BIOCHIMICA ET BIOPHYSICA ACTA
IRON-CHELATING ENZYME FROM RAT LIVER
YOSHIMASA YONEYAMA*, ASTUKO TAMAI, TAKAKO YASUDA AND HARUHISA YOSHIKAWA Department of Physiological Chemistry and Nutrition, Faculty of Medicine, University of Tokyo, Tokyo (Japan) (Received January roth, 1965)
Iron-chelating enzyme was extracted from rat-liver mitochondria. The measurement of the stoichiometry showed that no side reaction occurred. The reaction product was heme, not hemeprotein. Some other properties, such as pH response, K m for iron and protoporphyrin and effect of supernatant are reported.
In a previous communication, the authors showed in vitro enzymic incorporation of iron into heme in various organs using rat as an experimental aminal'. In liver it was further shown by the authors 2- 4 that mitochondria were the most active fraction to synthesize heme. The enzyme which incorporate iron into heme (protoheme ferrolyase EC 220.127.116.11), was studied further by LABBE et al.6- S and PaRRA AND JONES 9,l O. The authors ll - 15 recently purifed the enzyme from duck erythrocyte and studied the relationship with hemoglobin synthesis. In this communication, the purification of the enzyme from rat liver, the nature of the product, the stoichiometry of the reaction and other properties of the enzyme are described. MATERIALS AND METHOD
Preparation oj the enzyme Rats were decapitated under anesthesia with carotid bleeding. The liver was perfused with isotonic saline to remove hemoglobin. All subsequent procedures were done below 4°. Mitochondria were obtained with differential centrifugation in 10% sucrose or isotonic KCI. Mitochondrial paste was treated with ten volumes of distilled water and lightly homogenized. After the addition of one-ninth volume of II.5 % RCI and centrifugation at 6000 X g, the residue was extracted for 15 hat 4° with an equal volume of 0.05 Tris buffer (pH 8-4) made isotonic by addition of RCI containing r % sodium cholate. The clear supernatant after centrifugation at * Present Adress: Department of Biochemistry, School of Medicine, Kanazawa University, Kanazawa, Ishikawa (Japan). Biochim. Biopbys. Acta, 105 (196,5) 100-105
IRON-CHELATING ENZYME FROl'vI RAT LIVER
6000 X g was used as enzyme solution. This was diluted to a suitable protein concentration (usually I mg proteinjrnl) with 0.15 M Tris buffer (pH 8-4). 1ncttbation The incubation mixture contained 50 mzzmoles of protoporphyrin, 50 mzzmoles of FeCla ·6H 20 (approx. 105 counts/min of 59Fe), 2 ftmoles of cysteine, 0.5 ml of 0.15 M Tris buffer (pH 8-4) and 0.5 ml of enzyme (protein concentration I mgjrnl) in a final volume of 2 ml. Incubation was carried out aerobically for 30 min at 37°. Determination oj enzyme activity After incubation, the reaction mixture was cooled with ice water to stop the reaction and carrier hemoglobin solution and 50 ml of acetone were added. [59FeJhemin was crystallized according to CHU AND CHUI6, 59Fe was counted in a well-type scintillation counter, and the quantity of hemin was determined according to KING I7. The amount of heme synthesized was calculated from the amount of carrier hemoglobin added and the specific activity of [59FeJhemin. Protein was determined according to LOWRY et al. I B• RESULTS
Extracting conditions with sodium cholate Conditions of extraction were examined in the presence of 0.5% sodium cholate. The results are given in Table I. The effect of extracting time was also examined and overnight extraction was adopted. Purification. The rise of specific activity and yield of the enzyme are shown in Table II. A nearly S-fold increase was observed. TABLE I CONDITION OF EXTRACTION WITH CHOLATE
Mitochondrial debris was extracted with equal volume of extracting solution (made isotonic by addition of KCI) containing I % of sodium cholate for 15 hat 4°. After centrifugation at 6000 X g, the supernatant was diluted to a suitable concentration (usually I mg proteinjrnl) and used for enzyme assay. The results were expressed as relative activity.
Relative actiuity Expt;
Tris buffer (0.05 M)
KHCO.-KaCO. buffer (o.oy M)
8.0 8·S g.o
II2 176 IIO
g.o g.6 10.0
KHCO. (o.OS M)
Biochim. Biopbys, A eta, lOS (19 65) 100-105
TABLE II PURIFICATION OF ENZYME
The standard mixture, containing 0.5 mg of protein of each fraction, was incubated.
Protein (B) (mglml)
Hemin synthesized (C)
Relative total Yield activit" (A «:» X C'IO- 3 ) .
Whole homogenate of rat liver Homogenate of mitochondrial residue Cholate extract of mitochondrial residue
Stoichiometry The stoichiometry of the reaction during I h incubation is shown in Table III. Protoheme and protoporphyrin were measured according to DRESEL AND FALK 19 • The iron content of the mixture before and after the incubation was measured according to BRUECKMAN AND ZONDEK 2o. Protohemin was determined in acetic acidacetone solution after porphyrin extraction-I.P. No side reaction was observed. TABLE III STOICH:IOMETRY OF REACTION
The incubation mixture, containing iron, protoporphyrin, 10 ftmoles of cysteine, I ml of 0.15 M Tris buffer (pH 8.4) and 3.0 ml of non-diluted enzyme in a final volume of 4-5 ml, was incubated aerobically. Iron, protoporphyrin and hemin were determined before and after incubation.
