarming growth (O’Hara et al., 2000). P. mirabilis is an opportunistic pathogen (Griffith et al., 1976; Rosenstein et al., 1984; Griffith et al., 1973) and persist for long periods of time (Hennerr et al., 1982).  The free energy necessary to generate ATP is extrac-ted from the oxidation of NADH and FADH2 by electron transport chain, a series of four protein complexes, through which electrons pass from lower to higher stan-dard reduction potentials. The membrane protein cytochr-ome c plays the role of electron donation to the photo oxidized primary donor of the reaction center in electron transport chain. Cytochrome is a hemeprotein whose characteristic mode of action involves transfer of reducing equivalents associated with reversible change in oxida-tion state of the prosthetic group (Agalidis et al., 1999). Formally, this redox change involves a single-electron, reversible equilibrium between the Fe(II) and Fe(III) states of the central iron atom. It is evident from literature that cytochrome C and the reaction center have a lot of differences besides these prosthetic groups. Cytochro-mes can be roughly classified into four groups by the kind of heme present as  the  prosthetic  group;  group  a, b, c      *Corresponding authors E-mail: zea_chemist@yahoo.com. and d have heme a, protoheme, heme c and heme d or d1, respectively (Addison et al., 1977).  It has been shown by both NMR and crystallography that in cytochrome c, the low spin iron in both oxidation states has six ligands, four porphyrin pyrole nitrogen, one thio-ether and one histidine (Salero et al., 1990). Cytochrome c acts as the electron donor for cytochrome c oxidaze and is soluble except for cytochrome c of gram positive bacteria and has a molecular weight of 8,000 - 14,000. There are many cytochrome c are known which actually function or act as the electron donor for the cytochrome aa3 (Laemmli et al., 1970; Yamanaka et al., 1972; Yamanaka et al., 1973).   MATERIALS AND METHODS  Organism  The facultative anaerobic bacterium P. mirabilis strain (ATCC 29245), used in the present study was kindly provided by the PCSIR Laboratories, Lahore, Pakistan.   Growth profile of Proteus mirabilis  Nutrient broth was used as the growth medium for P. mirabilis in conical flasks and autoclaved at 121C for 15 min. After cooling the medium, it was inoculated from the agar aseptically. It was allowed to grow at 37C (Breitenbach et al., 1988) on shaker (C24 KC Refrigerated Incubator Shaker, USA). The shaker was operated at 250 rpm.  Growth  of  P. mirabilis  was   studied   after   the   regular  Zia et al.        1133    -20246810120 5 10 15 20 25 30timeO.D  Figure 1. Growth profile of Proteus mirabilis measured at OD595 after regular interval of 2 h.    interval of 2 h by taking the optical density by spectrophotometer (CECIL/UV-Visible Spectrophotometer, Hitachi U-2001) at 595 nm.   Collection of biomass  Sterilized nutrient medium was inoculated with P. mirabilis and allowed to grow at 37C (Breitenbach et al., 1988) for 10 h on shaker. The shaker was rotated at 250 rpm. Then the culture was prepared and it was centrifuged at 4000 rpm for 25 min for the collection of biomass.    Preparation of cell membrane proteins  Sample cells (25 g, wet weight) kept at 0C (Franks et al., 1993) were suspended in 100 ml of 100 mM phosphate buffer (pH 7.4) and 20 ml of 0.5 M EDTA. There have been many methods developed for the mechanical disruption of cell to isolate the cell membrane from P. mirabilis. Here we used the method of sonication (Kaback et al., 1969). For it, the suspension was then treated with a sonic oscillator (Soniprep 150 SANYO UK) at 10 - 12 KHz for total period of 15 min with interval of 1 min at 4C. The resulting suspension was centrifuged by centrifuge machine (HITACHI-CP 80 MX) at 15,000 rpm at 4C for 15 min. Supernatant obtained was consisted of membrane fraction as well as cytoplasmic fraction where unbroken cells were collected as pallets. Unbroken cells (pallets) were stored at -20C. The supernatant was then ultarcentrifuged at 35,000 rpm at 4C for 60 min. The reddish pallet obtained was of cell membrane whereas the supernatant was of cytoplasm (Qureshi et al., 1998). Then cell membrane pallet was suspended in 25 ml of 100 mM phosphate buffer (pH 7.4) with 2.5 ml EDTA and 4.2 ml of 20% (W/V) Triton-X 100 with final concentration of detergent is 3% (Penefsky et al., 1971; Helenius et al., 1975; Neugebaver et al., 1990; Jones et al., 1987). The resulting mixture was stirred using a magnetic stirrer for 1 h to solubilize the total cell membrane proteins. This suspension was then ultra-centrifuged at 45,000 rpm at 4C for 45 min. Reddish supernatant thus obtained was total membrane proteins while pallet was consisted of membrane lipids.   