Endogenous Hemin, Formed in the Liver, Eventually Also Appears In

THE ENZYMlIA TIC CONVERSION OF HEME TO BILIRUBIN BY 1HICROSOMAL HEME OXYGENASE* BY RAIMO TENHUNEN,t HARVEY S. AMARVER, AND RUDI SCHMID DEPARTMENT OF MEDICINE, UNIVERSITY OF CALIFORNIA (SAN FRANCISCO) Communicated by Julius H. Comroe, Jr., July 22, 1968 In the intact organism heme and hemoglobin are converted nearly quantita- tively to bilirubin.1 2 Although several model systems have been proposed, the exact mechanism and the individual steps of this conversion remain unknown. Lemberg3 suggested a coupled oxidation with ascorbate and molecular oxygen, which in vitro leads to formation of the bile pigment precursors choleglobin and verdohemochrome; on acidification, biliverdin is obtained. This nonenzymatic system is unlikely to reflect the physiologic mechanism of heme degradation because it results in formation of a mixture of biliverdin isomers,4 whereas in vivo only the a-isomer of the bile pigment is formed.5 Nakajima and co-workers claimed to have characterized and partially purified a soluble enzyme system that converted heme to a possible precursor of biliverdin. The enzyme, which they called heme a-methenyl oxygenase, was obtained from liver and kidney homogenate; as co-factors it required NADPH, ferrous iron, and an activator extracted from liver cell nuclei by boiling water.6 The system was unusual in its substrate specificity in that it acted only on pyridine hemochromogen, hemoglobin-haptoglobin complex, and myoglobin,7 whereas it was inactive with hematin, oxyhemoglobin, and methemoglobin. -Moreover, spleen and bone marrow, both tissues presumably active in hemoglobin degradation, were almost devoid of enzyme activity. Subsequent reports failed to confirm the existence of a soluble heme a-methenyl oxygenase system. Levin8 and M\ur- phy and co-workers9 showed that Nakajima's findings could be ascribed to a dialyzable and heat-stable factor of low molecular weight functioning as a reduc- ing agent. These observations and the peculiar substrate specificity of the system argue strongly against a physiologic role for "heme a-methenyl oxygenase" in the degradation of heme compounds. In a preliminary communication, Wise and Drabkin10 described a light-mito- chondrial system (obtained from the hemophagous organ of the dog placenta) which converted hemoglobin or heme to biliverdin; the system required NADP, NAD, oxygen, and ATP. Since this study was limited to an esoteric organ of unknown function which had long been known to contain biliverdin,'1 the rele- vance of this observation to the physiologic mechanism of bile pigment formation in the intact organism was not established. Recent findings indicate that labeled hemoglobin or hemin when injected into rats is converted to bile pigment largely in the liver.2 Furthermore, within the liver, the administered hemin is concentrated in the microsomal fraction; and endogenous hemin, formed in the liver, eventually also appears in the hepatic microsomes.12 Finally, in its oxidized form the prosthetic group of hemoglobin is easily detached from the globin,13 suggesting that hemoglobin-heme and unbound hemin may be degraded by the same mechanism. The microsomal fraction of the liver contains important pathways for the oxi- 748 Downloaded by guest on September 28, 2021 VOL. 61, 1968 BIOCHEMISTRY: TENHUNEN ET AL. 749 dation of drugs and endogenous steroids.'4 These systems usually involve a mixed-function oxidation that utilizes cytochrome P-450 as the terminal oxidase and requires NADPH and molecular oxygen.'4-'6 Information on oxidase activity in organs other than the liver is limited, but such activity at variable levels has been demonstrated in a number of tissues examined, including adrenal, kidney, spleen, lungs, gastrointestinal tract, and embryonal hamster cells.'4' 17, 18 The oxidized derivatives of compounds metabolized by these systems are fre- quently rendered more water-soluble by subsequent conjugation on hepatic microsomes. 14, 19 An enzymatic pathway of this type would account adequately for oxidative cleavage of the ferroprotoporphyrin ring of hemoglobin and the subsequent conjugation of the resulting bilirubin to its diglucuronide.20 Materials and Methods.-Recrystallized hemin was obtained from the Sigma Chemical Company, St. Louis, Mo. On spectroscopic analysis of the pyridine hemochromogen prepared with this material, the ratio of the maximal absorption in the a-band to the minimal absorption between the a and , bands was 3.45.21 In some instances the commercial hemin was recrystallized from pyridine.2' Bilirubin was purchased from the Pfanstiehl Chemical Company. Biliverdin was prepared by the method of Gray et al.;22 in methanolic solution this material displayed absorption maxima at 375 and 656 my with a ratio between the two bands of 3.13.22 Gas mixtures (cp) were purchased from the Matheson Company. Male Sprague-Dawley rats, weighing 300-350 gm, were fasted overnight and then de- capitated. The liver was