Manual control of catalytic reactions: Reactions by an apoenzyme gel and a cofactor gel

Enzymes play a vital role in catalysing almost all chemical reactions that occur in biological systems. Some enzymes must form complexes with non-protein molecules called cofactors to express catalytic activities. Although the control of catalytic reactions via apoenzyme–cofactor complexes has attracted significant attention, the reports have been limited to the microscale. Here, we report a system to express catalytic activity by adhesion of an apoenzyme gel and a cofactor gel. The apoenzyme and cofactor gels act as catalysts when they form a gel assembly, but they lose catalytic ability upon manual dissociation. We successfully construct a system with switchable catalytic activity via adhesion and separation of the apoenzyme gel with the cofactor gel. We expect that this methodology can be applied to regulate the functional activities of enzymes that bear cofactors in their active sites, such as the oxygen transport of haemoglobin or myoglobin and the electron transport of cytochromes.

for some substrates, such as peroxidase. Furthermore, when the gel assembly is separated by hand, individual gels did not exhibit catalytic behaviour (Fig. 1). This process was found to be repeatable.

Results
Preparation of the apoHRP and FePor gels. Polyacrylamide was selected as the main chain in this study because interactions of the -CONH groups with porphyrin or a protein are small when used in a gel electrophoresis. ApoHRP was prepared from HRP. HRP was dissolved in 100 mM sodium phosphate buffer (pH = 7.0). The pH of the solution was brought to pH = 1.9 by slowly adding 0.1 M HCl. The acidic HRP solution was extracted with 2-butanone to remove the heme, and it was then immediately neutralized with 1 M NaOH. The apoHRP in the neutral solution was obtained using dialysis with 50 mM sodium phosphate buffer (pH 7.0) 28 . FePor was prepared by mixing Por with FeCl 2 27 . The apoHRP and FePor gels were prepared by modifying apoHRP and FePor to the acrylamide/acrylic acid/N,N ′-methylene bisacrylamide terpolymer gel (MBAAm, cross-linker; Scheme S1 and S2). The poly(acrylamide) gel (a blank gel) was prepared using radical copolymerization of acrylamide and MBAAm that was initiated by ammonium persulfate in aqueous solutions (Scheme S3). In all gels, the MBAAm unit was fixed to 2 mol%. UV-Vis spectroscopy characterized the functional monomeric unit content of the apoHRP and FePor gels. Figure 1 depicts the chemical structures of the apoHRP(x) and FePor(y) gels. Here, x and y represent the mol% of the apoHRP and FePor units, respectively. The x mol% content of apoHRP was determined to be 0.22, 0.80, or 1.0 mol% in the apoHRP(x) gels (Fig. S1). Similarly, the y mol% content of FePor was determined to be 0.47 or 1.0 mol% in the FePor(y) gels (Fig. S2).
Interaction between the apoHRP and FePor units. Prior to the adhesion experiment between the apoHRP(x) and FePor(y) gels, the interaction between apoHRP and FePor was investigated in 4% DMSO and 50 mM sodium phosphate buffer (pH 7.0) (hereafter referred to as the buffer). Figure 2a shows the UV-Vis spectra of native HRP (orange), apoHRP (blue), FePor (black), and the apoHRP/FePor complex (red). Although the maximum absorption wavelength (λ max ) of FePor showed λ max = 398 nm, the λ max of the apoHRP/FePor complex was similar to that of native HRP (λ max = 410 nm). This red-shift is derived from complexation of L-histidine at active site of apoHRP and FePor by coordination bond 29,30 . The association constant (K a ) of apoHRP with FePor was determined to be 3.8 × 10 6 M −1 in buffer at 298 K (Fig. S3). These results indicate that FePor is properly accommodated in the heme pocket of apoHRP to form the apoHRP/FePor complex.
Based on the interaction between apoHRP and FePor, the complex formation of the apoHRP unit in an apoHRP(1.0) gel was investigated. The apoHRP(1.0) gel was immersed in a buffer with FePor (50 μ M) (Scheme S5). The apoHRP(1.0) gel with FePor (apoHRP(1.0) gel/FePor complex) was washed with excess buffer. The maximum absorption band corresponding to FePor shifted from 394 nm to 400 nm in the UV-Vis spectra of the apoHRP(1.0) gel/FePor complex ( Fig. 2b; Fig. S4, red). This result indicates that apoHRP in the apoHRP(1.0) gel forms a complex with FePor.
Adhesion of the apoHRP gel to the FePor gel through molecular recognition. Next, we investigated the adhesion of the apoHRP gel(x) to the FePor gel(1.0). These gels were cut into ca. 5 mm × 5 mm × 2 mm cuboids using a razor. One of the gels was placed on top of the other, and the stack was left undisturbed for 2 h at r.t. under humid conditions. The apoHRP(1.0) gel adhered to the  (Fig. S6). The adhesion strength of the assembled gels increased by increasing the molar content of apoHRP and FePor units in the gels, which is consistent with previous reports [25][26][27] . These results indicate that the adhesion between the apoHRP and FePor gels is due to complexation of apoHRP with the FePor unit on the gel surface (Fig. 2e).
