Bioconjugate Chem. 2006, 17, 236−240
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Encapsulation of Myoglobin with a Mesoporous Silicate Results in New Capabilities Tetsuji Itoh,*,† Ryo Ishii,† Takeo Ebina,† Takaaki Hanaoka,† Yoshiaki Fukushima,‡ and Fujio Mizukami† National Institute of Advanced Industrial Science and Technology (AIST), Nigatake 4-2-1, Miyagino-ku, Sendai, 983-8551, Japan, and Toyota Central R & D Labs. Inc., Yokomichi, Nagakute, Aichi 480-1192, Japan. Received August 5, 2005; Revised Manuscript Received October 4, 2005
A metmyoglobin (Fe3+), an oxidized form of myoglobin (Fe2+), was confined in nanospaces of about 4 nm in diameter in mesoporous silica (FSM; folded-sheet mesoporous material), forming a metmyoglobin (Fe3+)-FSM nanoconjugate. The spectral characteristics of metmyoglobin (Fe3+)- and myoglobin (Fe2+)-FSM show an absorption curve quite similar to that of native metmyoglobin, indicating that myoglobin retains its higher-order structure in the pores of FSM. The metmyoglobin (Fe3+)-FSM conjugate had not only a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and 2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfomic acid (ABTS) or guaiacol (a hydrogen donor) but also an advanced molecular recognition ability enabling it to distinguish between ABTS and guaiacol. Furthermore, the metmyoglobin (Fe3+)-FSM showed the peroxidaselike activity even in an organic media using benzoyl peroxide as the hydrogen acceptor and leucocrystal violet as the hydrogen donor. The simple immobilization of metmyoglobin (Fe3+) into FSM results in enhanced catalytic activity in organic media compared to that of native metmyoglobin (Fe3+).
INTRODUCTION For vital biological functions to occur, it is necessary that the components of which the organs consist are arranged on a nanoscale and assume highly ordered or, in other words, particular higher-order structures. In the biomimetic systems developed recently, different nanoparts, for instance, heme for the selective adsorption-desorption of O2, CO, or NO (1, 2), have been recognized and employed. However, we lack the means to organize nanoparts such as proteins, which, in the case of natural hemoglobin or myoglobin, is essential for their activation and enhancement. Myoglobin (Fe2+), which is a constituent of muscle that binds oxygen reversibly, is a single polypeptide (153 amino acids) protein (globin protein) of 18000 Da with one protoheme prosthetic group (3, 4). Its oxygenated form is called oxymyoglobin, MbO2, while it is known as carboxymyoglobin, MbCO, when the oxygen is displaced by carbon monoxide. The myoglobin (Fe2+) molecule has a compact spheroidal structure of 2.8 by 3.2 by 4.5 nm (5). Intercooperation between the heme and globin protein is likely to be important for it to achieve its functions. On the other hand, when the iron in myoglobin is oxidized from the ferrous (Fe2+) to the ferric (Fe3+), the compound is called metmyoglobin (Fe3+) and assumes a catalytic ability instead of losing its oxygenbinding capability, as well as a peroxidase-like activity in aqueous solution, as has been reported so far (6-8). Much attention has been devoted to mesoporous materials such as MCM (mobile crystalline material) (9) and FSM (foldedsheet mesoporous material) (10) having honeycomb (hexagonal) structures with ordered cylindrical channels of 2-10 nm in diameter, larger than the microporous cavities of conventional zeolites (0.6-1.2 nm). Enzymes, horseradish peroxidase (11, 12), cytochrome c (13, 14), or lysozyme (15), have been * Corresponding author. Phone: (+81)22-237-3097, FAX: (+81)22-237-5226, E-mail:
[email protected]. † National Institute of Advanced Industrial Science and Technology (AIST). ‡ Toyota Central R & D Labs. Inc.
introduced into mesoporous silica materials. We have also reported the incorporation of a large amount of chlorophyll a molecules into a FSM (16-18). The incorporation seems to increase the stability of the incorporated molecules, and such host-guest reactions should be useful for the arrangement and accumulation of the molecules and enzymes. The present paper deals with the preparation and characterization of FSM conjugated with myoglobin and the catalytic activity and chemical stability of myoglobin encaged in the nanopores.
