Effect of microwave radiation on the activity of catalase. decomposition of hydrogen peroxide under microwave and conventional heating

Abstract

The effect of microwave heating (MW) on the activity of a well-known enzyme (catalase) was elucidated by examining the catalase-assisted decomposition of hydrogen peroxide (H₂O₂ at various heating times (0 to 12 min)). For comparison, conventional water bath heating (WB) was also examined under identical temperature conditions. Microwave radiation had a positive effect on the activity of catalase only over a very short time (less than 3 min), presumably because of the possible disruption of the catalase structural integrity under microwave irradiation at longer times (a negative influence) as evidenced by Gel Permeation Chromatographic (GPC) and MALDI Time-Of-Flight-Mass-Spectrometric (MALDI-TOFMS) analyses. The effect of temperature on the catalase activity was also probed at 39, 37, and 25 °C. Results indicate that utilizing a hybrid heating approach with conventional heating (water bath) coupled to microwaves was more effective provided microwave irradiation was carried out for a short time (also less than 3 min). Moreover, it is demonstrated that microwave heating in degrading hydrogen peroxide was most effective when the enzymatic reaction was carried out at a lower temperature, particularly at 25 °C.

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Introduction

Recent years have witnessed an increasing number of studies in the biochemical field which used microwave radiation to effect processes.¹ Historically, the influence of microwaves on enzymes, proteins, genes,² and on enzymes and proteins in foodstuffs³ has been investigated in the last five decades. More recently, studies on the use of microwave radiation have spanned several different fields of science and engineering.⁴ For instance, Parker and coworkers^5,6 reported on organic syntheses involving microwave-assisted enzymatic reactions, while the accelerated proteolytic cleavage of proteins under controlled microwave irradiation was investigated by Pramanik and coworkers.⁷ Elhafi et al.⁸ examined microwave treatment as a satisfactory method for inactivating a virus in preserving nucleic acid for the polymerase chain reaction (PCR) identification. A novel microwave-assisted protein digestion method using trypsin-immobilized magnetic nanoparticles (TIMNs) has been developed by Lin and coworkers,⁹ while Shaw et al.¹⁰ performed the polymerase chain reaction (PCR) in a microfluidic device with microwave heating using 8 GHz microwaves.

Two recent articles have reviewed microwave-assisted enzymatic reactions^11,12 in which the impact of microwave radiation on enzymatic properties and their application in enzyme chemistry were highlighted: (a) shortening of reaction times and (b) the role played by specific microwave effects. In this regard, Kappe and coworkers¹² critically evaluated the microwave-assisted proteomic protocols, and on examining microwave versus conventional heating asserted that non-thermal effects (i.e. microwave specific effects) had no influence on the structure and enzymatic digest of proteins. They explicitly pointed out the issue of regulating the temperature measurements of samples exposed to microwave irradiation in many investigations. In this context, Horikoshi et al.¹³ recently reported on the enzymatic proteolysis of peptide bonds by a metalloendoproteinase under precise temperature controls using 5.8 GHz microwave radiation. The advantages of 5.8 GHz microwaves rather than the more commonly used 2.45 GHz microwaves were emphasized using a modular non-commercial apparatus for the proteolysis of the Arg-Arg-Leu-Ile-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Gly peptide at the amino side of the aspartic acid (Asp) using the Asp-N metalloendoproteinase enzyme. The microscale sample was subjected to the microwaves' electric field (E-field) and magnetic field (H-field) radiation; excellent temperature control of the samples at the microliter scale was achieved using an apparatus that emitted microwaves at the precise frequency of 5.800000 GHz.¹³ Heating the sample with the microwaves' magnetic field enhanced enzymatic activity.

Herein we examine the decomposition of hydrogen peroxide (2H₂O₂ → 2H₂O + O₂) using the common metalloenzyme catalase as our model ex situ reaction to explore the microwaves' influence on the activity of this particular enzyme. This reaction has been investigated extensively for nearly 100 years using catalase extracted from several sources.¹⁴

The four heme groups in catalase (Scheme 1) are pivotal to the reaction because of the oxidation of the hemes from Fe^III to the less common Fe^IV form (reaction (1)), followed by reduction of the latter by another H₂O₂ molecule yielding back the Fe^III form of the catalase (reaction (2)).

