Vol. 47, No.

5, May 1999 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

pages 899-907

PURIFICATION AND CHARACTERIZATION OF CYTOCHROME C FROM

CAMEL MUSCLE

Ali M. S. Gorban and Omar M. Izzeldin'

D e p a ~ n e n t of Biochemistr)~ College of Science, King Saud University, P. O. Box 2455,

Riyadh 11451, Saudi Arabia
"To whom correspondence should be addressed. Tel: +966 1 4675800; Fax: +966 1 4675791

Abbreviations: SDS-PAGE, sodium dodecylsulphate polyacrylamide gel electrophoresis

Summary

Cytochrome C was purified from camel skeletal muscles using ammonium

sulphate fractionation and gel filtration on Sephadex G25 and Sephecryl $200

columns. The preparations were pure by SDS-PAGE criteria, with molecular weight of

12300 Dalton. The electrophoretic mobility of camel cytochrome C was the same as

that of the horse heart. The spectral characteristics of the isolated cytochrome C were

also investigated.

Introduction

Cytochromes C are small electron-transfer proteins which are present in the

intermembrane space of all eukaryotic mitochondria where they fulfil a variety of

functions. While their primary role is that of an electron - transfer protein supplying

electrons to the terminal oxidase of the mitochondria respiratory chain.

Purification of cytochrome C started as early as 1930 by Keilin [1] who

identified and designated three cytochromes (a,b,c) on the basis of their absorption

spectra. Amberlite IRC 50[2] and cation exchange chromatography CM50 [3] were

used for the purification of cytochrome C from horse heart. Calcium phosphate column

chromatography, hydroxyapatite column and ammonium sulphate fractionation were

also used to purify cytochrome C from photosynthetic bacteria (Rhodopseudomonas

spaeroides) [4] and various forms of cytochrome C from theromphillic bacteria [5].

Copyright 9 1999 IUBMB

899 1039-9712/99 $12.00 + 0.00
Vol. 47, No. 5, May 1999 BIOCHEMISTRY a n d MOLECULAR BIOLOGY INTERNATIONAL

Crystallization of tuna ferricytochrome C was performed by Walter et al [6].

Physical and chemical characteristics were studied by many researchers [1,2,4].

Quantitative studies on the oxidized and reduced forms of cytochrome C were carried

out by Plummer [7] using potassium ferricyanide and sodium dithionite. Further

quantitative assays combining chromatography and spectrophotometry using rat tissue

were reported by Kim [8].

In the present work, we obtained pure preparations of camel skeletal muscle

cytochorme C using ammonium sulphate precipitation followed by fractionation on

Sephadex G25 and Sephecryl $200 gel filtration columns. Using the same procedure of

plummber for calculating the yield of reduced cytochrome C but changing the

wavelength from 550 nm to 410 nm a 6.7 times yield was obtained ofcytochrome C.

Materials

Trichloroacetic acid (TCA) and sodium hydroxide were obtained from Koch-

Light Laboratories (Colnbrook, Bucks England). Ammonium sulphate, coommassie

brilliant blue R250, N,N,N,N, Tetramethyl - aethylendiamin (TEMED)and sodium

dithionite were obtained from Fluka (Buchs, Switzerland), Merck (Darmstadt,

Germany), Technological Transfer (Cambridge, UK) and Riedel-De-Haen. All other

reagents were obtained from BDH Chemicals, (Poole, UK). Sephadex G25 and

Sephecryl $200 were obtained from Pharmacia Fine Chemicals. Pye Unicam Model SP

8-200 UV-VIS spectrophotometer was used for scanning of reduced and oxidized

cytochrome C.

Methods

Fresh camel muslce tissue was obtained from a local slaughter house

(RIYADH), and cleaned of fat and connective tissue. One Kg of cleaned tissue was

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Vol. 47, No. 5, May 1999 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

minced and blended for 2 minutes with 1500 mL of 0.145 N TCA. Stirred for 3 hours

at 4~ The homogenate was then filtered through muslin and its pH was adjusted to

7.3 using 10% NaOH. The homogenate was centrifuged for 15 minutes at 2000 rpm at

4~ To the supematant ammonium sulphate was added with continuous stirring, till

75% saturation, and the precipitate formed was filtered through large filter paper

(No.2). 85% ammonium sulphate was added to the filtrate which was left overnight at

4~ with continuous stirring. The supernatant was filtered again and the volume of

the filtrate was measured. 25 mL L ~ of 20% TCA was added to the filtrate and the

preparation was centrifuged at 10000 rpm for 15 minutes. The pellet was suspended in

100% ammonium sulphate and centrifuged again for 15 minutes at 15000 rpm at 4~

The pinky pellet containing cytochrome C was dissolved in 10 mM sodium phosphate

buffer pH 7.4, applied to Sephadex G25 column (2.5x40 cm), and eluted with the same

buffer. Fractions containing cytochrome C were collected, pooled and precipitated

with 20% TCA (25mL L-l). The precipitate formed was suspended in phosphate buffer

and applied to Sephecryl $200 column (2.5x40 cm)previously quilibrated with the

same buffer. Eluted cytochrome C was subjected to sodium dodecyl sulphate

polyacrylamide gel electrophroesis [SDS-PAGE] using horse heart muscle cytochrome