Pyridine spectrum of membrane proteins  The reaction mixture contained 2.5 ml membrane protein solution, 0.5 ml of 2 N NaOH, 0.5 ml pyridine and about 50 mg of Na2S2O4 (acting as a reducing agent) in the sample cuvette and scanned the sample between 380 – 650 nm ranges. Pyridine ferroheamchrome confirms the presence of covalently bound heme c in the cytoch-romes.   Redox spectra of membrane proteins  Absorption spectra were recorded with spectrophotometer (CICIL/UV-Visible Spectrophotometer, Hitachi U-2001), using a 1 cm light path cuvette at room temperature. Spectral Scanning was carried out between 380 – 650 nm. Reduced spectrum was taken by addition of a small amount of sodium dithionite.   RESULTS AND DISCUSSION  Growth profile of proteus mirabilis  Effect of incubation time on the growth of P. mirabilis was checked with every two hours up to total growth time. Log-phase was found between 6 - 12 h. Maximum growth was observed at around 12th h. Afterwards, there was a sharp decline in growth profile. Decline phase was spread up to 12 h duration, which shows decline in the nutrient component and the tension of growth condition as shown in Figure 1. We routinely collected the biomass at the 12th h of growth with maximum growth actively.   Reduced spectrum showing presence of cytochrome c  Figure 2 showed the absorption peak at 416.5 nm, 524 nm and 550.5 nm (Otten et al., 2001). These peaks showed the presence of cytochrome c in the membrane proteins. The membrane-bound c type cytochrome has a molecular weight of approximately in the range of 12 – 30 kDa. However, this can be confirmed by the electropho-retic data. In the functional sense cytochrome c is a group c cyto- 1134           Afr. J. Biotechnol.      Figure 2. Reduced spectrum showing presence of cytochrome c in membrane proteins.      Figure 3. Pyridine spectrum of membrane proteins showing the presence of heme c.   chrome which acts as the direct electron donor for cytochrome c oxidaze or for the enzyme which perform functions similar to those by the oxidase (Yamanaka et al., 1992). Pyridine spectrum of membrane proteins  When pyridine, NaOH (2 N) and a small amount of Na2S2O4 are added in turn to a solution of heam or  heam      protein, pyridine ferroheamchrome is formed. As theα peak position is characteristic of each kind of heam, we can identify the kind of heam by determining the α  peak position of pyridine ferroheamchrome. Reduced with dithionite pyridine spectra showed the peak at 498.4 nm, a small shoulder at 525 and 552 nm in Figure 3. These spectral properties suggest that the membrane proteins contain heam c (Qureshi et al., 1998). It is possible that cytochrome c-552 may serve as an electron donor for cytochrome c oxidase. Generally the c-type cytochromes function as physiological electron donors to the terminal respiratory enzyme complexes (Qureshi et al., 1996).   REFERENCES  Addison AW, Cullen WR, Dolphin D, James BR (1977). Biological aspects of inorganic chemistry (1st ed). John Wiley and Sons, New York, USA. pp. 78-95. Agalidis I, Othman S, Boussar A, Hussan R, Desbois (1999). Purification, redox and spectroscopic properties of the tetraheme cytochrome c isolated from Rubrivivax gelatinosus. Eur. J. Biochem. 261: 325-336.  Breitenbach JM, Hausinger RP (1988). Proteus mirabilis urease: Partial purification and inhibition by boric acid and boronic acids. Biochem. J. 250: 917-920.   Franks F (1993). 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Isolation and purification of membrane-bound cytochrome c from Proteus mirabilis

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Isolation and purification of membrane-bound cytochrome c from Proteus mirabilis

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