EXPERIMENTAL SECTION General. Myoglobin from horse heart, 2,2-azino-bis(3ethylbenzothiazoline)-6-sulfomic acid (ABTS), and ο-methoxyphenol (guaiacol) were purchased from Wako Pure Chemical Industries, Ltd (Osaka, Japan). Docosyltrimethylammonium chloride [C22H45N(CH3)3Cl] was kindly donated by Lion Corporation (Tokyo, Japan). Kanemite (layered polysilicate) was kindly donated by Tokuyama Siltech Co., Ltd (Yamaguchi, Japan). Leucocrystal violet was obtained from Tokyo Kasei Co., Ltd. (Tokyo, Japan). Benzoyl peroxide was obtained from Nacalai Tesque, Inc. (Osaka, Japan). Mesoporous materials, FSM-16 and -22 materials, 2.7 and 4 nm in pore diameter, respectively, were prepared from kanemite (layered polysilicate) using alkyltrimethylammonium [CnH2n+1N+(CH3)3] with different alkyl-chain lengths (n ) 16 and 22), according to the method reported by Inagaki et al. (10). Other reagents were of analytical grade. Characterization. Absorption spectra were measured with a Shimadzu (Kyoto, Japan) spectrophotometer, MPS-2400. Nitrogen adsorption isotherms were measured at 77 K on automatic gas adsorption apparatus, a Belsorp 28SA (BEL Japan, Inc. (Osaka, Japan). Specific surface areas were calculated by the BET method using adsorption data ranging from P/P0 ) 0.05 to 0.30, and the pore size distributions were determined by analyzing the adsorption branch by the BJH method (19). Preparation of Metmyoglobin (Fe3+)-FSM. A metmyoglobin (Fe3+)-FSM conjugate was prepared as follows. To 5.0
10.1021/bc050238i CCC: $33.50 © 2006 American Chemical Society Published on Web 12/20/2005
Technical Notes
mL of metmyoglobin (Fe3+) obtained by the oxidation of myoglobin (Fe2+) (0-30 mg) dissolved in phosphate buffer (pH 6.9) was added 100 mg of FSM-16 or FSM-22. The suspension was then shaken for 10 h at 25 °C to establish an adsorption equilibrium. The metmyoglobin (Fe3+)-FSM conjugate was collected by centrifugation. The amount of metmyoglobin (Fe3+) adsorbed into the pores of the FSMs was spectrophotometrically determined by measuring the absorbance of the supernatant obtained after centrifugation at 420 nm, which is characteristic of the absorption band of metmyoglobin (Fe3+). Reaction of Carbon Monoxide and Myoglobin (Fe2+)FSM. The metmyoglobin (Fe3+)-FSM conjugate (2.57 mg/ mL: 0.57 mg as metmyoglobin) was reduced by the addition of sodium dithionite (10 mg) in water (10 mL), to yield a myoglobin (Fe2+)-FSM conjugate. Carbon monoxide was reacted with myoglobin (Fe2+)-FSM by bubbling CO gas in the aqueous solution containing the conjugate, after which the UV-visible spectra of 3 mL portions (3 mL: 0.077 mg/mL: 0.017 as metmyoglobin) of the solution were taken with a Shimadzu (Kyoto, Japan) spectrophotometer, MPS-2400. Peroxidase-Like Activity of Metmyoglobin-FSM in Aqueous Solution. The experiments were conducted by measuring the peroxidase-like activity of the metmyoglobin-FSM using guaiacol and 2,2-azino-bis(3-methylbenzthiazoline)-6-sulfomic acid (ABTS), respectively, as the substrates. When the substrate was guaiacol (0.18 mmol), the metmyoglobin-FSM (0.95 mg: 0.21 mg as metmyoglobin) or metmyoglobin (0.2 mg) was suspended in 3 mL of phosphate buffer (pH 6.9) containing hydrogen peroxide (0.1%). In the case of ABTS acid (2.9 µmol), the metmyoglobin-FSM (0.0476 mg: 0.0093 mg as metmyoglobin) or metmyoglobin (0.009 mg) was suspended in 3 mL of phosphate buffer (pH 6.9) containing hydrogen peroxide (0.02%). The peroxidase activity of metmyoglobin (Fe3+)-FSM in the aqueous solution was spectrophotometrically determined by measuring the amount of oxidized guaiacol and ABTS using the respective molar extinction coefficients of at 435 nm ) 2.55 × 104 M-1 cm-1 (20) and at 410 nm ) 3.6 × 104 M-1 cm-1 (21). Measurement of Peroxidase-Like Activity of Metmyoglobin-FSM in Toluene. The peroxidase activity of metmyoglobin (Fe3+)-FSM was tested by the modified method of Ajima et al. (22). The experiment was conducted in 3 mL of toluene, using metmyoglobin (Fe3+)-FSM (2 mg/mL: 0.4 mg as metmyoglobin) and metmyoglobin (Fe3+)(0.4 mg/mL). Benzoyl peroxide (0.28 mM) and leucocrystal violet (8 mM) were employed as a hydrogen acceptor and donor, respectively. The reaction was carried out for 200 s at 25 °C. The peroxidase activity of metmyoglobin(Fe3+)-FSM in the organic solvent was spectrophotometrically determined by measuring the amount of oxidized crystal violet using the molar extinction coefficient of at 604 nm ) 1.2 × 105 M-1 cm-1 (22).