C as a standard. The prepartion showed a single band with the same mobility of that of

horse heart cytochrome C. Total cytochrome C was determined based on absorption

spectra of reduced cytochrome C. 1 mL of sodium dithionite saturated solution was

added to 1.9 mL of sodium phosphate buffer (0.1 mol L "1 pH 7.4). and 1 mL of 0.17

mgmL~) cytochrome C. Concentration of cytochrome C was determined at two

wavelengths 550 nm and 410 nm using molar extinction coefficient of2.77x104 and

10.6x10 4 respectively. SDS-PAGE was done according to the procedure of Lammaeli

[9]. Protein concentration was determined by the method of Peterson [10].

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Vol. 47, No. 5, May 1999 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

Results and Discussion

The results offractionation ofcytochrome C using fraction collector by using

Sephadex G25 and Sephecryl $200 are shown in (Fig 1 and Fig 2).

0.6s ~
.48 0.60 ~ ~
f ro E
.44 0.60 E
o
.40 O.SO

.36 0.4S " ec

32 0,0 :~ ~
E ~ o
= .E E
~ .~ 0.35 E = o
o.- L:
0.30 2 m o =
o
~.~.
~ .20, 0.25 ~ ~ ~E
0.20 ~

"~ .12 i

I"
O.lO ~ i

.os ~ +

10 20 30 40 50 60 70 S0 90 100 110 120 130 140 150

Effluent Volumt {ml)

Fig 111Elution o f camel cytochrome C on Sephadex G25 column. 2 mL sample

containing 10 mg protein (muscle) was applied on 2.5x40 em column. The column was

eluted with 10 mM sodium phosphate buffer pH 7.4 contains 0.05% NAN3. The flow

rate was 2 mL rain-~ and void volume of the column was 58 m L

.S2 .52

946

,&&
,40
\ .40 ~ .~
.G

""ii
3G

J~2
J .32
.2~ uo e =
~ 24

16 .~6 ~* :~.~

+ ,~
.08
.o~ i J
04 .o, & !
10 20 30 40 50 G0 70 I)O 90 100 110 120 130 140 150 160 170 180 Ig0 20O
E111uent Volume (ml)

Fig [2] Eluction o f camel cytochrorne C on Sepbecryl $200 column. Elution conditions

including column characteristics were the same as described in Fig. l.

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Vol. 47, No. 5, May 1999 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

Oxidized cytochrome C exhibited et band at 550 nm, 13 band at 521 nm and 5

band at 415 nm. Upon reduction, the absorption of the ct band shifted to 548 nm, the 13

at 521 nm, remained the same. While the ~5410 nm band decreased in magnitude (See

Fig. 3).

---- ,~550 n m
.... /t 521 n m Oxidized
.... ~415 nm

g
0,9 0 [I $21 Reduc~
i
0,80

0.70
u
o &60 t
=
= O.SO

< 0.~0
idlzed
0.30

0.20

0,1C
i
-- ._.o. "% . . . . . . t~O0" '
6O0 500 3~'0
Wav~ l.~nOth (n rn)

Fig 13] Absorption specmlm o f purified camel muscle cytochrome C. Tracings show

the absorption of oxidized ............. and reduced _ _ forms of camel cytochrome

C. The wavelength ofcq 13 and 8 peak are shown. The spectra were measured at 1 nm

sec4 uslng pYE-UNICAM, Model SP 8-200 UV/VIS spectrophotometer,

Table 1 shows summary of protein purification. Based on the extinction

coefficient reported by Margoliash [11], the yield of cytochrome C at 550 nm was 0.07

mgmL 4, while the yield at 410 nm was 0.47 mgmL 4. The total yield was 20.30 mgs

Kg 4 (2.5 p.mole Kg 4 ) from camel skeletal muscles. We obtained 26.48% yield and 24

purification fold. Comparatively, the yield from camel skeletal muscle was less than

reported from human skeletal muscles (4 ~tmole Kg "1 ) [2] or human cardiac muscle

(16-20 I.tmole Kg 4 ) [12]. Camel cardiac muscle yielded 10 ~tmole Kg 4 (unpublished

data).
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Vol. 47, No. 5, May 1999 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

Table [1] Purification Chart of Camel M u s c l e Cytochrome C [blonitered at 4 1 0 nm]

Purification Volume Yield Protein Specific ToLal Total Purification Recovery

Step mL (reduced) cone. activity protein yield fold
mg mgmL-I mg/mgmL-t mg mg

Homogenate 1458 0.35 1.26 0.28 1837.08 514.38 I00 I00

~mmoui~m sulphaze 1849 0.16 0.38 0.42 702.62 295,10 1.5 57.37
75%
%~m~onium sulphate 1890 0. I0 0.36 0.27 680,40 183,71 0.96 35.71
]5%
~ephadex G25 3t0 0.50 0.56 0.89 173.60 154.50 3.18 30.04

~ephecryl S200 290 0,47 0.07 6.71 20.30 136.21 23.96 26.48

- OuT of I Kg of camel muscles, the purified protein was 20.3 mgs.