H₂O₂ + Fe^III–enzyme → H₂O + O=Fe^IV–enzyme (1)

H₂O₂ + O=Fe^IV–enzyme → H₂O + O₂ + Fe^III–enzyme (2)

From the little that is known mechanistically,¹⁵ it would appear that upon entering the heme cavity the peroxide is severely sterically hindered and must interact with His⁷⁴ and Asn¹⁴⁷; the first stage of the catalysis apparently takes place at this position. Proton transfer from one oxygen of the peroxide to the other (via His⁷⁴) elongates and polarizes the O–O bond which ultimately breaks up heterolytically as a peroxide oxygen is coordinated to the iron center followed by displacement of water and formation of O=Fe^IV and a heme radical species. The latter rapidly degrades by another electron transfer but leaves the heme ring intact. In the second stage, a similar two-electron transfer process occurs in which the O=Fe^IV reacts with a second hydrogen peroxide to yield the original Fe^III–enzyme, another water molecule and molecular oxygen.

Scheme 1 Tetrameric structure of catalase; from the Protein Data Bank, Japan (PDBj; http://www.pdbj.org).

In the present study, the enzymatic reaction was performed using both the microwaves' magnetic field (H-field) and electric field (E-field) from a single-mode apparatus. A commercial microwave chemical equipment with multi-mode operation was also used. We show that the effect of microwave heating in this catalase-assisted reaction was to enhance the degradation of the peroxide and thus maintain or increase the catalase activity for a short time (<3 min), while at longer times the activity of the enzyme was subdued, likely the result of the microwave-induced degradation of catalase. Accordingly, features of this catalase-assisted reaction were examined under both microwave heating and conventional (water bath) heating together with a hybrid heating method that involved both microwave and conventional heating to probe the effect(s) of the microwaves on catalase.

Experimental

Methodology and analysis

The enzymatic activity of catalase (from bovine liver; Wako Pure Chemical Industries) in the degradation of H₂O₂ in aqueous media was investigated by introducing 2 mg of catalase in an aqueous Na₃PO₄ buffered solution (50 mM; 15 mL). Three microliters (3 μL) of this catalase solution were subsequently added to a quartz tube reactor (dia., 12 mm; internal dia., 10 mm; height, 100 mm) followed by addition of 30 mL of an aqueous Na₃PO₄ buffered solution (50 mM; pH 8.0; 29.8 mL) containing 0.2 mL of 30% H₂O₂ (quantity of H₂O₂ in the resulting solution was 0.2%). According to the data sheet supplied by the vendor, the optimal reaction temperature at which the catalase enzyme displays its greatest activity is 37 °C. The reactor containing a magnetic stirring bar (25 mm) was then positioned in the microwave equipment (microwave heating) or in a water bath (conventional heating) with temperature kept at 37 °C. After a given reaction time (3, 6, 9, or 12 min), the solution was immediately put into boiling water bath for 1 min to arrest the activity of the enzyme. No changes occurred in the H₂O₂ concentration during this 1 min. The quantity of H₂O₂ remaining in solution was monitored with a UV/Vis spectrophotometer (JASCO V-760) at 190 nm. Analogous conditions of thermometer, reactor, and the same solution prevailed under both conventional and microwave heating.