RESULTS AND DISCUSSION Adsorption of Metmyoglobin (Fe3+) into the Pores of FSMs. The adsorption amounts of metmyoglobin (Fe3+) onto FSM-16 and -22 powder in phosphate buffer (pH 6.9) were spectrophotometically measured with respect to the equilibrium concentration of metmyoglobin (Fe3+) (Figure 1). The adsorptions of metmyoglobin (Fe3+) onto the FSM-16 and -22 of 2.7 and 4.0 nm in pore diameter, respectively, are shown by A and B, respectively. When the pores are large, the adsorption proceeds efficiently with an increase in the equilibrium concentration of metmyoglobin (Fe3+) and reaches a constant level at 1.2 mg/mL (curve B). In this case, 24 mg of the 30 mg of metmyoglobin (Fe3+) was adsorbed onto FSM (100 mg) in phosphate buffer (pH 6.9). On the other hand, the amount of metmyoglobin (Fe3+) adsorbed onto FSM-16, the pore size of
Bioconjugate Chem., Vol. 17, No. 1, 2006 237
Figure 1. The adsorption of metmyoglobin (Fe3+) onto FSM-16 (curve A) and FSM-22 (curve B) in phosphate buffer (pH 6.9), spectrophotometrically measured with respect to the equilibrium concentrations of metmyoglobin (Fe3+) and metmyoglobin (Fe3+)-FSM.
Figure 2. Pore size distributions obtained from the adsorption branch by the BJH method. Curves A, B, C, and D correspond to FSM-16, metmyoglobin-(Fe3+)-FSM-16, FSM-22, and metmyoglobin (Fe3+)FSM-22, respectively.
which is smaller than that of FSM-22, was only slight (curve A). This difference in the adsorption behavior of metmyoglobin (Fe3+) between FSM-16 and FSM-22 suggests that metmyoglobin (Fe3+) is absorbed more easily into the pores than onto the outer surfaces. The corresponding pore size distributions of FSM-16 and -22 before and after metmyoglobin (Fe3+) loading are shown in Figure 2. The pore volume of FSM-16 was little changed before (curve A) and after (curve B) metmyoglobin (Fe3+) loading. The pore volume of FSM-22 dramatically decreased after the immobilization treatment (compare C and D). From the results, it can be seen that metmyoglobin (Fe3+) molecules do not occupy the mesopore spaces of FSM-16 but exist on the outer surfaces of the particles, where as FSM-22, on the other hand, absorbs metmyoglobin (Fe3+) molecules into the pores. This would further indicate that in the case of FSM, the pores of which are large enough to accommodate metmyoglobin (Fe3+) or protein, such protein tends to be much more easily immobilized in the pores rather than on the outer surface, because the molecular size of metmyoglobin (Fe3+) (2.8 by 3.2 by 4.5 nm) lies between the pore sizes of FSM-16 and -22, and the amount of metmyoglobin (Fe3+) adsorbed onto FSM-16 was only slight. The nitrogen adsorption isotherm of metmyoglobin (Fe3+) was examined for FSM-22 (Figure 3a). With an increasing amount of metmyoglobin (Fe3+) (0, 4.3, 9.2, 17, and 24 mg) confined into the pores of FSM-22 (100 mg), the amount of nitrogen adsorbed onto the metmyoglobin (Fe3+)-FSM decreased, as shown by curves A, B, C, D, and E, respectively. A steep increase in nitrogen uptake at the relative pressure of 0.4 for FSM is probably due to capillary condensation (curve A). On the other hand, the nitrogen uptakes by the metmyoglobin (Fe3+)-FSM (curves B-E) were lower than that of FSM. In the case of curve E, the pore volume of metmyoglobin (Fe3+)FSM was only ca. 46% that of FSM-22. In line with the BET surface areas and pore volumes of FSM (Table 1), the pore peaks (Figure 3b) decreased with an increase in the amount of metmyoglobin (Fe3+) adsorbed into the pores of FSM (Table 1). These results indicate that the pores of FSM were occupied
238 Bioconjugate Chem., Vol. 17, No. 1, 2006
Technical Notes