- TheconcentrationSpeclfic
acs equal to yield of Cytochrome C at 410 nm (in mgs) divided by the pros

Fig [41 SDS polyacrylamide gel electrophoresis of camel skeletal muscle. The figure

shows the various stage of cytochrome C purification. Lane (A) homogenate fraction;

(B) 75% ammonium sulphate

(C) 85% ammonium sulphate

(D) Sephadex G25

(E) and (F) SephecPyl $200

(G) Horse heart cytoehrome C (standard)

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Vol. 47, No. 5, May 1999 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

Following the procedure of Lammaeli [9] of SDS-PAGE electrophoresis using

a low molecular weight calibration kit (12300-78000) Daltons, the electrophoretic

mobility of camel muscle cytochrome C was the same as that of the horse heart

cytochrome C, both having a molecular weight of 12300 (see Fig. 4 & 5). This was

confirmed by laser densitometric tracing of SDS-PAGE of the purified camel muscle

cytochrome C, using horse heart cytochrome C as a reference. Both camel cytochrome

C and horse heart cytochrome C standard have the same retention time of 0.23 minutes

(see Fig. 6 & 7). Also the camel cardiac muscle had the same molecular weight as

camel's skeletal muscle or horse cardiac muscle cytochrome C (unpublished data).

~ i 84184

Fig [5]Purity of cylochrome C. SDS-PAGE (sodium dodecyl. Sulphate -

polyacrylamide gel electrophoresis ) of different amounts of camel skeletal muscle

cytochrome C, with lanes (A),(B),(C) and (D) contained 7, 8.5, 10.7, 14.2 ugs of pure

eytochrome C respectively. Lanes (E) and (F) contained 10, 15 ugsofhorse heart

cytochrome C (standard). Lane (G) and (H) contained t5 and 20 ug of molecular

weight calibration kit proteins (12300-78000). Gels were stained coommassie blue.

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Vol. 47, No. 5, May 1999 BIOCHEMISTRY a n d MOLECULAR BIOLOGY INTERNATIONAL

I0
10 ~ ~C tin

IO
umin umin
SO

&o

arbcmir a nhydrase

_a

oglol)in

~rn~, . . . . . . ~ Cytechrome

10 IIl I I i 9
10 ,l& IS ts tO
~ t a l monomer concentration {% T}

Fig [61 Typical calibration line for molecular weigh marker electran. MWt range range

12300-78000. 10% gradient gel.

o ~
<~
|

f
.s 1.o \ 1.s
Relative mobility

Fig [7 IDensitornetric tracing of SDS-PAGE of purified camel skeletal muscle

cytocbrome C. Panel (A) shows proteins of molecular weight calibration kit 12300-

78000. Panel (B) shows horse heart cytochrome C, and panel (C)shows the camel

muslce cytochrome C. The measurements were conducted on LKB 2202 Ultroscan

Laser Densitometer using Helium Neon Lasser as light source and chart speed of 2mm

rain-I

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Vol. 47, No. 5, May 1999 BIOCHEMISTRY and MOLECULAR BIOLOGY INTERNATIONAL

References

1. Keilin, D., 1930. Proc. Roy. Soc. London. B. Biol., 106, 418-420.

2. Margoliash, E. and Walask, O.F. 1967. Methods in Enzymology., Vol X, 339-

348.

3.. Brautigan, D.L., Miller, S., Margoliash, E. 1978. Methods in Enzymology.,

Vol L[I] Part D, 128-134.

4. Yu, C.A., Mei, Q.C., Yu, L. 1984. Biochem Biophys. Res. Commun.,

118,964-969.

5. Yoshida, T., Lorence, R.M., Choc, M.G., Tarr, G.E., Finding, K.L., Fee, J.A.

1984. J. Biol. Chem, 259, 112-123.

6. Walter, M.H., Westbrook, E.M., Scott, T., Andrew, M. and Margoliash, E.

1990. J. Biol. Chem, 265, 4177-4180.

7. Plummer, D.T. 1978. An Introduction to Practical Biochemistry. Mc Graw-Hitl

Book Company (U.K.), Second Edition, 151-154.

8. Kim, I.C. 1989. Anal. Biochem., 181, 140-144.

9. Lammaeli, U.K. 1979. Nature., 227, 680-685.

10. Peterson, G.L. 1977. Anal. Biochem., 83, 346-356.

11. Margoliash, E., Frohwirt, N., Wiener, E. 1959. The Biochem. J., 71, 559-571.

12. King, T.E. 1978. Methods in Enzymology Vol : LIII Part D, 181-191.

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