Quantity (m/1-moles) at
Iron Protoporphyrin Hemin
57·3 3 6.8 43·7
47·3 27. 6 54·5
10,0 9·5 10.S
Reaction product As rat-liver mitochondria contained water-insoluble cytochromes such as cytochrome oxidase, cytochrome b and cytochrome Cl, direct identification of the product was impossible. Measurement of difference spectra was employed. The difference spectra between oxidized and reduced formes of mitochondrial debris before and after incubation are shown in Fig. I. The a-band of cytochrome a and cytochrome b (and/or cytochrome cl ) was recorded before incubation, while after incubation the increase at 550-555 mf.J, was observed, which corresponded to the a band of cytochrome band lor cytochrome Cl' As cytochrome b and cytochrome cl are reported not to combine with CO, the difference spectra between reduced and reduced + CO Biochim. Biopbys. A eta, 105 (1965) 100-105
IRON-CHELATlNG ENZYME FROM RAT LIVER
+ 40 , - - - - - - - - - - - - - - - ,
Wave length (m)d
Wave length (% I
Fig. 1. Difference spectra between oxidized and reduced forms of mitochondrial debris. Before incubation ( - - - ) and after incubation ( ). Incubation mixture contained 100 mumoles of protoporphyrin, 100 mrzmoles of FeCI.' 6H.O, 4 ftmoles of cysteine, 2 ml of suspension of mitochondrial debris and 2 ml of o. 15M Tris buffer (pH 8.4) in a final volume of +6 ml. Incubation was carried out anaerobically in a Thunberg-type tube for 2 h. Half of incubation mixture was oxidized with aeration, while other reduced with dithionite. Fig. 2. Difference spectra between reduced and CO-reduced forms of mitochondrial debris. Before incubation ( - - - ) , after incubation ( ) and with addition of 30 m,t moles of protohemin and without incubation (.-.-.-.-). Incubation was carried out as in Fig.!. Incubation mixture was reduced with dithionitc, and half of it bu bbled with CO gas.
forms were further examined. The results are shown in Fig. 2. The difference spectra showed an increase of CO-combining pigment after incubation, which virtually coincided with the addition of protohemin to the incubation mixture as shown in Fig. 2. These results suggested an increase of protoheme, not of protohemeprotein. 10.0
Fig. 3. Time course of reaction.
Some properties of the enzyme
The time course of the reaction is shown in Fig. 3. Up to 45 min, the reaction was linear with time. With regard to enzyme concentration, the reaction velocity was linear with enzyme concentration in the range tested (Fig. 4). The optimum pH Biochim. Biophys. A cia,
105 (1965) 100-105
.2 ~ 0
Proleln concenlrgtion of enzyme (mg/0.5ml)
Fig . 4. Effect of enzyme concentra tion on reaction v elocity. Th e incubation mixture, containing 0.5 ml of different en zyme con centration, was incubated in t he us ual way. Fig. 5. pH act ivity curve.
for the enzyme was found to be about 8.4, as shown in Fig. 5. The influence of the supernatant, which was reported to acti vate the ery throcyt e enzy me, was examined: no stimulatory effect was obser ved . The effect of reducing subst ances and anaerobic incubation was investigated : anaerobic conditions favored heme synthesis, while some differences were found am ong reducing substances (Tabl e IV). The Michaelis constant for ferrous iron was 6· 10- 5 M, while that for protoporphyrin was 1'10-4 M. T ABLE I V EFFECT OF REDUCTANT AND ANAEROBIC IN CUBATION
In cubation was carried out in usual way. Anaero b ic incubation was made in a T hunberg-t yp e tube und er N1
R elative activity
(Il mol es) A erobic
Cysteine Glutathione Ascorba te
42 48 4 7 44
74 87 32
80 93 15 8
DI SCUSSIO N
The iron-chelating enzyme from rat liver had considerable resemblance with t hat of fowl erythrocyte. They are both associated with particles situated in mitochondria, and are only solubilized with the aid of detergent. Furthermore, as shown Biochim . Biophys. Ac ta, 105 (19 65 ) 100-105
IRON-CHELATING ENZYME FROM RAT LIVER
for duck erythrocyte preparation, no side reaction such as destruction of heme and protoporphyrin was observed in the rat-liver preparation. The enzymic destruction of hemoglobin is assumed by YAMAOKA et al. 21 to take place in liver cells. Our results showed no direct relation of heme destruction with heme synthesis in liver. Mammalian liver mitochondria contain protohemeproteins such as cytochrome b and catalase (Ee 18.104.22.168). With our preparation, however, the reaction product was found spectrophotometrically to be protoheme, not hemeprotein. Thus similar results were obtained as with duck erythrocytes-t. For erythrocyte preparations, the activating effect of the supernatant of erythrocyte hemolysate has been reported from several laboratories 13,22, and the phenomenon was discussed from the standpoint of hemoglobin formation. No stimulatory effect was observed in liver preparations by addition of liver supernatant or liver-mitochondrial-soluble fraction. The results were quite different from those for erythrocytes. The lack of apoprotein or other factors might be responsible for the difference. The Michaelis constant-" for iron of the liver preparation was nearly the same as that for the fowl erythrocyte preparation, while that for protoporphyrin of the liver somewhat ten times greater than that of the erythrocyte. We have no explanation for this difference. REFERENCES
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Biochim, Biophys, Acta, 105 (1965) 100-105