Competitive experiments confirmed the formation of the gel assembly based on the complexation of apoHRP with FePor. The addition of apoHRP-containing buffer (350 μ M) led to the dissociation of the apoHRP(1.0) gel/FePor(1.0) gel assembly (Fig. S7a, Supplementary Movie 3). This result is attributed to apoHRP in buffer competitively forming a complex with the FePor unit of the FePor(1.0) gel instead of the apoHRP unit in the apoHRP(1.0) gel because the concentration of apoHRP in buffer (1.4 μ mol) is 1,500 times higher than in the apoHRP gel (0.95 pmol, Fig. S7b). These results indicate that the apoHRP/ FePor complexation is responsible for the formation of the apoHRP(x) gel/FePor(y) gel assembly.
Catalytic behaviour of the apoHRP/FePor complex. 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) was used as a typical substrate oxidized by HRP (Fig. 3a). We investigated the oxidative catalytic activities of the apoHRP/FePor, apoHRP(x) gel/FePor, and apoHRP /FePor(y) gel complexes. The absorbance of the oxidized product of ABTS (ABTS +• ) was measured in buffer using UV spectroscopy while stirring at 10 °C. Although ABTS was not oxidized by either apoHRP (Fig. 3b, blue diamonds) or FePor (Fig. 3b, black dots), the apoHRP/FePor complex (Fig. 3b, red squares) oxidized ABTS. The apoHRP/FePor complex oxidized other HRP substrates such as pyrogallol (Fig. S8a), 2-aminophenol (Fig. S8b), o-phenyldiamine (Fig. S8c), o-dianisidine (Fig. S8d), and hydroquinone (Fig.  S8e). These results indicate that the apoHRP/FePor complex has the same catalytic system as native HRP. We investigated the catalytic activity of the apoHRP(1.0) gel/FePor complex and the apoHRP/ FePor(1.0) gel complex for oxidizing ABTS. In addition, the oxidative reaction of ABTS in the presence of the apoHRP(1.0) gel/FePor complex and the apoHRP/FePor(1.0) gel complex was recorded using UV spectroscopy while stirring at 10 °C. The apoHRP(1.0) (Fig. 3c, blue diamonds), FePor(1.0) (Fig. 3c, black dots), and blank ( Fig. 3c green triangles) gels did not cause oxidation of ABTS. However, the apoHRP(1.0) gel/FePor complex (Fig. 3c red squares) and the apoHRP/FePor(1.0) gel complex (Fig. 3c, purple crosses, Scheme S6) caused oxidation. The change in the UV-Vis spectra of ABTS in the presence of the apoHRP(1.0) gel/FePor complex was similar to that in the presence of the apoHRP/FePor complex ( Fig. S9 and S10). These results indicate that the apoHRP and FePor units in the gels exhibited catalytic activity. An increase in the apoHRP unit molar content of the apoHRP(x) gel/FePor complex from 0.22 to 1.0 mol% increased the oxidation rate of ABTS (Fig. S11). This result indicates that the oxidation rate is based on the apoHRP unit molar content in the gel. Therefore, the oxidation rate can be regulated by changing the molar content of the apoHRP and FePor units in the gels.
The oxidative rate was correlated with the adhesive surface area of the apoHRP(1.0) gel/FePor(1.0) gel assembly. The oxidative rate of ABTS in the presence of the apoHRP(1.0) gel/FePor(1.0) gel assembly increased when the adhesive area of the gel assembly increased from 6.2 to 25 mm 2 (Fig. S15). This result indicated that the number of apoHRP with FePor complexes at the gel adhesive surface increased when the adhesive area of the apoHRP gel/FePor gel assembly increased.
Although it has been previously described, it is worth mentioning that the apoHRP(0.80) gel did not adhere to the FePor(1.0) gel and that the contact object of the apoHRP(0.80) gel/FePor(1.0) gel showed catalytic activity. A 5 g weight was placed on top of the contact object of the apoHRP gel/FePor gel to prevent dissociation of the contact object of the apoHRP gel/FePor gel in buffer (Fig. S16a). Fig. S18b shows the absorbance change in the oxidative reaction of ABTS in the presence of various gel concentrations. The contact object of the apoHRP(0.80) gel/FePor(1.0) gel oxidized ABTS in buffer, which suggests that the apoHRP and FePor units form complexes when gel surfaces contact without apoHRP gel/FePor gel adhesion.
The oxidative rate, in the presence of the contact object of the apoHRP(0.80) gel to the FePor(y) gel, increased when the molar content of the FePor unit increased from y = 0.47 to 1.0 mol% (Fig. S16b  red squares and blue diamonds). However, the oxidative rate in the presence of contact object of the apoHRP(0.22) gel to the FePor(y) gel remained the same when the molar content of the FePor unit in the gel increased from y = 0.47 to 1.0 mol% (Fig. S16b black dots and purple crosses). This occurs because the apoHRP binding sites at the gel interface are fully occupied with FePor. These results indicate that by changing the gel size and molar content of the apoHRP and FePor units we can control the catalytic oxidation rate.
Control of catalytic reactions. Additionally, we investigated the change in the production of ABTS oxidation product when the apoHRP(0.80) gel/FePor (1.0) gel was separated and manually re-adhered in buffer containing 0.5 mM ABTS and 5.0 mM H 2 O 2 (Fig. 4a). Notably, separation of the apoHRP(0.80) gel/FePor(1.0) gel suspended ABTS oxidation (Fig. 4b blue area). However, upon readhesion of the apoHRP(0.80) and FePor(1.0) gels (Fig. 4b red area), the oxidation reaction accelerated. This process is repeatable. We obtained similar results using pyrogallol (Fig. S17). These results indicate that catalytic activity is switched on and off via adhesion and separation of the apoHRP and FePor gels, respectively (Fig. 4c).

