A Protein of the Z Class of Liver Cytosolic Proteins in the Rat That Preferentially Binds Heme*

A low molecular weight protein purified from rat liver cytosol was observed to bind heme with an affinity higher than that for other organic anions. Purification was achieved by two procedures, one employing affinity chromatography on oleic acid-agarose, and the other using sequential ion-exchange and gel filtration chromatography after initial removal of aprotinin-sen-sitive proteases. Removal rather than inhibition of proteases improved the yield four times. Both procedures produced a stable protein. The purified protein binds heme with a higher affinity (Kd 0.15 p ~ ) than any other organic anion tested including other (metallo)porphyrins, bilirubin, and oleic acid. Based on its molecular weight, amino acid composition, immunological properties, and the in- crease of its tissue levels in response to the administration of hypolipidemic agents, the protein was identified as being related to proteins of the Z class, whose mem- bers include fatty acid binding protein and sterol carrier protein. Like other Z proteins, our protein exhibits several forms on electrofocusing, but differs from fatty acid-binding protein and sterol carrier protein in that its major form exhibits a PI of 7.4. In view of its distinct isoelectric focusing pattern, its higher affinity for heme than for oleic acid, and its apparent inability to bind cholesterol and steroids, we cannot identify this protein as any of the above-mentioned proteins of the Z class. Consequently we have provisionally desig- nated it

A low molecular weight protein purified from rat liver cytosol was observed to bind heme with an affinity higher than that for other organic anions. Purification was achieved by two procedures, one employing affinity chromatography on oleic acid-agarose, and the other using sequential ion-exchange and gel filtration chromatography after initial removal of aprotinin-sensitive proteases. Removal rather than inhibition of proteases improved the yield four times. Both procedures produced a stable protein.
The purified protein binds heme with a higher affinity (Kd 0.15 p~) than any other organic anion tested including other (metallo)porphyrins, bilirubin, and oleic acid. Based on its molecular weight, amino acid composition, immunological properties, and the increase of its tissue levels in response to the administration of hypolipidemic agents, the protein was identified as being related to proteins of the Z class, whose members include fatty acid binding protein and sterol carrier protein. Like other Z proteins, our protein exhibits several forms on electrofocusing, but differs from fatty acid-binding protein and sterol carrier protein in that its major form exhibits a PI of 7.4. In view of its distinct isoelectric focusing pattern, its higher affinity for heme than for oleic acid, and its apparent inability to bind cholesterol and steroids, we cannot identify this protein as any of the above-mentioned proteins of the Z class. Consequently we have provisionally designated it heme-binding protein.
The mechanism(s) of intracellular heme distribution are not yet defined (1). One possible mechanism involves the participation of proteins in the transport of heme from the mitochondria, the site of completion of heme synthesis, to other parts of the cell. In order to establish that proteins are involved in this process, in vivo experiments were conducted in which two subfractions of cytosol were shown to exhibit a particularly high heme turnover (2). As this finding was consistent with the participation of proteins in heme transport, we endeavored to purify proteins from cytosol that could be identified as heme carriers.
We now report on the purification in high yield of a stable low molecular weight, heme-binding protein from rat liver cytosol. This protein also binds, with a lower affinity, other porphyrins and metalloporphyrins as well as bilirubin and a variety of additional organic anions. Its molecular weight, In one experiment male rats were fed chow containing 0.25% clofibrate for 2 weeks prior to killing. Livers were perfused with ice-cold pH 7.5, containing 1 mM EGTA, 1 mM EDTA, and M PMSF normal saline and homogenized in 3 volumes of 20 mM Tris buffer, (buffer A). The homogenate was centrifuged at 100,000 X g for 90 min, and the supernatant was aspirated carefully to avoid contamination with the fat layer.
