Optimalization of preparation of apo-cytochrome b5 utilizing apo-myoglobin

Cytochrome b5 (cyt b5), a component of endoplasmic reticulum membrane, plays a role in modulation of enzymatic activity of some cytochrome P450 (CYP) enzymes. The effect of apo-cytochrome b5 on this enzymatic system has not been investigated in details, because preparation of cyt b5 as a pure protein failed in many laboratories. In order to prepare the native apo-cytochrome b5 in a large scale we utilized a protein with higher affinity toward the heme; the apo-myoglobin from the equine skeletal muscle. In the first step, we extracted heme moiety from the native myoglobin by butanone extraction. Than the effect of pH on spontaneous heme release from both proteins was investigated: purified rabbit cyt b5 as well as equine skeletal muscle myoglobin. The prepared apo-myoglobin was incubated with the cyt b5 and heme transfer was monitored as a shift of absorption maximum from 413 to 409 nm in pH varying between 3–6 (10 mM KH2PO4, pH 3–6). Here, we obtained 43 mg of the equine skeletal muscle apo-myoglobin (43% yield). The optimal pH range for heme transfer from cyt b5 into apo-myoglobin was between 4.2 and 5. Native apo-cytochrome b5 was successfully prepared using procedure described here.

b 5 (lacking the electron transferring cofactor -heme) on Sudan I oxidation is necessary to be evaluated.
The affinities of apohemoproteins for heme are very large, showing equilibrium dissociation constants in the 10 -10 -10 -15 M region (Hargrove et al., 1996;Miksanova et al., 2006). In the holoproteins, the heme prosthetic group appears to be stabilized by a large number of hydrophobic (van der Waals) and electrostatic contacts. The vinyl groups are pointing toward the protein interior and surrounded by nonpolar aliphatic and aromatic side chains, whereas the propionates point toward the solvent and interact with a variety of charged or polar amino acids. These interactions and the extremely small dissociation constants imply a high degree of specificity in the binding process (Hargrove et al., 1994). However, a variety of experimental evidence suggests that the association of heme with apoglobin is little affected by globin structure (Hargrove et al., 1996).
In the present time there are just two methods for the apohemoproteins preparation in a large scale. (i) First it is a butanone extraction method (Rossi-Fanelli et al., 1959). (ii) Second it is a expression of recombinat hemoprotein in the system without heme precursor -δ-aminolevulic acid (Miksanova et al., 2006). However, the first method requires nonphysiological conditions such as pH 2.5 and

Introduction
Cytochrome b 5 (cyt b 5 ), a component of endoplasmic reticulum membrane, is a heme protein with molecular weight of 16,800. It is composed of two functional domains, a soluble heme-containing core, and a short hydrophobic C-terminal tail, which anchors the protein into the microsomal membrane (Schenkman and Jansson, 2003). Cyt b 5 has been shown to stimulate, inhibit or have no effect on cytochrome P450 (CYP)-mediated reactions. There are two major theories explaining this effect: (i) direct electron transfer from cyt b 5 to CYP and (ii) conformational effects of cyt b 5 on CYP without contribution of electron transfer (Yamazaki et al., 2001). Recently, we found that cyt b 5 isolated from rabbit liver microsomes and reconstituted with CYP1A1 and NADPH:CYP reductase modulates the oxidation of carcinogenic azo dye Sudan I by this system (Stiborova et al., 2006).
To study the mechanism of this modulation, not only the effect of native cyt b 5 , but also that of the apo-cytochrome oRIGINAL ARTICLe Also available online on setox.eu/intertox Interdisciplinary Toxicology. 2008; Vol. 1(2): [190][191][192] Copyright © 2010 Slovak Toxicology Society SETOX for apohemoproteins preparation. Most important are the physiological conditions of the new method and the negligible amount of the residual holohemoprotein.

Preparation of the apo-myoglobin from the equine skeletal muscle
In the first step, heme moiety was extracted from the native myoglobin by butanone extraction (Rossi-Fanelli et al., 1958). pH of the myoglobin solution (100 mg of the equine skeletal muscle myoglobin in 40 ml dist. H 2 O) adjusted to pH 2.5 by 1 M HCl. The solution was moved to the separation funnel and equal volume of 2-butanone was added. Mixture was slowly shaked 3 times for 5 minutes and than placed for 10 minutes into the cold room (8 °C). Aqueous phase was dialyzed at 8 °C against 2 l of water, than 2 days against 2 l of 10 mM Tris, pH 8.0. The solution was concentrated in an Amicon stirred cell using a PM-10 membrane. In order to prevent the precipitation of the apo-myoglobin, pH was adjusted to 5 using 2 M CH 3 COOH before it was stored at -80 °C.

Protein analysis
Protein concentration was determined by the standard bicinchoninic acid protein assay (Wiechelman et al., 1988). Bovine serum albumin was used as a standard.