immediately perfused through the portal vein with cold, iso- tonic saline, and the liver, spleen, and kidneys were homogenized in 2-3 vol (w/v) of 0.1 M potassium phosphate, pH 7.4, or in a comparable volume of 0.25 M sucrose. Sub- cellular fractions of these organs were prepared by the procedure of Schneider.23 Homoge- nates in sucrose were employed for preparation of mitochondria and lysosomes; in most other instances, phosphate buffer was used for homogenization. Methemalbumin was prepared by dissolving 13 mg of hemin in 2.5 ml of 0.1 N NaOH containing 12 mg of Tris base. This solution was mixed with 5 ml of 2% human albumin and the pH was adjusted to 7.4 with 1 N HCl. The final concentration of methemalbumin was 2.5 mM. Difference spectra were determined with a Shimadzu MPS-50L split-beam recording spectrophotometer. The rate of conversion of heme to bilirubin was monitored in a Gil- ford model 2000 spectrophotometer equipped with a constant-temperature cuvette chamber. Formation of bilirubin was determined from the increase in optical density at 468 mu, at which wavelength the pigment absorbed maximally in the incubation mixture used. The extinction coefficient of bilirubin was measured by adding weighed amounts of the crystalline pigment (dissolved in small volumes of 0.05 N NaOH) to aliquots of the incubation mixture so that the final pigment concentration ranged from 5 to 10 mg %. Trhe extinction coefficient of biliverdin was determined similarly by addition of a methanolic solution of the pigment to aliquots of the incubation mixture. For bilirubin the milli- molar extinction coefficient, at 460-470 m1u, ranged from 27.7 to 31.7; and for biliverdin, at 650-660 minA, from 7.3 to 8.0. Bilirubin in the incubation mixture was quantitated also by spectrophotometric determination of the pigment in a chloroform extract.24 For ex- traction, the incubate was mixed with '/6 vol of formic acid and with sodium fluoride to a final concentration of 0.1 M. Bilirubin and biliverdin were also identified by thin-layer chromatography on silica gel, using a solvent system consisting of methylethylketone, propionic acid, and water (20:5:5).25, 26 Heme was determined as the pyridine hemochromogen.2' NADPH consumption in the presence and absence of methemalbumin was estimated by the decrease in optical density at 340 miu. Methemalbumin-dependent oxygen consumption was measured with a Clark-type oxygen electrodeY Measurements were made with an elec- trode obtained from the Yellow Springs Instrument Company, together with an Oxygraph Downloaded by guest on September 28, 2021 7.50 BIOCHEMISTRY: TENHUNEN ET AL. PROC. N. A. S. model KM manufactured by Gilson Medical Electronics. Microsomal carbon monoxide- binding cytochrome was determined by the method of Omura and Sato.28 Acid phospha- tase was assayed using ,3-glycerophosphate as substrate.29 Phosphorus was measured by the method of Chen et al.30 Cytochrome oxidase was determined by the procedure of Wharton and Tzagoloff.3" All enzyme activities were calculated from maximum reaction rates. Protein was quantitated by the method of Lowry et al.32 Additional details of experimental procedures are given in the legends to the tables and figures. Results.-Incubation of methemalbumin with a 10,000 X g supernatant or a microsomal fraction of liver (Fig. 1), spleen, or kidney resulted in the appearance of a new absorption band in the 460-470 my region. A second, much smaller, band appeared between 650 and 660 mu; this was thought to represent biliverdin.3 The reaction required the presence of molecular oxygen and of NADPH (Fig. 1, and Tables 1 and 2). Liver Enzyme + + NADPH minus Liver Henme+ Hem.e FIG. 1.-Conversion of heme to bilirubin by liver Enzyme microsomes. The difference spectra demonstrate bilirubin formation in the 10,000 X g supernatant of >- 0.200 - - ZERO TIME liver homogenate after 30 min of incubation at 370C. AFTER 30 min. - The incubation mixture (3.85 ml) consisted of 10,000 X g supernatant (70 mg of protein), 170 1AM heme, 1.4 0.100 \\& mM NADP, 4 mM glucose-6-phosphate, 6.6 mM MgCl2, and 90 mM potassium phosphate buffer (pH 7.4). In the control incubation, NADP and glucose- o - 6-phosphate were replaced by 0.1 M potassium phos- * | phate buffer, pH 7.4. Difference spectra were ob- 400 450 500 550 tained after diluting the incubation mixtures 2.75 WAVELENGTH mp times with potassium phosphate buffer, pH 7.4. The following evidence indicated that the reaction product exhibiting an absorption maximum at 460-470 my was bilirubin formed from the added substrate, heme: (a) When crystalline bilirubin was added to the incubation mixture used, a similar absorption band at 460-470 mgu was observed. (b) When a methanolic solution of diazotized sulfanilic acid24 was added directly to a 10,000 X g supernatant fraction of liver or spleen containing NADPH, a red azoderiv- ative characteristic of bilirubin24 33 was formed only after incubation with heme in air.

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