Purification of the Heme-binding Protein Four-step Procedure-Aprotinin-sensitive proteases were removed from the liver cytosol of 5 rats by affinity chromatography on 50 ml of aprotinin-agarose preequilibrated with buffer A. The remaining proteins (cytosol minus proteases) were subjected to anion exchange chromatography (batch procedure) on 300 ml of DEAE-cellulose in 10 mM Tris, pH 8.0, 0.5 mM EGTA, and M PMSF (buffer B). Proteins that did not bind to DEAE-cellulose were concentrated to 80 ml by ultrafiltration (Amicon PM-10) and dialyzed overnight against two changes of 20 mM Na phosphate buffer, pH 6.0, 20% glycerol, 0.5 mM EGTA and 10" M PMSF (buffer C). The dialysate was applied to a column of 200 ml (3 X 29 cm) of SPLSephadex preequilibrated with buffer C. The unbound fraction, which contained HBP, was collected by washing the column with buffer C and concentrated to 2 ml in a Micro-ProDiCon while dialyzing against 10 The abbreviations used are: FABP, fatty acid-binding protein; HBP, heme-binding protein; EGTA, ethylene glycol bis(/3-aminoethyl ether)-N,N,N',N'-tetraacetic acid; PMSF, phenylmethylsulfonyl fluoride; ANS, 8-anilino-1-naphthalenesulfonic acid; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; SCP, sterol carrier protein. of Rat Liver Cytosol mM Tris, pH 7.4, 0.1 M NaCl, 0.5 mM EGTA, and lo-' M PMSF (buffer D). The proteins were subjected to gel filtration on 180 ml of Sephadex G-75 (2 X 60 cm). The second peak obtained from this column corresponded to pure HBP. The purification of HBP was monitored by subjecting all protein fractions, including liver cytosol, to SDS-PAGE and by determining spectrophotometrically the hemebinding capacity of each fraction. The apparent molecular weight of HBP was estimated by comparing its relative mobility on SDS-PAGE and its retention time on Sephadex G-75 column with values obtained for molecular weight standards.
One-step Procedure-Oleic acid was complexed to 10 ml of aminoethylamino-agarose according to the method of Peters et al. (13).
The binding capacity of the affinity gel was determined to be about 150 mg of bovine serum albumin, i.e. 0.2 pmollml. A small volume (-10 ml) of the 100,000 X g supernatant of rat liver homogenate was mixed with oleic acid-agarose previously equilibrated in PBS. (In one experiment the cytosol was first delipidated by incubating with hydroxyalkoxypropyl dextran at 37 "C for 1 h as described by Glatz et al. (14).) Unbound proteins were removed by washing with PBS and bound proteins were eluted with 100 ml each of 5, 10, 25, and 50% ethanol in 75 mM Na phosphate, pH 6.0, and then with 100 ml of 50% ethanol in 75 mM Na phosphate, pH 2.4. The pH of the buffer was chosen based on observations by others (10, 13) and results of preliminary experiments of ours in which the gel was eluted with 50% ethanol in Na phosphate buffer of three different pH values, 2.4,6.0, and 7.0. The ethanol content of the pH 6.0 buffer (found to be more effective than the other buffers) was then increased from 5 to 50% in 5% increments. All protein fractions were concentrated in a Micro-ProDiCon while dialyzing against PBS and subjected to SDS-PAGE and isoelectrofocusing.

Electrophoresis and Isoelectrofocusing
SDS-PAGE was performed in vertical slab gels according to the method of Laemmli (15) using a 14% developing gel. In separate experiments, urea (8 M) and NaCl (1 M) were included. in the SDS-PAG to test their effect on the appearance of the HBP single band. Analytical electrofocusing was performed using LKB Ampholine polyacrylamide gel plates of pH range 3.5-9.5. A mixture of PI makers was applied and a standard curve constructed by plotting the distance of each PI marker band from the cathode uersus its PI value; this curve was then used to estimate the PI value of the three main HBP bands. Preparative electrofocusing was carried out on a thin layer of Ultrodex prepared in a solution containing LKB ampholytes, pH 3.5-10, and 5 mg of purified HBP. Electrofocusing was performed at 8 watts for 17 h at 10 "C. The gel was divided into 20 fractions, each of which was eluted with 10-20 ml of distilled water and concentrated to 0.5 ml in a Micro-ProDiCon while dialyzing against Na phosphate buffer, pH 7.4, with 20% glycerol. Protein was detected in fractions 5 (pH 5.5), 8 (pH 6.1), 9 (pH 6.3), 11 (pH 7.1), 12 (pH 7.5), and 13 (pH 7.7), all of which were subjected to analytical electrofocusing. Fractions 11,12 and 13 were mixed (about 1 mg of total protein) and were subjected to a second preparative electrofocusing run. Protein was detected in fractions 8 and 12, and these were analyzed by electrofocusing.

