THE ENZYMlIA TIC CONVERSION OF TO BILIRUBIN BY 1HICROSOMAL * 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 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 , 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 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 , 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 hemo- chromogen, hemoglobin-haptoglobin complex, and ,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 sys- tem 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 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 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 pre- pared 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 metha- nolic 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 metha- nolic 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 con- sumption 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 biliver- din.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 ab- sorption maximum at 460-470 my was bilirubin formed from the added sub- strate, heme: (a) When crystalline bilirubin was added to the incubation mix- ture 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. (c) After incubation with heme, a yellow pigment was extractable that had the solubility and spectrophotometric properties of bilirubin and gave a positive diazo reaction24 33 (Table 3). (d) On chromatography with silica gel, this yellow chloroform-soluble pigment had an Rf identical with that of authentic bilirubin. (e) A major fraction of the heme that disappeared on incubation was accounted for by recovered bilirubin (Table 3). (f) Radioactive material having the properties of bilirubin was crystallized' from incubation mixtures containing C'4-heme. Formation of bilirubin diglucuronide could not be demonstrated in the present system.25' 26 The presence of microsomes in the incubation mixture appeared to be essential for the conversion of heme to bilirubin. Small and variable enzyme activity in other subcellular fractions was attributable to microsomal contamination. In all preparations studied, enzyme activity closely paralleled the activity of micro- somal carbon monoxide-binding cytochrome, whereas a comparable correlation was not observed with cytochrome oxidase or acid phosphatase activity. Downloaded by guest on September 28, 2021 VOL. 61, 1968 BIOCHEMISTRY: TENHUNEN ET AL. 751

TABLE 1. The role of NADPH and oxygen in the conversion of heme to bilirubin by spleen microsomes. Bilirubin formation (mjtmoles/10 mg Conditions protein/10 min) In air with NADPH 9.32 In 96% N2 +4% 02with NADPH 8.79 In 50% CO + 46% N2 +4% 02with NADPH 0.63 In air without NADPH 0.26 The reaction mixture (3.0 ml) consisted of 10,000 X g supernatant (6 mg of protein), 17 AM heme, 0.5 mM NADP, 1.3 mM glucose-6-phosphate, 1.9 mM MgC12, and 90 mM potassium phosphate buffer (pH 7.4). The cuvettes were gassed with the indicated gases for 2 min before and during the assay. The rate of bilirubin formation was constant for at least 15 min. TABLE 2. Heme-dependent oxygen, NADPH consumption, and bilirubin formation in the 20,000 X g supernatant of rat spleen. Mymoles/10 mg protein/min Per mole bilirubin Oxygen consumption 2.51 3.0 (2.12-3.08) (2.4-3.4) NADP consumption 3.71 4.4 (2.94-5.08) (4.1-4.7) Bilirubin formation 0.84 (0.69-1.12) The reaction mixture (3.0 ml) used for the measurement of oxygen and NADPH consumption and of bilirubin formation was similar to that described in Table 2 except that the 20,000 X g super- natant (9-13 mg of protein) and heme in concentration of 51 .M were employed. The mean and range of values of five individual determinations are shown. Although microsomal preparations alone were active in converting heme to bilirubin, this activity was enhanced considerably by the addition of a 105,000 X g supernatant fraction. Only a trace of bilirubin was formed when heme was incubated with the supernatant alone (Table 4). The factor in the 105,000 X g supernatant fraction responsible for the enhanced bilirubin formation was heat- labile and nondialyzable. Human or rat albumin in concentrations ranging from 0.2 to 3.5 mg per ml was inactive when substituted for the supernatant fraction. When the supernatant was omitted from the incubation mixture containing microsomes and methemalbumin, the minor absorption peak appearing in the 650-660 my band was enhanced as compared to incubation of the complete system. With the complete system, containing 10,000 X g supernatant of liver or spleen in the concentrations indicated, the rate of bilirubin production from heme was maximal and constant for the initial 15 minutes of incubation (Fig. 2). The concentration of heme used in these experiments was saturating, exceeding the apparent Km of the system four to six times.34 When the amount of 10,000 X g supernatant added was varied, bilirubin production was a linear function of the enzyme concentration, at least within the range of 1.0-3.5 mg of protein per milli- liter of incubation mixture. The presence of NADPH or an operational NADPH- generating system was an absolute requirement for formation of bilirubin (Figs. 1 and 2); NADH in concentrations up to 200 AiM or 10 mM ascorbic acid were ineffective as substitutes. The system was completely inhibited by carbon monoxide or by the absence of molecular oxygen (Fig. 2 and Table 2). Downloaded by guest on September 28, 2021 752 BIOCHEMISTRY: TENHUNEN ET AL. PROC. N. A. S.