A possible change in the molecular structure of the catalase enzyme or its integrity under microwave irradiation was investigated by Gel Permeation Chromatography (GPC) and by MALDI-time-of-flight-mass-spectrometric (MALDI-TOFMS) method. The MALDI-TOFMS apparatus was a Shimazu AXIMA Confidence. Each 2 μL of the matrix (α-cyano-4-hydroxycinnamic acid) and the aqueous sample taken after the different reaction times were introduced into the microfuge tube, following which the mixed solution was placed onto a μFocus MALDI plate; the sample was then dried overnight under atmospheric conditions followed by the MALDI-TOFMS analysis. The molecular weight of catalase was determined using a JASCO liquid chromatograph (HPLC) equipped with a JASCO UV-2070 UV-Vis diode array multi-wavelength detector, and either a Tosoh Bioscience TSKgel G3000SWxl GPC column (for weights in the range 10 to 500 kDa; flow rate, 0.5 mL min⁻¹; injection volume, 20 μL; column temperature, 25 °C) or a Superdex 75 10/300 GL GPC column (for weights between 3 kDa and 70 kDa; flow rate, 0.1 mL min⁻¹; injection volume, 20 μL; column temperature, 25 °C). The mobile phases were 50 mM tris(hydroxymethyl)aminomethane and 150 mM NaCl in ion-exchanged water. The molecular weight markers were cytochrome-C (11.7 kDa), carbonic anhydrase (2.9 kDa), and albumin (6.6 kDa).

Microwave equipment

The microwave irradiation setup consisting of a single-mode TE₁₀₃ cavity (transverse electric 103 mode) and schematically illustrated in Fig. 1a, also included a short plunger, an iris, a three-stub tuner, a power monitor and an isolator.

Fig. 1 (a) Details of the experimental setup and positioning of the samples in the single-mode microwave resonator; (i) maximal position of the electric field (E-field) density and (ii) maximal position of the magnetic field (H-field) density. (b) Photograph of the single-mode microwave resonator and the 2.45 GHz semiconductor microwave generator; the photograph also shows the actual position of the sample at the H field maximum. Reproduced from ref. 16; Copyright 2012 by the International Microwave Power Institute.

Continuous microwave radiation was generated from a microwave semiconductor generator (Fuji Electronic Industrial Co. Ltd.; GNU-201AA; maximal power, 200 W) that emitted microwaves at the very precise frequency of 2.450000 GHz. The resonance of the microwaves was adjusted with the iris and the plunger at 1.5 cycles. Heating the enzyme solution was achieved by locating the quartz tube reactor in the single-mode microwave apparatus of Fig. 1a and b within the waveguide at positions either of maximal electric field (position (i)) or magnetic field (position (ii)) density. Temperatures of the solutions were measured at 3 s intervals with an optical fiber thermometer (FL-2000, Anritsu Meter Co. Ltd.).

The wavelength of propagation of the microwaves in the TE₁₀₃ mode within the waveguide was 14.78 cm (estimated from eqn (3)):¹⁶

where λ is the wavelength in the waveguide; λ_{o(2.45 GHz)} (= 12.24 cm) is the wavelength in vacuum given by c/f, {c being the speed of light, 2.9979 × 10¹⁰ cm s⁻¹, and f being the microwave frequency 2.45 × 10⁹ s⁻¹ for the 2.45 GHz microwaves used}; and b is the height of the waveguide, 10.92 cm (other dimensions of the apparatus are singled out in Fig. 1b). The maximal position of the E-field from the iris was located at 3/4 the wavelength of the standing wave in the waveguide (i.e., at 11.09 cm). An electric field monitor (Fuji Electronic Industrial Co. Ltd.) was used to maintain the sample tube at the maximal position of the E-field density, as the reproducibility of such experiments is often diminished if such operations are neglected. Under our conditions, no significant positional changes of the electric field were necessary.

The temperature profile of microwave heating was nearly identical with conventional heating (temperature profiles are reported below). A microwave input power of 1–3 Watts was used to achieve 25 °C, and 37 °C reaction conditions in the single-mode system. Ambient temperature was maintained at 24 °C for the reaction carried out at 37 °C, and 15 °C for the 25 °C reaction. The microwave input power was otherwise similar for both E-field and H-field irradiation.

A commercial microwave apparatus with multi-mode applicators was also used (Milestone StartSYNTH). In this case, the microwave input power was ca. 60–75 W to achieve a temperature of 37 °C, and 33–37 W for 25 °C controlled by the PID (proportional-integral-derivative controller) system available on the apparatus. Again, reactor, magnetic stirring, and solution were the same as those used when the reaction was carried out under single-mode microwave heating and conventional heating. All experiments for microwave heating and water bath heating were repeated no less than six times; the average of the data is reported.