Protein Determination
During purification, protein was detected by measuring the absorbance at 280 nm and was quantitated by the method of Bradford (16) and/or Lowry et al. (17) using bovine serum albumin as a standard. Both of these methods overestimated the actual aminoacyl mass of purified HBP, as determined from the amino acid analysis, by a factor of 1.6. All stated values for purified protein (but not for impure fractions) are corrected by this factor.

Immunochemical Studies
antibody to purified HBP in rabbits. The antigen, in solution or in Several different immunization methods were used for raising slices of polyacrylamide gel obtained after SDS-PAGE, was mixed with complete Freund's adjuvant and injected in multiple intradermal sites. Some of the rabbits were sensitized with lipopolysaccharide prior to injection of antigen. All of these methods yielded weak, nonprecipitating antibodies. A stronger, yet not precipitating antibody as judged by Ouchterlony double imunodiffusion and rocket immunoelectrophoresis, was obtained by injecting the antigen mixed with complete Freund's adjuvant intraperitoneally.
The presence of antibody in the serum was determined by solid-phase radioimmunoassay. Purified antigen (1 pg in 100 pl PBS) was coated to polpinyl chloride 96-well plates (4 "C overnight), and excess binding sites were blocked with a 3% bovine serum albumin solution in PBS (37 "C, 1 h). The wells were then incubated (37 "C, 1 h), first with duplicates of five different dilutions (1:lOO to 1:10,000) of the antiserum or preimmune serum and then with goat anti-rabbit '9-labeled IgG (200,000 cpm/well). Nonspecific binding to the wells was minimized by washing with PBS containing 0.2% Tween-20 after each incubation. The wells were dried, excised, and counted in a ycounter. Maximum binding to the antigen was obtained with a 1:1,000 dilution of the antiserum. Using this dilution of the antiserum a standard curve was constructed by varying the HBP concentration. The lower and upper limits of detection of the assay were 3 and ,100 ng of HBP in a 100-pl sample. The standard curve was then used to estimate the HBP concentration in liver cytosol.

Western Blots
Purified HBP (15 pg/lane) was subjected to analytical isoelectrofocusing and the various protein bands were transferred electrophoretically (100 V for 1 h) from the gel onto nitrocellulose paper. Following transfer the nitrocellulose paper was immersed for 1 h in 0.2% gelatin solution in PBS ( t o block remaining binding sites on the paper) and then incubated first with a 1:lOO dilution of the rabbit antiserum and subsequently with goat anti-rabbit lZ5I-labeled IgG fraction. The effect of heme on the binding of the antibody was examined by incubating the transferred protein with 0.14 mM heme solution prior to antibody incubation. Nonspecific binding was minimized by washing thoroughly with PBS containing 0.2% Tween-20. The nitrocellulose paper was air-dried and subjected to autoradiography.

Amino Acid Analysis
Samples of purified HBP were hydrolysed in duplicates in 6 M hydrochloric acid for 24 and 48 h. Amino acid analysis, except for tryptophan, was carried out by a single-column ion-exchange procedure (18).

Circular Dichroism Studies
Measurements were made with a Cary 60 CD spectrometer using a 0.1-cm cell. HBP solutions were prepared in 0.1 M Tris, pH 8.0, and spectra were recorded after addition of heme and bilirubin at a final concentration equal to that of HBP.

Binding Studies
Fluorescence spectroscopy was used to determine the interaction of purified HBP with heme, protoporphyrin IX and its zinc and copper derivatives, deuteroporphyrin I X and its zinc derivative, hematoporphyrin, uroporphyrin, coproporphyrin, bilirubin, ANS, oleic acid, clofibrate, retinol, retinal, retinoic acid, progesterone, cholesterol, @-estradiol, corticosterone, and taurocholate. This method was chosen over equilibrium dialysis because of the tendency of heme and porphyrins to bind to membranes and because the long periods of time required for equilibration through the dialysis membrane would lead to ligand and possibly protein degradation (19). An attempt to determine the equilibrium binding constant of oleic acid by the method of Colowick and Womack (20) was not successful. At least 25% of the radiolabeled oleic acid bound nonspecifically to the membrane used to separate free oleic acid from that bound to HBP.