Figure 3. Changes in nitrogen adsorption isotherms of metmyoglobin (Fe3+)-FSM conjugates (a) and the corresponding pore size distributions obtained from the adsorption branch by the BJH method (b). Curves A, B, C, D, and E indicate the amounts of metmyoglobin (Fe3+) adsorbed to the pores of FSM (100 mg), 0, 4.3, 9.2, 17.0, and 24.0 mg, respectively. Table 1. Physicochemical Properties of the Myoglobin-FSM Conjugate myoglobin adsorbed (mg/100 mg FSM)
pore volume (cm3 g-1)
specific surface area (m2 g-1)
0 4.3 9.2 17.0 24.0
1.39 1.11 0.99 0.78 0.64
1110 886 856 722 561
by metmyoglobin (Fe3+) and support the opinion expressed in the preceding paragraph. Reaction of Myoglobin (Fe2+)-FSM with Carbon Monoxide. The absorption spectrum of the myoglobin (Fe2+)-FSM conjugate in water, shown in Figure 4 (curve B), differs considerably from that of the metmyoglobin (Fe3+)-FSM conjugate (curve A). Upon reducing the conjugate with dithionate, the metmyoglobin (Fe3+)-FSM spectrum (curve A in Figure 4) changed remarkably, the Soret shifting from 420 to 431 nm and a sharp single peak appearing in the visible region at 557 nm (curve B in Figure 4). The single adsorption band (λmax ) 557 nm) in the R, β region compared favorably with the wavelengths of deoxymyoglobin (23). The addition of carbon monoxide to the myoglobin (Fe2+)-FSM conjugate caused the formation of a CO-myoglobin-FSM having absorption maxima at 567, 539, and 420 nm (Figure 4: curve C). The spectrum has two clearly split peaks (R- and β-bands) in the visible region and a Soret peak. It is well-known that deoxymyoglobin changes easily into carboxymyoglobin in the presence of carbon monoxide with a spectral change. Thus, the change of spectrum from B to C, that is, the band shift from 557 and 433 nm to 567, 539 and 420 nm, respectively, corresponds to the change of myoglobin (Fe2+)-FSM to CO-myoglobin (Fe2+)-FSM. In addition, CO-myoglobin (Fe2+)-FSM shows an absorption curve quite similar to that of CO-heme coupled with native myoglobin (23). From the results above, it is deduced that myoglobin retains its higher-order structure in the pores of FSM. Peroxidase-Like Activity of Metmyoglobin (Fe3+)-FSM. Figures 5a and 5b show the peroxidase-like activity of the metmyoglobin (Fe3+)-FSM conjugate against incubation time in the reactions using hydrogen peroxide (a hydrogen acceptor) and guaiacol (23) or ABTS (25) (hydrogen donors) in aqueous solution (Scheme 1). When guaiacol was the hydrogen donor, the metmyoglobin (Fe3+)-FSM conjugate (Figure 5a, curve A) showed activity similar to that of the native form (Figure 5a, curve B). The reaction rate with the metmyoglobin (Fe3+)FSM and the native form were 1.59 × 10-6 and 1.26 × 10-6 M/min, respectively. In the absence of myoglobin-FSM in the reaction systems, no oxidation of organic substrate occurs (date not shown). This clearly indicates that the metmyoglobin does
Figure 4. Absorption spectra of metmyoglobin (Fe3+)-FSM (curve A), deoxymyoglobin (Fe2+)-FSM (curve B), and CO-myoglobin (Fe2+)-FSM (curve C) in phosphate buffer (pH 6.9). Curve B in the spectrum of deoxymyoglobin (Fe2+)-FSM in phosphate buffer (pH 6.9) was obtained by adding dithionite to metmyoglobin (Fe3+)-FSM. The concentration of metmyoglobin (Fe3+)-FSM was 0.077 mg/mL (0.017 as metmyoglobin). The peak positions of the R, β, and Soret bands for metmyoglobin (Fe3+)-FSM change from 640, 538, and 420 nm to 557 and 432 nm, respectively. Curve C is the spectrum of COmyoglobin (Fe2+)-FSM obtained by adding carbonmonoxide,CO, to deoxymyoglobin (Fe2+)-FSM. The peak positions of the R, β and Soret bands for deoxymyoglobin (Fe2+)-FSM change from 557 and 432 nm to 567, 539, and 420 nm, respectively.