TABLE 3. Correlation between heme disappearance and bilirubin formation. Heme breakdown Bilirubin formation Bilirubin recovered (mpmoles/10 mg (mtmoles/10 mg in per cent of calculated Tissue protein) protein) heme breakdown Spleen, 10,000 X g 15.68 12.17 77.6 supernatant (12.90-17.07) (8.09-15.02) (62.7-88.0) Liver, 10,000 X 9 2.00 0.54 27.0 supernatant (1.86-2.28) (0.42-0.71) (22.4-31.2) The conditions of incubation were the same as described in the legends to Figs. 1 and 2. Bilirubin formed on incubation was extracted with chloroform and determined as described in the text. The mean and range of values of five individual experiments are shown.

TABLE 4. Conversion of heme to bilirubin by spleen microsomes in the presence and absence of 105,000 X g supernatant. Bilirubin formed (mMmoles/10 mg of System microsomal protein/min) Microsomes 1.07 Supernatant (105,000 X g) Trace Microsomes + supernatant 1.68 Microsomes + dialyzed supernatant 1.55 Microsomes + Sephadex-treated supernatant 1.51 Microsomes + boiled supernatant 0.97 The reaction mixture contained spleen microsomal suspension in 0.25 M sucrose (2 mg of protein), 105,000 X g supernatant either untreated or treated as indicated below equivalent to 5 mg of protein, 17 uM heme, 90 ,uM NADPH, 1.9 mM MgCl2, and 90 mM potassium phosphate buffer (pH 7.4). In the control cuvettes, NADPH was replaced by 0.1 M potassium phosphate buffer, pH 7.4. Various treatments of the 105,000 X g supernatant included dialysis against 0.01 M potassium phosphate buffer (pH 7.4) for 12 hr; chromatography on Sephadex G-25 fine using the void volume; boiling for 2 min.

With the complete system containing 10,000 X g supernatant of spleen, ap- proximately 3 moles of molecular oxygen and 4-5 moles of NADPH were con- sumed per mole of bilirubin formed (Table 2). When spleen microsomes were used without the 105,000 X g supernatant, 3-4 moles of molecular oxygen and 7 moles of NADPH were utilized per mole of bilirubin produced. Enzyme activity varied widely in the various tissues studied; spleen was much more active than liver and kidney. Per unit of protein, comparative enzyme activity in spleen, liver, and kidney was 10-20, 1, and 0.3. Discussion.-These observations provide evidence for an enzymatic mechanism in microsomes that is capable of catalyzing the oxidative cleavage of ferriproto- IX to form bilirubin. That the reaction is enzymatic in nature is attested to by (a) its inhibition by carbon monoxide in the presence of oxygen; (b) its absolute requirement for NADPH; and (c) its heat lability. The product formed under the specified conditions of incubation was identified as bilirubin by spectrophotometric, chromatographic, and chemical means. The enhance- ment of bilirubin formation by a 105,000 X g supernatant fraction (Table 4) may be ascribed to biliverdin reductase, which is reported to be present in this fraction in high concentration.35 In the complete system, biliverdin reductase did not appear to be rate-limiting. However, biliverdin was increased when the 105,000 X g supernatant fraction was omitted from the incubation mixture. With the complete enzyme system, bilirubin and the small amount of biliverdin Downloaded by guest on September 28, 2021 VOL. 61, 1968 BIOCHEMISTRY: TEANHUNEN ET AL. 753