Protein fluorescence spectra were recorded on a Perkin-Elmer MPF-44A fluorometer; the excitation wavelength was 278 nm and the emission spectrum exhibited a maximum at 305 nm, typical of tyrosine-containing class A proteins. Unlike tryptophan fluorescence, the fluorescence of tyrosine in class A proteins is not affected by changes in the protein conformation (21), and calculations of equilibrium binding constants are therefore not complicated by conformational changes. Binding was determined by adding small volumes (5-10 ml) of ligand stock solutions to 3.0 ml of HBP solution in 50 mM Na phosphate, pH 7.4, until quenching of the protein fluorescence was complete. Heme binding was also determined by difference absorption spectroscopy by adding aliquots of heme solutions to 1.0 ml of HBP solution in the sample cuvette and to 1.0 ml of buffer solution in the reference cuvette. Heme additions were made until no further increase in absorption at 405 nm was observed. Fluorescence and difference absorption spectra were recorded within 5 min after ligand addition. That equilibrium was reached was confirmed by showing that the spectra recorded 5,15, and 30 min after ligand addition were identical. Heme, bilirubin, and porphyrin stock solutions were freshly prepared and protected from light; a few grains of the solid compound were dissolved in 2 drops of 0.1 N NaOH and then diluted with 50 mM Na phosphate, pH 7.4. Concentrations of stock solutions were determined spectrophotometrically using published values for molar absorptivities (22-24). Stock solutions of all other compounds were made in 95% ethanol and their concentrations determined gravimetrically. Addition of small volumes of ethanol had no effect on protein fluorescence.
The number of moles of ligand bound per mole of protein, n. was calculated from the total ligand concentration minus the concentration of bound ligand (d X protein concentration). Data points for which the fraction of bound to total ligand was significantly less than 1 were used in the calculation of the dissociation constants. Values of equilibrium dissociation constants (&) obtained from Scatchard plots were refined by nonlinear regression analysis.
Competitive binding was determined in the presence of excess ANS (ANS:protein molar ratio about 50) by measuring the decrease in the fluorescence of the HRP-ANS complex at 460 nm after each addition of ligand solution (the excitation wavelength was 390 nm). Competition with heme for the HBP binding site was also studied spectrophotometrically by measuring the decrease in the absorption of the heme-HBP complex a t 405 nm after addition of the competing ligand. Data were analyzed by the method of Steinhardt and Reynolds (28) as described by Ketterer et al. (4). Using linear regression analysis the data were fitted to the equation Kz = (dZKtLf)/d&, where suhscript 1 refers to the ligand in excess and subscript 2 to the competing ligand. HBP-In preliminary studies concerned with the identification and purification of heme-binding proteins from rat liver cytosol, we used affinity chromatography on heme-agarose (29) and heme-sepharose (30). Several proteins were found to bind to these gels but in small amounts, making it difficult to purify any particular protein. This approach was therefore not pursued further. Instead, a fourstep procedure, outlined in Table I, was developed for the purification of one of these heme-binding proteins, referred to here as HBP. Ita purification was followed by measuring the heme-binding ability of the various protein fractions using difference absorption spectroscopy.

Purification of
As a first step, aprotinin-sensitive proteases were removed by affinity chromatography producing a 4-fold increase in H B P yield over the yield obtained when proteases were inhibited by the addition of aprotinin, PMSF and the Ca2+ chelators EDTA and EGTA. After removal of proteases the cytosol TARLE I Four-step procedure for the purification of rat liver HRP HBP was purified from the hepatic cytosol (100,OOO X supernatant of perfused liver homogenate) from 5 animals each of three groups: male, clofibrate-induced male, and female rats. The table lisu the protein content of the cytosol and of the HRP-containing fractions a t each of the four stages of purification. The last line gives the amount of pure HBP obtained by gel filtration on Sephadex (3-75, corrected by a factor of 1.6 as described under "Experimental P m edures." Comparison of columns 1 and 2 shows the 4-fold increase in HBP yield produced by removing proteases initially from the cytosol on aprotinin-agarose rather than inhihiting them with the addition of aprotinin to the homogenization buffer.  (15) wan followed. using a 14% developing gel. Lanes 2. 3, and 4 contain liver c-ytosol from female, clofibrate-induced male, and male rats. respectively. L a w s 5-8 contain HBP purified by the four-step procedure shown in Tahle I. h n e n 5 and 6 contain 4 and 10 pg. respectively, of HRP from the liver cytosol of male rats. Lanes 7 and H contain 10 pg of HRP purified hy the four-step procedure from liver cytosol of clofihratr-induced male and uninduced female rats. Law 9 contains 10 pg of HRP purified by the one-step procedure shown in Tahle  was subjected to ion-exchange chromatography firut on DEAE-cellulose and then on SP-Sephadex. Since only 5% of HBP bound to DEAE-cellulose, while no sipificant binding to SP-Sephadex was detected, batch rather than column chromatography could be used.