Figure 5. The peroxidase-like activity spectrophotometrically determined by measuring the absorbance at 435 and 410 nm for the respective oxidations of guaiacol (a) and 2,2-azino- bis(3-methylbenthzthiaoline)-6-sulfomic acid (ABTS) (b). (a) Oxidation of guaiacol (0.18 mmol) with hydrogen peroxide (0.1%) in 3 mL of phosphate buffer (pH 6.9) catalyzed by metmyoglobin-FSM (0.95 mg: 0.21 mg as metmyoglobin) (curve A) or metmyoglobin (0.2 mg) (curve B). (b) Oxidation of ABTS (2.9 µmol) with hydrogen peroxide (0.02%) in 3 mL of phosphate buffer (pH 6.9) catalyzed by myoglobin-FSM (0.0476 mg: 0.0093 mg as metmyoglobin) (curve A) or metmyoglobin (0.009 mg) (curve B). Scheme 1
not lose its activity by encapsulation into the pores of FSM and probably maintains its nominal higher-order structure. However, when ABTS was the hydrogen donor, the amount of oxidized hydrogen donor increased with time for both the metmyoglobin (Fe3+)-FSM and the native form (Figure 5b, curve B), but oxidation did proceed slightly with metmyoglobin (Fe3+)-FSM (Figure 5b, curve A). The reaction rate with the metmyoglobin (Fe3+)-FSM and the native form were 0.9 × 10-6 and 3.8 × 10-6 M/min, respectively. Generally, in a process catalyzed by an enzyme, the reaction proceeds with the enzyme first recognizing the substrate and/or reactant to form
Technical Notes
Bioconjugate Chem., Vol. 17, No. 1, 2006 239
Scheme 2
a certain activation complex with them. Thus, the difference between the activities of metmyoglobin (Fe3+)-FSM conjugate for guaiacol and ABTS indicates that the metmyoglobin could not form such an activation complex with ABTS but could with guaiacol. In other words, it can be said that by the encapsulation, the metmyoglobin obtained an advanced molecular recognition ability that can distinguish between ABTS and guaiacol. Although it is not completely clear how the new ability comes about at present, it may be due to the limited flexibility in the higher-order structure of metmyoglobin and the selective adsorption properties (16-18) of metmyoglobin-FSM caused by the encapsulation. Peroxidase-Like Activity of Metmyoglobin (Fe3+)-FSM in Toluene. The finding shown above suggested that we examine the action of metmyoglobin (Fe3+)-FSM conjugate in an organic solvent. Thus, the catalysis of metmyoglobin (Fe3+)-FSM was investigated for the oxidation of leucocrystal violet (26) (AH) with benzoyl peroxide in toluene as shown below (Scheme 2). The absorption spectrum of metmyoglobin (Fe3+)-FSM in toluene is shown as a solid line in Figure 6 and shows a sharp Soret band at 410 nm. The absorption spectrum of metmyoglobin (Fe3+), the dashed line in Figure 6, shows a broad Soret band at approximate 420 nm, clearly indicating an agglutinated form of metmyoglobin (Fe3+). From the results obtained above, it can be seen that metmyoglobin (Fe3+) (native) is easily deactivated in the organic media, but the metmyoglobin (Fe3+) encaged in the pore of FSM is not denatured. Figure 7 shows the oxidation of leucocrystal violet (AH) with benzoyl peroxide catalyzed by metmyoglobin-FSM in toluene. The peroxidase activities of metmyoglobin (Fe3+)FSM and metmyoglobin (Fe3+) against incubation time correspond to curves A and B, respectively. The activity of the metmyoglobin (Fe3+)-FSM increased with time, while the native form showed almost no activity. On the basis of the above results, we can point out the following. Although the aggregation of metmyoglobin is accelerated in toluene, it can be prevented by encapsulation with FSM. Another role of encapsulation is to retain metmyoglobin (Fe3+) in its active form or, in other words, at the nominal higher-order structure, as can be seen from the high activity of metmyoglobin(Fe3+)-FSM for the reaction mentioned above. As for the reasons why the metmyoglobin (Fe3+) encaged in FSM retains the nominal higher-order structure and acts as a catalyst even in toluene, various factors can be considered. First of all, if the pores of FSM are highly hydrophobic, they can expel toluene molecules. If so, toluene
Figure 6. Absorption spectra of metmyoglobin (dashed line) and metmyoglobin-FSM (solid line) in toluene.