Spectrophotometric measurements
Spectrophotometric measurements were performed on a Hewlett Packard 8453 UV spectrophotometer with 1 ml samples. 10 µl of purified rabbit cyt b 5 or equine skeletal muscle myoglobin was diluted to 990 µl of 10 mM KH 2 PO 4 , pH 3-5. Afterwards the cyte b 5 was incubated with the apo-myoglobin in the molar ratio 1:1.5. The heme transfer in various pH was monitored as a shift of absorption maximum of heme from 413 to 409 nm. The exact pH values of individual incubation mixtures were measured after the spectrophotometric measurement.

Results
The effect of pH on spontaneous heme release from rabbit cyt b 5 and equine skeletal muscle myoglobin was investigated. Figure 1 shows the low pH induced dissociation of heme from cyt b 5 . The absorption maximum of heme shifts, during incubation at pH 3.9, from 413 nm to 382 nm indicating release of the free heme cofactor. This process was fast, cyt b 5 lost its heme completely during the first minute of incubation. On the contrary, the heme bound in molecule of cyt b 5 is stable at pH between 5.2-5.9 (absorption maximum stays at 413 nm).
Under the more acidic conditions (pH 3.6-3.8) heme cofactor readily dissociated from the molecule of myoglobin. This process was observed as a shift of heme absorption maximum from 409 to 397 nm within 30 s of the incubation (Figure 2). No changes in absorption maximum of myoglobin were detected for pH 4.4-4.9 (Figure 2).
Significant changes in UV/VIS spectra were observed during incubation of cyt b 5 with apo-myoglobin in various pH. The initial spectrum eliciting maximum at 413 nm corresponds to pure cyt b 5 . Both proteins are loosing heme spontaneously at pH 3.7, therefore the heme transfer at this pH was not observed (Figure 3), and the resulting spectrum resembles spectrum of the free hemin.
The absorption maximum of heme chromophore in the incubation mixtures (pH 4.2, 4.3 and 5) was shifted from 413 nm to 409 nm (Figure 3). This finding indicates that heme transfer from cyt b 5 to apo-myoglobin occurs. Detail spectral changes, observed in incubation mixture pH 4.2, are shown in Figure 4. The resulting spectrum resembles the holomyoglobin spectrum (maximum 409 nm) (Figure 5), this is indicating that equilibrium state was achieved. Although, there was only slight excess of myoglobin over cyt b 5 (molar  ratio 1:1.5), the heme transfer should be nearly complete ~99%, due to a significantly higher affinity of myoglobin to heme cofactor, ~2 orders of magnitude more than cyt b 5 . The heme transfer from cyt b 5 to apo-myoglobin is much slower compared to release of free hemin observed in more acidic conditions (pH 3.9 and 3.8, for cyt b 5 and myoglobin, respectively). This heme transfer is also extremely sensitive to pH. It tooks 10 minutes at pH 4.2 and 4.3, but at slightly less acidic conditions (pH 5), the transfer was completed in 30 min ( Figure 3). Besides, heme transfer from cyt b 5 to apo-myoglobin at pH 5.25 is extremely slow and required 6 hours to complete (data not shown).

Discussion
In this study we utilized the principle of the assay for hemin dissociation rate constant (Hargrove et al., 1994;1996) for the preparation of the apohemoprotein in a large scale. The method employs the fact that apomyoglobin has a very high affinity for heme when compared to others hemoproteins (Hargrove et al., 1996;Miksanova et al., 2006). For example, heme dissociation rate constant for myoglobin is 8.4 × 10 -7 /s (Hargrove et al., 1996), however for the bovine cyt b 5 , which is highly homologous (83.5% identity) to rabbit cyt b 5 used in this study, is 7.7 × 10 -5 /s (Altuve et al., 2004). Considering the fact that association of heme with apoglobin is little affected by globin structure (Hargrove et al., 1996), the heme equilibrium dissociation constant for myoglobin is at least two orders of magnitude smaller when compared to cyt b 5 , showing much higher affinity to heme.
In order to prepare the apo-cytochrome b 5 in a large scale we utilized a protein with high affinity toward the heme; the apo-myoglobin from the equine skeletal muscle. We obtained 43 mg of the equine skeletal muscle apo-myoglobin (43% yield). We investigated the effect of pH on spontaneous heme release from both proteins: purified rabbit cyt b 5 as well as equine skeletal muscle myoglobin. These realize shows that the optimal pH for heme transfer from cyt b 5 into apo-myoglobin is 4.2-5.0.  (1) spectrum of the equine skeletal muscle apo-myoglobin, (2) spectrum of hemin with maximum absorbance at ~385 nm, (3) spectrum of purified rabbit cytochrome b 5 with maximum absorbance at 413 nm, (4) spectrum of the equine skeletal muscle myoglobin with maximum absorbance at 409 nm.