A SP-Sephadex column was used in the third step so as to elute differentially the fractions containing heme-binding proteins other than HBP. The purity of HRP obtained from the last step (gel filtration) was determined by SDS-PAGE. As seen in Fig. 1, HRP purified from the liver cytosol of male and female rats exhibited a single band with a M, of about 14,000. A single band was obtained when urea and NaCl were added to the SDS-containing gel (data not shown). The HRP purified from liver cytosol of clofibrate-induced male raM showed traces of a high molecular weight impurity, suggesting of Rat Liver Cytosol that 50 mg is probably the maximum amount of HBP that can be purified using the conditions specified in this paper for ion-exchange and filtration chromatography. Also, it was calculated that the per cent recovery of HRP from the cytosol of male rats decreased from 60 to 38% when the rats were induced with clofibrate and that the recovery from cytosol of female rats was 35%, even though the actual amount of HBP was greater in both cases. The lower recovery is probably due to the higher cytosolic content of HBP in livers of female and induced male rats, estimated by radioimmunoassay a t 5.3 and 5.0 as opposed to 2.5% (in uninduced male rats) mg of HBP/ mg of total protein.
After HBP was purified and found to bind oleic acid, a onestep purification procedure was developed using oleic acidagarose. Proteins bound to this gel were eluted with different amounts of ethanol in the Na phosphate buffers. Table I1 shows the results of a representative experiment in which cytosol from clofibrate-induced male rats, containing approximately 1 pmol of HBP as determined by radioimmunoassay, was used. Although the total binding capacity of the gel was 2 pmol, only about 0.3 pmol of HBP was obtained after stepwise elution and concentration/dialysis of the eluates. Delipidation of cytosol on hydroxyalkoxypropyl dextran did not increase the yield. Still, the 30% yield obtained is comparable to the yield of the four-step purification procedure. The total amount of protein purified by affinity chromatography was of course much less than that purified by the four-step procedure, the small volume of the affinity gel limiting its proteinbinding capacity.
Pure HBP was eluted from the affinity gel with 10-25% ethanol in Na phosphate buffer at pH 6.0. The SDS-PAGE of the 25% eluate is shown in Fig. 1, lane 9. Higher-pH buffers were not as effective in HBP purification, whereas lower pH values resulted in minor degradation of the protein. A few fatty acid-binding proteins other than HBP were eluted with 5% ethanol, and additional proteins were eluted by increasing the ethanol content of the buffer to 50% and also by lowering the pH to 2.4, indicating that most of these proteins have a higher affinity than HBP for oleic acid. Judging by the total amount of protein eluted from oleic acid-agarose, these higher molecular weight proteins should constitute more than 30% of the total cytosolic fatty acid-binding proteins. Ockner et al.
(10) estimated that up to 40% of cytosolic fatty acids are bound to proteins other than FABP. All of the proteins with lower affinity and some of those with higher affinity than HBP for oleic acid were eliminated when cytosol was treated with aprotinin-agarose prior to oleic acid-agarose incubation (data not shown).