Temperature conditions of the sample under each heating method

The temperature distribution of the aqueous sample subsequent to being exposed to microwaves and conventional heating after a reaction time of 6 min was determined by thermography (Fig. 2) after taking out the quartz reactor with the sample from either the waveguide or the water bath; the color from the thermography ascertained the temperatures. Ambient temperature was 24 °C. Thermography measurements could not be done after heating with the multi-mode applicator because of the configuration of the microwave equipment.

Fig. 2 Temperature distribution in a reactor containing the solution exposed to maximal electric field (E-field) or maximal magnetic field (H-field) density for microwave heating; also shown is the distribution under conventional water bath heating (WB).

Temperature profiles were assessed at the center of the solution using an optic fiber thermometer. Results for E-field, H-field and multi-mode microwave heating, and for water bath heating to 37 °C are displayed in Fig. 3. The temperature difference between E-field/H-field heating and conventional heating was less than 1 °C, while the temperature difference at the solution center for multi-mode heating were substantively lower than for conventional heating up to about 90 s.

Fig. 3 Temperature profiles at the center of the solutions under E-field (blue dashed line) and H-field (red solid line) microwave heating, multimode microwave heating (red dashed line), and water bath heating (WB; black solid line).

The relative dielectric loss factor (ε′′_r = 7.95) and the relative dielectric constant (ε′_r = 50.08) of the aqueous sample were determined using an Agilent Technologies E5071 C Network Analyzer with a 200 mm slim probe. Also of interest is the penetration depth (D_p) of the microwaves that refers to the depth that the microwaves pervade into a material at which the power flux has fallen to 1/e (= 36.8%) of its surface value; D_p was estimated from eqn (4):¹⁷

In the present instance, the penetration of the 2.45 GHz microwaves into the solution was estimated to be ca. 17 mm at 37 °C (internal dia. of quartz reactor, 10 mm). Accordingly, the microwaves permeated sufficiently into the center of the solution, and thus temperature fluctuations in the solution were negligible as the sample was continually stirred (magnetic bar).

Results and discussion

Catalase-assisted degradation of H₂O₂ under microwave and/or water bath heating

The decrease in the concentration of hydrogen peroxide (H₂O₂) at 37 °C as a result of the catalase-assisted reaction subjected to various conditions is displayed in Fig. 4 (note that each experiment was repeated eight times).

Fig. 4 Residual quantity of H₂O₂ in the catalase-assisted degradation reaction under microwave heating (E-field and H-field) and conventional heating at a temperature of 37 °C at which the enzyme is most active.

Compared to conventional heating, the degradation of H₂O₂ at 37 °C was enhanced 1.2-times and 1.3-times under the microwaves' E-field heating and H-field heating, respectively, in the first 3 min. Under conventional heating, the degradation of H₂O₂ progressed continuously after this time, while the degradation of H₂O₂ stopped after the 3 min period under microwave heating (E-field and H-field). This suggests that the activity of catalase was considerably affected by the microwave irradiation at times longer than 3 min. With multi-mode microwave heating, the degradation of H₂O₂ tended to be less efficient than with conventional heating throughout the 12 min heating period, indicating a decreased activity of the catalase with the former heating method, while catalase seems less affected by conventional heating. Regardless of the above inferences, it is relevant to query further the degradation of H₂O₂ in the presence of catalase, especially under microwave heating in contrast to conventional heating: (i) why was the H₂O₂ reaction enhanced by catalase in the first 3 min, but (ii) considerably muted at times longer than 3 min?

Why was the reaction enhanced by catalase in the first 3 min under microwave heating?