Figure 7. Oxidation of crystal violet with benzoyl peroxide catalyzed by metmyoglobin-FSM (curve A) and metmyoglobin (curve B) in toluene. Benzoyl peroxide (0.28 mM) and leucocrystal violet (8 mM) were used as a hydrogen acceptor and donor, respectively, in 3 mL of toluene. The reaction was carried out for 200 s at 25 °C. Curve A: metmyoglobin (Fe3+)-FSM (2 mg/mL: 0.4 mg as metmyoglobin). Curve B: metmyoglobin (Fe3+)(0.4 mg/mL). The reaction system is the same as that of curve A.
cannot do any damage to the metmyoglobin (Fe3+). Thus, the adsorption isotherm of toluene into metmyoglobin (Fe3+)-FSM (20 mg of metmyoglobin /100 mg of FSM) was measured for the purpose of examining whether toluene molecules could enter into the pore cavity. The pore volume of metmyoglobin (Fe3+)FSM calculated on the basis of the toluene adsorption isotherm was the same as that obtained from the corresponding nitrogen adsorption isotherm. Accordingly toluene molecules can enter the pore cavity of metmyoglobin (Fe3+)-FSM, yet they do not deactivate the metmyoglobin (Fe3+). This may be due to the limited flexibility of metmyoglobin (Fe3+) in the pores of FSM. Note that Wang et al. (27) have found that hemoglobin immobilized between titanate layers does not lose its activity in organic solvents; they postulate that water between the layers plays an important role in retaining the activity of the enzyme in its higher-order structure. Furthermore, the interlayer water is presumed to prevent organic solvents from taking water molecules which the enzyme originally possesses, resulting in retention of the activity and higher-order structure though the interaction between the interlayer water and the enzyme-bound water molecules. A similar mechanism may be considered relevant in this work. In conclusion, the encapsulation of myoglobin into the silicate mesoporous increases not only the molecular recognition ability of myoglobin but also the resistance to organic solvents, or surroundings. Thus, the encapsulation of enzymes with mesoporous materials is quite useful for establishing new functions and will have wide application to different chemical processes.
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Technical Notes (18) Itoh, T., Yano, K., Inada, Y., Kajino, T., Itoh, S., Shibata, Y., Mino, H., Miyamoto, R., Inada, Y., Iwai, S., and Fukushima, Y. (2004) Nanoscale organization of chlorophyll a in mesoporous silica: efficient energy transfer and stabilized charge separation as in natural photosynthesis. J. Phys. Chem. B. 108, 13683-13687. (19) Barret, E. P., Joyner, L. G., and Halenda, P. P. (1951) The determination of pore volume and area distributions in porous substance. Computation from nitrogen isotherms. J. Am. Chem. Soc. 73, 373-380. (20) Bergmeyer, H. U. (1974) Methods of Enzymatic Analysis, 2nd ed., pp 494-495, Academic Press, New York. (21) Be´douet, L., Adenier, H., Pulven, S., Bedel-Cloutour, C., and Thomas, D. (2004) Recovery of the oxidative activity of caged bovine haemoglobin after UV photolysis. Biochem. Biophys. Res. Commun. 320, 939-944. (22) Ajima, A., Cao, S. G., Takahashi, K., Matsushima, A., Saito, Y., and Inada, Y. (1987) An attempt to determine lipid peroxidase with polyethylene gliycol-modified hemin. Biotechnol. Appl. Biochem. 9, 53-57. (23) Mahler, H. R., and Corde E. H. (1969) Biological Chemistry, pp 583-589, Harper & Row, London. (24) Doerge, D. R., Divi, R. L., and Churchwell M. I. (1997) Identification of the colored guaiacol oxidation product produced by peroxidases. Anal. Biochem. 250, 10-17. (25) Wolfenden B. S., and Willson R. L. (1982) Radical-cations as reference chromogens in kinetic-studies of one-electron transferreactions - pulse-radiolysis studies of 2,2′-azinobis(3-ethylbenzothiazoline- 6-sulfomate). J. Chem. Soc., Perkin Trans. 2. 805812 (26) Safarikova, M., and Safarik, I. (2002) Magnetic solid-phase extraction of target analytes from large volumes of urine. Eur. Cells Mater. 3, 192-195 (27) Wang, Q., Gao, Q., and Shi, J. (2004) Enhanced catalytic activity of hemoglobin in organic solvents by layered titanate immobilization. J. Am. Chem. Soc. 126, 14346-14347. BC050238I