One-step procedure for t h e purification of rat liver HRP
The 100,000 X g supernatant from the homogenate of perfused rat liver of a clofibrate-induced rat was incubated with 10 ml of oleic acid-agarose a t 4 "C for 1 h. Round proteins were eluted with different amounts of ethanol in Na phosphate buffer. Samples were dialyzed against PRS and concentrated. The results of a representative experiment are given. Isoelectrofocusing-Purified HBP appeared homogeneous on SDS-polyacrylamide gel electrophoresis but was shown to be heterogeneous on electrofocusing. As shown in Fig. 2 it exhibited several bands focused in three clusters with PI 7.4 (major component), 6.2, and 5.7. Protein purified from the cytosol of uninduced and clofibrate-induced male rats exhibited the same pattern, indicating that clofibrate induces the synthesis of each isomer equally. The PI 7.9 band present in the protein from clofibrate-induced male rats is probably identical to the high molecular weight impurity detectable on SDS-PAGE. Protein purified from the cytosol of female rats exhibited a somewhat different pattern in that the second band was centered around pH 6.4 rather than pH 6.2 as in the male.
HBP eluted from oleic acid-agarose with 10% ethanol appeared to consist mostly of the PI 6.2 isomer (Fig. 2, lane 8) indicating that this isomer may have a lower affinity than the others for oleic acid. HBP eluted with 25% ethanol exhibited the same electrofocusing pattern as HBP purified by anion exchange and gel filtration chromatography.
HBP was subjected to preparative electrofocusing on Ultrodex, and three fractions corresponding to the three isomeric clusters were eluted from the gel and subjected to analytical electrofocusing (not shown). Rands with PI values 7.4 and 6.2 redistributed to the original three bands, whereas the band with PI 5.7 focused into two bands with PI 6.2 and 5.7. The major band (PI 7.4) was subjected to a second preparative electrofocusing, and two protein-containing fractions were eluted. When subjected to analytical electrofocusing the fraction eluted from nearest the anode exhibited one major band with PI 6.2, while the more basic fraction redistributed to the three original bands. Protein A (4) and a protein homologus with protein Z (31) have also been reported to exhibit similar redistribution phenomena upon repeated electrofocusing.

Interaction of HBP with Heme and Other OrRank Anion.9-
Purified HBP was tested for binding of heme, heme precur- sors, and other porphyrins and metalloporphyrins as well as bilirubin, the heme degradation product. In order to compare the binding properties of HBP with those of homologous proteins described in the literature, several organic anions such as oleic acid, progesterone, @-estradiol and cholesterol, retinoids, andkclofibrate were tested. Binding was measured by difference absorption spectroscopy (the HBP-heme complex had a maximum at 405 nm) and by quenching of the protein fluorescence at 305 nm. Organic anions which did not quench the fluorescence of the protein, such as oleic acid, progesterone, @-estradiol, cholesterol, taurocholate and clofibrate were tested for their ability to displace ANS from HBP and hence to quench the fluorescence of the HBP-ANS complex, which had an emission maximum at about 460 nm. Oleic acid binding was confirmed by incubating the protein with [14C]oleic acid and removing unbound oleic acid on hydroxyalkoxypropyl dextran as described by Glatz et al. (14) (data not shown). Absorbance and fluorescence titration data were analyzed to obtain the number of moles of heme and other porphyrins bound per mole of HBP. This was accomplished by plotting relative intensity of fluorescence or absorbance against the ligandHBP molar ratio as shown for heme and mesoheme in Fig. 3. The titration curves are consistent with a 1:l protein-to-ligand interaction. Dissociation constants were obtained using the data from the curved portion of the graphs; Scatchard plots of these data yielded straight lines consistent with the presence of only one binding site. Representative Scatchard plots for the interaction of heme, mesoheme, and bilirubin are shown in Fig. 4. Dissociation constants for these, other metalloporphyrins and naturally occurring porphyrins as well as ANS, oleic acid, and retinoids are listed in Table 111.  Absorbance (AIA-) where A , is the final absorbance at the end of the titration. Fluorescence (F) was monitored at 305 nm, the emission maximum of the tyrosine-containing HBP excited at 278 nm, and it was expressed as Relative Fluorescence, FIF,, F O representing the protein fluorescence before ligand addition. Hemelprotein is the ratio of the molar concentration of total heme (or mesoheme), which was varied, to total protein, which was kept constant within each titration but varied between titrations. Absorption data: 13, heme, 0-15.1 FM, HBP 3.55

TABLE I11 Equilibrium dissociation constants (Kd) of HBP complexes with heme, other porphyrins, and organic anions