The data reported in Fig. 4 reveal that microwave H-field heating was slightly more effective than E-field heating in degrading H₂O₂, suggesting that E-field heating had a greater (negative) effect on catalase activity as evidenced by the continuous trend throughout the reaction period. Catalase is a hemeprotein that embodies iron(III)-porphyrins and so is a paramagnetic substance that would be influenced by a magnetic field.¹⁸ Studies that examined the effects of a magnetic field on enzymes have shown that applying a heterogeneous magnetic field (6 T) on catalase increased its activity by nearly 52%;¹⁹ the activity was also proportionally dependent on the magnetic field strength.²⁰ It is worth noting, however, that the magnetic field of the microwaves is an alternating current magnetic field and thus different from a direct current magnetic field used in the earlier studies.^19,20 Nonetheless, despite the different nature of the microwaves' magnetic field, the activity of catalase appears to be affected significantly by this H-field. Yet the data of Fig. 4 show that in the first 3 min microwave (E-field and H-field) heating had a lesser negative effect on the enzymatic activity of catalase than did both multi-mode microwave heating and conventional heating.

To further clarify the above inferences, the decomposition of H₂O₂ was examined at a fixed degradation time for a total of 9 min using a combination (hybrid) of microwave heating and water bath heating. More precisely, the degradation of H₂O₂ was investigated for 3, 6 and 9 min with the results illustrated in Fig. 5, which confirm the tendency of the degradation of H₂O₂ at each of these times to parallel the observations seen in Fig. 4.

Fig. 5 Residual quantity of H₂O₂ from the catalase-assisted degradation reaction under microwave heating and water bath heating for 9 min; hybrid microwave/water bath heating at 37 °C at various times of 3, 6, and 9 min. The horizontal dashed line is our frame reference with which to compare other means of heating the reaction components.

The aqueous sample was first heated for 3 min with microwaves, followed by water bath heating for 6 min (MW3-WB6), which resulted in only 20% of H₂O₂ decomposed, indicating that the activity of catalase was significantly lower than when the reaction was carried out with microwave heating for 9 min (MW9), which led to ca. 30% of H₂O₂ decomposed. Next, the catalase reaction was carried out by water bath heating for 6 min followed by microwave heating for 3 min (WB6-MW3). This resulted in about 50% peroxide decomposition indicating a higher activity of catalase compared with water bath heating for 9 min (WB9) that led to only ∼38% of hydrogen peroxide being decomposed. Thus, the activity of catalase with the WB6-MW3 method enhanced the degradation of H₂O₂ by factors of 1.6 and 1.3 compared with the MW9 and the WB9 methods, respectively. Moreover, carrying out the reaction with microwave heating for a short time (≤3 min) after water bath heating seemed to affect the catalase activity the least at 37 °C, which led to the next question as to why the decomposition of hydrogen peroxide was considerably muted by microwave heating for times longer than 3 min.

Why was the catalase-assisted reaction muted after 3 min?

Enzyme inactivation can be achieved by physicochemical and environment factors such as pH, temperature, pressure, concentration of the enzyme, and concentration of the substrate,^14,21 with the temperature factor playing an important role in this regard that may be changed either by microwave and/or conventional (water bath) heating. When an enzyme is subjected to heating beyond its optimal temperature at which the enzyme displays its greatest activity, the activity tends to decrease.¹³ Accordingly, the catalase-assisted degradation of H₂O₂ was examined at both 37 °C (optimal temperature) and at a slightly higher temperature (39 °C) under conventional heating. Results summarized in Fig. 6 show that at the latter temperature the degradation efficiency was significantly lower (0.58 times that at 37 °C) after a heating period of 12 min. However, the fact that there was no temperature variation between microwave heating with a single-mode system and conventional heating (see Fig. 2 and 3) calls into question selective heating of catalase in the aqueous sample by the microwave heating method.

Fig. 6 Residual quantity of H₂O₂ in the catalase-assisted degradation of hydrogen peroxide in aqueous solution at 37 and 39 °C subjected to conventional heating (water bath).