z LU 0 FIG. 2.-Conversion of heme to bilirubin > 2.0 by spleen microsomes as a function of time. E Reaction mixture (3.0 ml) consisted of 0 1.6 10,000 X g supernatant equivalent to 6 mg E of protein, 17 /AM heme, 0.5 mAM NADP, E 1.2 - 1.3 mMI glucose-6-phosphate, 1.9 mM zo MgCl2, and 90 mM potassium phosphate < buffer (pH 7.4). In the control cuvette, £ 0.8 NADP and glucose-6-phosphate were re- placed by 0.1 M potassium phosphate buf- Z 0.4 fer, pH 7.4. / 15 30 45 60 75 90 TIME IN MIN. formed did not appear to account for all of the heme that had disappeared at the end of the incubation period (Table 3). Although heme injected into experimental animals in vivo is converted almost quantitatively to bilirubin,2 in vitro the ferriprotoporphyrin is relatively unstable and undergoes oxidative breakdown to various derivatives.36 Porra et al. demonstrated that heme is de- graded aerobically in the presence of a mitochondrial extract and thiols;37 the reaction products have not been characterized. Fractional recovery of bilirubin was much higher with splenic microsomal preparations than with the liver system (Table 3); this suggests that formation of nonbilirubin derivatives is more active with hepatic microsomes. It is unknown whether this is due simply to the higher activity of microsomal heme oxygenase in the spleen, or whether it reflects metabolic properties of hepatic microsomes which in vitro favor degradation of heme by nonbilirubin pathways. The latter explanation is attractive because of the recent observation that in rats poisoned with allylisopropylacetamide a significant fraction of the heme degraded in the liver is not converted to bili- rubin.38 39 The conversion of heme to bilirubin is localized in microsomes, requires NADPH and oxygen, and is strongly inhibited by carbon monoxide (Tables 1 and 2). These are all characteristics of microsomal mixed-function oxidases,40 but the present findings provide only indirect evidence that the reaction is cata- lyzed by an enzymatic mechanism of this type. Although the stoichiometry of the reaction can only be approximated, consumption of about 3 moles of oxygen per mole of bilirubin formed (Table 2) is consistent with the concept of a mixed- function oxidase reaction. Theoretically, 1.5 moles of oxygen should be con- sumed for each mole of bilirubin formed; i.e., 1 mole is inserted on cleavage of the protoporphyrin ring and 1 atom is utilized in the oxidation of the a- methene carbon to carbon monoxide. In a mixed-function oxidation, the additional 1.5 moles of oxygen would be used to oxidize NADPH.14 16, 40 It is apparent, how- ever, that if the conversion of heme to bilirubini is expressed as a mixed-function oxidatioti, the 4-) moles of NADPH utilized (Table 2) (ould niot provide all of the reducing equivalents necessary for this reaction. Four reducing e(luivalents are needed for the formation of bilirubin and at least an additional 3 moles of NADPH would be consumed in the production of water. It is possible that the Downloaded by guest on September 28, 2021 70a47BIOCHEMISTRY: TENHUNEN ET AL. PROC. N. A. S.

additional reducing equivalents are derived from the 105,000 X g supernatant fraction, since it was noted that 7 moles of NADPH were consumed when heme was incubated with microsomes in the absence of the 105,000 X g supernatant. Among the various tissues assayed, heme oxygenase activity per gram tissue protein is highest by far in the spleen. However, the weight of the spleen of the rat is only about '/20 that of the liver. Thus, enzyme activity per whole organ probably is highest in the liver. This is consistent with the concept that red cell hemoglobin is degraded primarily in the spleen, with the liver playing a secondary role. The only hydroxylase previously identified in the spleen is benzpyrene hydroxylase, but in comparison with other tissues such as the intestine, adrenal, and liver, its splenic activity is extremely low.'7 Since in most mixed-function oxidations the carbon monoxide-binding cytochrome P-450 is the terminal oxi- dase,'4'-6 it is of interest that the spleen now may be added to the list of tissues containing this enzyme.4' 1\Iicrosomal heme oxygenase presumably represents an enzyme system capable of catalyzing the oxidative cleavage of the heme moiety of hemoglobin and of other . However, the intact need not be a substrate for the enzyme; heme easily dissociates from native globin" as well as from altered or denatured protein. The activity of microsomal heme oxygenase in the spleen as determined in the present assay system (4 mumoles/spleen/min) is in good agreement with the kinetic requirements for hemoglobin turnover in the whole organism. In a 350-gm rat, 2.2 mjumoles of hemoglobin heme are degraded per minute; this assumes a mean erythrocyte life span of 60 days,42 a hemoglobin concentration of 140 mg per milliliter of blood, and a blood volume of 6 per cent of body weight.42 Summary.-Evidence is presented for a previously undescribed enzyme system in microsomes capable of degrading heme to bilirubin. The system has an abso- lute requirement for molecular oxygen and NADPH and is inhibited by carbon monoxide; this suggests that the reaction involves a mixed-function oxidation. The activity of the enzyme per gram tissue protein and its anatomic and sub- cellular localization indicate that it may play a major role in heme turnover in the intact animal. Abbreviations: NAD, nicotinamide-adenine dinticleotide; NADPH, reduced nicotinamide- adenine dinucleotide phosphate; ATP, adenosine 5'-triphosphate; Tris, tris(hydroxymethyl) aminomethane. Heme refers to iron protoporphyrin IX complexes collectively. The specific heme compounds employed are identified in the text. * Supported in part by USPHS grants AM-11275 and ANI-11296. t On leave from Helsinki University Central Hospital, Helsinki, Finland. 'Ostrow, J. D., J. H. Jandl, and R. Schmid, J. Clin. Invest., 41, 1628 (1962). 2 Snyder, A. L., and R. Schmid, J. Lab. Clin. Med., 65, 817 (1965). 3Lemberg, R., Rev. Pure Appl. Chem., 6, 1 (1956). 4Petryka, Z., D. C. Nicholson, and C. H. Gray, Nature, 194, 1047 (1962). 5 Fischer, H., and H. Orth, Die Chemie des Pyrrols (Leipzig: Akademische Verlagsgesell- schaft, 1937), p. 409. 6 Nakajima, H., J. Biol. Chem., 238, 3797 (1963). 7Nakajima, H., T. Takemura, 0. Nakajima, and K. Yamoaka, J. Biol. Chem., 238, 3784 (1963). 8 Levin, E. Y., Biochim. Biophys. Acta, 136, 155 (1967). 9 Murphy, R. F., C. O'hEocha, and P. O'Carra, Biochem. J., 104, 6C (1967). Downloaded by guest on September 28, 2021 VOL. 61, 1968 BIOCHEMISTRY: TENHUNEN ET AL. 755