Binding was measured as outlined in the legends to Figs. 3,4, and 5. Equilibrium dissociation constants were obtained by nonlinear regression analysis of the data from at least two fluorimetric or absorptimetric (heme only) titrations. All data could be fitted to a one-binding-site model except for the hematoporphyrin data, which could not be analyzed. The constants in parentheses were obtained by competitive binding in the presence of excess heme (absorption) or ANS (fluorescence) and were calculated as explained under "Experimental Procedures." The purified protein had an %fold higher affinity for heme than for oleic acid, the fatty acid for which FABP has supposedly the highest affinity (4,6). This was determined fluorometrically by competition studies between ANS and oleic acid, as shown in Fig. 5, and also by difference absorption spectroscopy where a 30-fold excess of oleic acid was required to displace bound heme. Still, our binding constant for oleic acid is higher than that reported by Ketterer  Clofibrate, a hypolipidemic drug which increases the levels of HBP, did not quench protein fluorescence nor did it displace bound ANS. Other chemicals failed to quench the fluorescence of the protein-ANS complex as follows (the highest ligandprotein molar ratio used is given in parentheses): cholesterol (601), progesterone (35:1), @-estradiol (30:1), corticosterone (33:1), taurocholate (37:l). Cholesterol and @-estradiol also did not displace bound heme when used in 50 and 100 X molar excess. (Their limited aqueous solubility prevented testing with excesses larger than 100 x.) Circular Dichroism Studies-The CD spectra of the complexes of HBP with heme and bilirubin are shown in Fig. 6. Heme bound to HBP generated a positive band at 419 nm and a slightly smaller negative band at 386 nm. The spectrum of the HBP-bilirubin complex is similar to that of the bilirubin complex with protein Z (32) and protein A (33), exhibiting a minimum at 415 nm and a maximum at 465 nm. The general pattern of the CD spectra of these HBP complexes is similar to that exhibited by albumin-bilirubin complexes, which has been interpreted in terms of excitation splitting of the ligand chromophores (34).
Amino Acid Composition and Some Immunochemical and Physicochemical Properties of HBP-The amino acid composition of HBP is very similar to that of aminoazodye-binding protein A (41, protein Z (7), FABP (10) and SCP (121, all of which lack tryptophan. Using a solid-phase radioimmunoassay and Ouchterlony immunodiffusion it was determined that HBP cross-reacts with protein A and FABP. Moreover, the antibody raised against HBP was shown, by a combination of electrofocusing and Western blotting techniques, to recognize all protein isomers. Incubation with heme seemed to increase binding of the antibody to all protein isomers, indicating that heme binds equally well to all isomers. The purified protein is very stable and can be stored in dilute solution at 4 "C for several days and at -70 "C for at least 1 year without any sign of degradation, as determined by SDS-gel electrophoresis and isoelectric focusing as well as evaluation of its ligand and antibody-binding properties.

DISCUSSION
A low molecular weight protein was purified from rat liver cytosol, characterized, and identified to be a distinct member of the Z class of proteins first described by Ketterer and coworkers (3, 4) and Arias and co-workers (5-7). Included in this group are aminoazodye-binding protein A (3, 4), protein Z (5-7), FABP (8-lo), and SCP (11, 12), all of which are present in liver in significant amounts and have been reported to occur also in several other tissues (6, 8, 35, 36). These proteins share antigenic determinants and a similar amino acid composition. The identity and origin of the extrahepatic FABPs has been investigated by Gordon et al. (36), who found mRNA corresponding to hepatic FABP only in liver, small intestine, and colon. Tissues such as heart, lung, and adrenals contained mRNA corresponding to another form most abundant in intestines but present only in trace amounts in liver. Thus, the tissue expression of hepatic FABP and intestinal FABP is differently regulated, and they may have distinct ligand affinities and physiological functions.
The heme-binding protein we have purified and provisionally designated HBP has several characteristics making it unique within the Z class of proteins. HBP does not bind cholesterol or steroids, setting it apart from SCP (11,12), but like FABP it does bind oleic acid although less strongly than it binds heme. The method of fluorescence quenching used in this study does not exclude the possibility of a separate binding site for cholesterol and steroids. Using competitive binding studies, Ketterer et al. (4) detected by equilibrium dialysis a binding site on protein A which had a high affinity for fatty acyl-CoA but did not bind free fatty acids. The binding of fatty acyl-CoA esters to protein Z has also been reported (37). In view of the low affinity of HBP and other proteins of the Z class (4,6) for free fatty acids at a site where heme and other anions are bound, it is reasonable to conclude that it is their affinity for fatty acyl-CoA esters that is responsible for their suggested role in fatty acid metabolism (37-40). To accommodate the proposed role for this protein in cholesterol and steroid metabolism (11,41-43) a third, high steroid affinity binding site would have to be postulated.