To confirm whether or not selective heating of catalase might occur under microwave irradiation necessitated the determination of the appropriate dielectric parameters of the reaction components. In this regard, the most important parameters are the dielectric constant (ε′), the dielectric loss factor (ε′′), and the dissipation factor (tan δ).¹⁷ The dielectric constant (ε′) depends on the frequency of the microwave radiation, which in the present instance was 2.45 GHz. The dielectric loss factor represents the quantity of input microwave energy that is lost to the sample by being dissipated as heat; it is a useful index of the generation of heat because of the interaction of the components with the microwave radiation field. The ratio of the dielectric loss to the dielectric constant, tan δ (= ε′′_r/ε′_r), is also an important parameter in that it determines the heating rate of the microwaves. The ability of a substance to convert electromagnetic energy into heat is determined by this dissipation factor. Comparison of dielectric factors requires that they be accessed under otherwise identical temperature conditions and microwave frequency. The presence of ionic species (as well as humidity) can greatly impact the dielectric parameters. Table 1 summarizes the relative dielectric constants (ε′_r), the relative dielectric loss factors (ε′′_r), and the dissipation factors (tan δ) of the various components at the microwave frequency of 2.45 GHz and at constant 37 °C.

Table 1 Relative dielectric constants (ε′_r), relative dielectric loss (ε′′_r) and dissipation factors (tan δ) for ion-exchanged water, the buffered solution, 0.2% of H₂O₂ in the buffered solution, catalase in the buffered solution and in the aqueous sample. Microwave frequency, 2.45 GHz; temperature, 37 °C

Conditions	Relative dielectric constants (ε′r)	Relative dielectric losses (ε′′r)	Dissipation factors (tan δ)
Ion-exchanged water	76.36	7.65	0.100
Buffered solution	77.20	12.03	0.156
H2O2 (0.2%)-containing buffer solution	56.53	8.88	0.157
Catalase in buffered solution	76.89	11.36	0.148
Sample solution	50.08	7.95	0.159

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The dissipation factor (tan δ) of the buffered solution was nearly 1.6 times greater than for the ion-exchanged water, a result of the presence of ionic species (Na⁺ and/or PO₄³⁻) that lead to microwave Joule heating. The presence of hydrogen peroxide and/or catalase in the buffered solution resulted in no enhancement of the heating efficiency by the microwaves, probably because of the small amounts of H₂O₂ and catalase.

It needs to be emphasized that microwave and conventional heating involve the same heat energy. However, the mechanisms by which this thermal energy is generated and transmitted to the reaction components are significantly different. Conventional heating occurs by heat conduction, whereas microwave radiation delivers the energy directly to the various substrates. Under microwave irradiation, heat was generated through different physical phenomena from ions (Joule effect) or from water in the buffered solution.¹⁶ Such subtle mechanistic differences may be responsible for some of the observations on the enzymatic reaction. For instance, changes of temperature at the macroscopic scale can bear significantly on the activity of catalase.^14,21 Accordingly, we probed the activity of catalase in the degradation of H₂O₂ under microwave heating at a temperature lower (25 °C) than the optimal temperature of 37 °C at which catalase usually displays its maximum activity. The concentration profiles of H₂O₂ in the catalase-assisted reaction carried out at the constant temperature of 25 °C are reported in Fig. 7.

Fig. 7 Residual quantity of H₂O₂ in the degradation process assisted by the catalase enzyme subjected to microwave heating and conventional heating at 25 °C.

Results at the lower temperature of 25 °C show that after 3 min into the catalase-assisted reaction, the degradation of hydrogen peroxide was 2.1-times and 2.4-times more efficient, respectively, under the microwaves' E-field and H-field heating than with conventional heating (WB). Indeed, the microwave enhancing effect at 25 °C was greater than at 37 °C. Use of the microwave multi-mode system to degrade H₂O₂ led to parallel observations to those from the single-mode microwave irradiation. At this lower temperature (25 °C; Fig. 7), the enhancing effect of microwave heating continued, and catalase activity was little affected at least up to 12 min into the reaction.

Examination of the integrity of the catalase structure under microwave irradiation

The activity of catalase in the degradation of hydrogen peroxide carried out at the constant optimal temperature of 37 °C was considerably affected after 3 min under microwave heating (see Fig. 4). Inactivation of catalase is expected if its three-dimensional structure is somehow disrupted and/or the catalase molecule is decomposed to lower fragments as was evidenced by Gel Permeation Chromatographic (GPC) and by MALDI-Time-Of-Flight Mass Spectrometric analyses. The GPC analysis of catalase exposed to microwave irradiation for 6 min (without H₂O₂) using the single-mode microwave apparatus revealed a molecular weight of catalase of ca. 240 kDa determined using a molecular marker (Fig. 8a); the GPC analysis also revealed the peak of catalase to occur at the retention time of 10.9 min.