Discussion
We successfully constructed a system with controllable catalytic activity via the adhesion of gels at the macroscale. The oxidation of substrates for HRP is accelerated via adhesion of the polyacrylamide gel modified with apoHRP and FePor. The catalytic oxidation rate of these substrates is increased by increasing the adhesive area of the apoHRP gel/FePor gel assembly or the molar content of apoHRP and FePor in the gels. Thus, by changing the gel size or molar content of apoHRP or FePor in the gel, we can control the catalytic reaction. Furthermore, adhesion and separation of the apoHRP and FePor gels turns on and off the catalytic activity, respectively. We expect these findings to be applicable for various materials, such as drug carriers that release the drug upon gel adhesion.  28 . The resultant gel was thoroughly rinsed with a 0.1 M MES buffer (pH 5.6) and was soaked in excess buffer (Scheme S1). The mol% of apoHRP, x, was determined using the Lambert-Beer law from the absorbance of apoHRP in the gel with a thickness of 100 or 50 μm (Fig. S1).

Methods
Preparation of the FePor gel. AAm, MBAAm, and N-succinimidyl acrylate were dissolved in DMSO. After purging with dry argon for 3 h, 2-ketoglutaric acid was added to the monomer solution, which was subsequently irradiated using UV light with a wavelength of 365 nm for 8 h. The resultant gel was soaked in a DMSO solution containing FePor 26 . The FePor gel was washed with DMSO and was soaked in excess buffer (Scheme S2). The mol% of FePor, y, was determined using the Lambert-Beer law from the absorbance of FePor in the gel with a thickness of 10 μm (Fig. S2). The UV-Vis spectra were obtained using a JASCO V-650 spectrophotometer.
Switching of the catalytic activity. Substrate oxidation (2.0 mM pyrogallol or 0.5 mM ABTS) was examined in the presence of the apoHRP (0.80) gel-FePor (1.0) gel assembly and the separated apoHRP and FePor gels. The changes in absorbance of λ max for the oxidation product of each substrate were monitored using a UV-Vis spectrometer.