Separation of hemoglabin and cytochrome c by electrophoresis and by chromatography on DEAE-Sephadex

500

SHORT COMMUNICATIONS

sc ILO78 Separation of hemoglobin and cytochrome c by electrophoresis and by chromatography on DEAE-Sephadex In the course of studies on the heme proteinsX, ~ a complete removal of hemoglobin from myoglobin and cytochrome c preparations was found to be essential. Several procedures for the use of cellulose ion-exchange columns have been developed for the heme proteins, and a method for the chromatography of myoglobin on DEAEcellulose has been described 3. This report describes procedures for the separation of hemoglobin and cytochrome c by the method of membrane-filter electrophoresis 4 and for the chromatography of these proteins on DEAE-dextran columns. Cytochrome c from horse heart, Batch No. i i , 0.42% Fe, 92% purity, was obtained from Seravac Laboratories, Cape Town, South Africa and Colnbrook, Great Britain. Hemoglobin used for chromatography was prepared in the met(ferri-)state, in our laboratory by the method of ROSsI-FANELLI5. In electrophoresis experiments washed and hemolysed erythrocytes were applied directly to the strips. The applied mixture contained about 20 #g of total protein. Cellulose acetate electrophoresis strips (Oxoid, Oxo Ltd, London) 9 × 2.5 cm were used. The electrophoresis was carried out by the method of KOHN4, using a veronal buffer (pH 8.6), and alcoholic amidoblack for staining. The chromatographic separation was carried out on DEAE-Sephadex A-5o (Pharmacia, Uppsala, Sweden). The Sephadex material was repeatedly suspended in Tris-buffer (pH 8.6) and decanted after 15 inin. After packing into a 0. 9 × IO cm tube the column of Sephadex was washed again with Tris-buffer until the pH was constant. A sample containing cytochrome c and a relatively large amount of hemoglobin (about 20 mg of total protein) was allowed to

Time(h)

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Fig. I. Micro-electrophoresis of a m i x t u r e of c y t o c h r o m e c a n d h e m o g l o b i n . Applied v o l t a g e I5 ° V. P h o t o g r a p h (negatives) o b t a i n e d b y c o n t a c t p r i n t i n g f r o m stained, u n c l e a r e d strips.

Biochim. Biophys. Acta, 77 (1963) 500 5Ol

SHORT COMMUNICATIONS

501

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Fig. 2. C h r o m a t o g r a p h y o f a m i x t u r e o f c y t o c b r o m e c a n d h e m o g l o b i n . 280 m / t ; , a b s o r b a n c y a t 530 m / ~ .

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percolate into the column by gravity. Cytochrome c was eluted with 0.05 M Tris buffer (pH 8.6). Hemoglobin was retained at the top of the column and was removed by 0.05 M Tris buffer (pH 8.6) in 0.2 M NaC1. Flow rate was about I ml/min and 5-ml samples were collected. The concentrations of proteins in the effluent fractions were assayed by measurement of absorbancy in a Pulfrich photometer with Elpho-Zusatz 53o-m# filter, and ultraviolet absorbancy at 280 m/z in a Zeiss spectrophotometer VSU I. Similar procedures may be used for the separation of hemoglobin from myoglobin 6. Our thanks are due to Doc. Dr. E. LOZA and Mr. S. TARKOWSKIfor the sample of cytochrome c, to Mrs. B. KRUPA for her invaluable technical assistance and to Dr. K. MURAWSKI for his helpful suggestions.

Department of Analytical Biochemistry, University of L6d~, L6d~ (Poland)

WA~DA LEYKO ROMAN GONDKO

x W . LEYKO, Lodz. Towarz. Nauk. Wydzial I I I , No. 61 (1959). 2 W . LEYKO, A. DMOCHOWSKI, L. BUCZEK AND R. GONDKO, Zeszyty Nauk. Univ. Lodz. Set. II, i 2 (1962) 373 W . D. BROWN, J. Biol. Chem., 236 (1961) 2238. 4 j . KOHN, Clin. Chim. Acta, 3 (1958) 45 TM 5 A. ROSSI-FANELLI AND E. ANTONINI, Arch. Biochem. Biophys., 58 (1955) 498. 6 R. GONDKO AND M. SCHMIDT; r e p o r t e d at 2rid Polish Conf. Chromatog., Lublin, Sept. 1963, in p r e p a r a t i o n .

Received April I7th, 1963 Biochim. Biophys. Acta, 77 (I963) 5 0 0 - 5 o l