Fig. 8 GPC chromatograms (Tosoh Bioscience TSK gel G3000SWxl GPC column; 10–500 kDa) of the catalase enzyme solution after 6 min (a) without H₂O₂, and (b) after degradation of H₂O₂ using microwave heating (red line; H-field; 9 min), water bath heating (blue line, 9 min), and catalase solution without H₂O₂ (control, black line); (c) same as (b) but with retention time axis between 14 and 20 min – note that the intensity axis was also expanded.

The full GPC spectrum of the sample solution recorded after the degradation of H₂O₂ at 37 °C under microwave heating for 9 min is reported in Fig. 8b. The concentration of catalase decreased to 0.87 times its initial concentration, while under conventional heating no change in peak intensity was seen. Moreover, a new peak was also observed under microwave heating at the retention time of 18.3 min (Fig. 8c), thus a new species, for which the molecular weight was estimated to be less than 1 kDa.

The unusual behavior of catalase under microwave irradiation, as exemplified by the GPC spectrum of Fig. 8c, infers a change in the molecular integrity of the enzyme. Accordingly, such a change or changes were examined by a MALDI-TOFMS analysis. Species with molecular weights of 223 kDa (assigned to catalase) and 198 kDa were detected in the mass spectrum of an aqueous catalase solution (Fig. 9a). Subsequent to microwave H-field heating for 9 min during the degradation of H₂O₂, the spectrum displayed several additional peaks in the mass range 160–240 kDa (Fig. 9b) confirming considerable changes in the molecular integrity of the catalase structure. The tetrameric catalase structure could break up under microwave irradiation into a monomer (ca. 60 kDa), a dimer (ca. 120 kDa), and/or a trimer (ca. 180 kDa). With the present available data, it is not possible to infer the nature of the species observed by the mass spectral method, nor species with other mass peaks. It is relevant to emphasize, however, that under conventional heating of a catalase aqueous solution no such additional peaks were observed in contrast to those displayed in Fig. 9b.

Fig. 9 MALDI-TOFMS spectra of (a) the catalase enzyme solution without H₂O₂, and (b) after degradation of H₂O₂ under microwave heating (H-field; 9 min). Mass units are in kDa.

Concluding remarks

This article examined the influence of microwave heating in a catalase enzymatic reaction carried out ex situ in which microwave radiation alone had a deleterious influence on the enzymatic reaction unless some control was placed on microwave heating. In this regard, inactivation of an enzymatic reaction in a living body by microwave heating was reported nearly three decades by Ikarashi and coworkers²² who noted that microwave heating could inactivate the enzyme in the brain of the rat, but that such inactivation caused no tissue disruption. The present study confirms the effect of microwaves on the catalase enzyme and shows that indiscriminate use of microwave radiation as just another heat source in carrying out ex situ enzymatic reactions is not advisable. However, a combination of conventional heating followed by microwave heating for a short period of time was more effective than microwave irradiation alone as the enzyme was inactivated for times longer than 3 min at 37 °C as seen in Fig. 4. Moreover, performing enzyme-assisted reactions at near-ambient temperatures (25 °C) proved more effective as observed from the results reported in Fig. 7. Therefore, we deduce that carrying out ex situ protein reactions or organic syntheses with microwave irradiation in the presence of an enzyme at lower temperatures and for short times might also be advantageous, particularly if the compounds are heat-sensitive.

Acknowledgements

We are grateful to the Japan Society for the Promotion of Science (JSPS) for financial support through a Grant-in-aid for Scientific Research (No. C-25420820). We would also like to thank Sophia University for a grant from the Sophia University-wide Collaborative Research Fund and Advanced Materials (ADAM) at the Research Institute for Sustainable Humanosphere, Kyoto University to S.H. One of us (N.S.) is grateful to Prof. Albini of the University of Pavia (Italy) for his continued hospitality in his PhotoGreen Laboratory.

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