'0Wise, C. D., and D. L. Drabkin, Federation Proc., 24, 222 (1965). "Lemberg, R., J. Barcroft, and D. Keilin, Nature, 128, 967 (1931). 12Marver, H. S., in First International Symposium on Microsomes and Drug Oxidations, in press. 13 Bunn, H. F., and J. H. Jandl, J. Biol. Chem., 243, 465 (1968). 14Conney, A. H., Pharmacol. Rev., 19, 317 (1967). 1" Cooper, D. Y., S. Levine, S. Narasimhulu, 0. Rosenthal, and R. W. Estabrook, Science, 147, 400 (1965). 16Omura, T., R. Sato, D. Y. Cooper, 0. Rosenthal, and R. W. Estabrook, Federation Proc., 24,1181 (1965). 17 Wattenberg, L. W., and J. L. Leong, Cancer Res., 25, 365 (1965). "I Alfred, L. J., and H. V. Gelboin, Science, 157, 75 (1967). 19 Brodie, B. B., J. Am. Med. Assoc., 202, 600 (1967). 20 Schmid, R., in The Metabolic Basis of Inherited Disease, ed. J. B. Stanbury, J. B. Wyn- gaarden, and D. S. Fredrickson (New York: McGraw-Hill, Inc., 1966), 2nd ed., pp. 872-874. 21 Falk, J. E., and Metalloporphyrins; Their General Physical and Coordination Chemistry, and Laboratory Methods (Amsterdam: Elsevier Publ. Co., 1964), pp. 181-182. 22Gray, C. H., A. Lichtarowicz-Kulczycka, D. C. Nicholson, and Z. Petryka, J. Chem. Soc., 2264 (1961). 23Schneider, N. C., J. Biol. Chem., 176, 259 (1948). 24Malloy, H. T., and K. A. Evelyn, J. Biol. Chem., 119, 481 (1937). 25Schmid, R., J. Biol. Chem., 229, 881 (1957). 26Tenhunen, R., Ann. Med. Exptl. Biol. Fenniae (Helsinki) 43, Suppl. 6 (1965). 27 Clark, L. C., Trans. Am. Soc. Artificial Internal Organs, 2, 41 (1956). 28Omura, T., and R. Sato, J. Biol. Chem., 239, 2370 (1964). 29 Palade, G. I., Arch. Biochem., 30, 144 (1951). 30 Chen, P. S., Jr., T. Y. Toribara, and H. Warner, Anal. Chem., 28, 1756 (1956). 31 Wharton, D. C. and A. Tzagoloff, in Methods in Enzymology, ed. R. W. Estabrook and M. E. Pullman (New York: Academic Press, Inc., 1967), vol. 10, pp. 245-246. 32Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall, J. Biol. Chem., 193, 265 (1951). 33 Lemberg, R., and J. W. Legge, Hematin Compounds and Bile Pigments (New York: Inter- science Publishers, Inc., 1949), pp. 114-123. 34Tenhunen, R., H. S. Marver, and R. Schmid, manuscript in preparation. "5 Singleton, J. W., and L. Laster, J. Biol. Chem., 240, 4780 (1965). 36Siedel, W., in Handbuch der physiologisch und pathologisch-chemischen Analyse, ed. K. Lang, E. Lehnartz, and G. Siebert (Berlin, G6ttingen, Heidelberg: Springer-Verlag, 1960), 10th ed., vol. 4, p. 920. 37 Porra, R. J., K. S. Vitols, R. F. Labbe, and N. A. Newton, Biochem. J., 104, 321 (1967). 38 Marver, H. S., L. Kaufman, and J. Manning, Federation Proc., 27, 774 (1968). 39 Callahan, E. W., Jr., S. A. Landaw, and R. Schmid, Clin. Res., 16, 280 (1968). 40 Mason, H. S., Advan. Enzymol., 19, 79 (1957). 41Marver, H. S., and J. Manning, unpublished observations. 42 Robinson, S. H., M. Tsong, B. W. Brown, and R. Schmid, J. Clin. Invest., 45, 1569 (1966). Downloaded by guest on September 28, 2021