An alternative to the presence of three different binding sites on such a small molecule would be to assume that there are multiple forms of this protein. Thus, the preparations obtained by other investigators and ourselves could consist of multiple forms in differing amounts depending on the purification method. This would explain the observed differences in isoelectric point value(s) and binding properties. Although multiple forms of protein A (4) and protein Z (7) have already been isolated, it is generally believed that they differ mostly in the types and amount of endogenous fatty acids bound to them (10, 31). Since purified proteins A and Z have a low affinity for free fatty acids these endogenous acids would have to be bound in the form of their CoA esters in order to remain bound to the protein. The fact that HBP eluted from oleic acid-agarose with 25% ethanol exhibited the same pattern on electrofocusing as HBP purified by ion-exchange chromatography also argues against the presence of endogenous free fatty acids.
The preferential affinity of HBP for heme may mean that it functions in intracellular heme distribution (43, 44), although binding and transport function cannot be equated. An observation in support of HBP being involved in heme transport is that higher levels of both FABP and HBP in female and clofibrate-treated male rats can be correlated with peroxisomal proliferation and an associated increase in catalase levels. Catalase is known to be assembled inside the peroxisomes from apoprotein and heme (45). Elevated sex steroid levels and colfibrate administration could therefore produce an increased demand for the transport of heme into peroxisomes and cause levels of heme carrier(s) to increase. Why certain hypolipidemic drugs do and others do not induce catalase is unknown. Benzafibrate, for example, induces the activity of the enzyme system catalyzing fatty acid oxidation but, unlike clofibrate, causes no proliferation of the organelles and does not affect catalase levels (46). Probucol and niceritrol, which have been observed not to increase FABP levels (47), may have mechanism(s) of action similar to that of benzafibrate. In this context, it is of interest. to note that rat liver cytosol contains a protein which binds nafenopin and clofibrate (48), whereas we found that HBP does not bind clofibrate.
Reported equilibrium constants for the binding of heme and oleic acid to aminoazodye-binding protein A (4, 44) and protein Z (6) are lower than those of HBP. It was also reported that partially purified FABP (10) and aminoazodye-binding protein A (4) bound fatty acids with higher affinity than the purified protein, and it was suggested that this was due to residual ampholytes bound to the protein (10) or to conformational changes produced during its purification by electrofocusing (4). We have found that most of the liver cytosolic proteins that bind free fatty acids do so with a higher affinity than HBP. It is possible, therefore, that the decrease in fatty acid affinity observed upon purification of protein A and FABP is due to the removal of these contaminating proteins rather than to the presence of residual ampholytes. On the other hand, methods for the purification of protein A, protein Z, and FABP included as the last step electrofocusing, which could cause irreversible conformational or other changes in the protein molecules reflected in their lower affinity than HBP for all ligands. Stability problems have been reported for protein Z (7). We have found that HBP is very stable and that its affinity is retained at 4 "C for 1 week, after storage at -70 "C for 1 year and after 2-3 freezelthaw cycles. A slight tendency to precipitate was observed in the absence of added glycerol but only at low pH (56.0) and not at all pH values as reported for protein Z purified by ion-exchange chromatography (7).
Dempsey et al. (12) suggested that the acidic forms of protein A and Z and FABP observed on electrofocusing may represent degraded forms. We found, however, that HBP, which is very stable, exhibits two acidic bands and, when left at room temperature for 1 day, its electrofocusing pattern does not change. Also HBP eluted from oleic acid-agarose with 10% ethanol exhibited predominantly an acidic band, whereas the major band of HBP eluted with 25% ethanol was slightly basic. Alternatively, the increased stability of our purified protein could be due to the removal of cytosolic proteases as a first step in our four-step purification procedure and to the minimum handling it undergoes in our one-step affinity chromatographic method. Both are simple, high-yield methods, and should make it possible to purify and systematically characterize these small molecular weight proteins from liver and other tissues.