New colorimetric method to determine catalase mimic activity

A new colorimetric method was used to determine catalase mimic activities of manganese dioxide (MnO2), iron oxide (Fe2O3) nanoparticles were prepared by a hydrothermal method (autoclave), and its composite. The MnO2 nanoparticles were annealed at different temperatures (250–700 °C), while MnO2: Fe2O3 composite in the mole ratio of 3:1 annealed at 400 °C. The structures and surface morphology were characterized by FT-IR measurements, x-ray diffraction (XRD), Scanning Electron Microscopy (SEM), and Atomic Force Microscope (AFM). This new method succeeds to determine catalase mimic activity, and found the activity of the composite was lower than its activity of MnO2 alone, in the same annealing temperature.


Introduction
Catalase (CAT) is a common enzyme (metalloprotein) that presents in all oxygen metabolizing cells. This enzyme exists in a soluble state in erythrocyte, and human erythrocytes are normally rich in catalase, so the catalase activity of blood is practically all due to the erythrocyte [1]. Hydrogen peroxide (H 2 O 2 ) is produced naturally in the aerobic organisms and human body. It is a strong oxidizing (one of reactive oxygen species) and bleaching agent and can be converted to highly reactive hydroxyl radicals, which are extremely toxic and can cause damage to dopaminergic neurons. Increased lipid peroxidation, elevated iron levels, expanded creation of reactive oxygen species (ROS) and diminished degrees of weakened glutathione have also been identified in the substantial nigra of patients suffering from Parkinson's disease [2]. Catalase stops the accumulation of hydrogen peroxide and defends cellular organelles and tissues from destruction by peroxide, which is constantly shaped by many metabolic reactions. Catalase pauses H 2 O 2 into H 2 O and O 2 and defends organisms from free radicals. It also has manufacturing uses to avoid certain pollutants in food and as a disinfectant for interaction lenses and a cleansing agent in some other yields.
The catalase action can be estimated by deciding the lessening of H 2 O 2 absorption (at λ=240 nm) [3,4]. The complications combined with this method, caused by using great levels of the substrate about (5-50 mM) to acquire adequate absorbance [5]. Furthermore, the excessive levels of hydrogen peroxide (H 2 O 2 ) lead to the realization of small bubbles in the solution which cause an error in the measurements [6]. CAT activity can be measured by other approaches such as the titration method, which is the sample does not allow in the spectrophotometer method when the precipitation or pigmentation was formed [1,7]. Goth [8] used other colorimetric methods for CAT by measuring spectrophotometrically the unreacted H 2 O 2 by reacting a compound with the ammonium molybdate. Another method used by Sinha and Hadwan [9,10] which are the hydrogen breakdown of H 2 O 2 measured (spectrophotometrically) by the reaction of dichromate and acetic acid as a reagent to formation complex. The titration method considered as a method for measurement of catalase activity, this method used when precipitation or pigmentation was formed, these do not allow in the UVspectrophotometric method [5]. manganese oxides (MnO 2 ) , for many years, with various crystal is giving a lot of attention, since their different chemical and physical properties, which are used in the different applications, such as catalysts, biosensors, water treatment, molecular sieves, and supercapacitors [11]. MnO 2 is n-type Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. semiconductor oxide [12]. Iron oxide (Fe 2 O 3 ) prepared by different methods such as oxidation precipitation, co-precipitation, Sol-Gel, and thermal decomposition. The physical and chemical properties are affected by the type method used for preparation of nanoparticles, for example uniformity, crystallinity, and size [13,14]. Fe 2 O 3 has different crystal structure for example wustite, hematite, maghemite, and magnetite which summarize as FeO, α-Fe 2 O 3 , β-Fe 3 O 4 and ɣ-Fe 3 O 4 respectively [15][16][17]. Fe 2 O 3 nanoparticles have the advantage of low cost and environmental harmlessness, MnO 2 and Fe 2 O 3 , two very low-cost metal oxides and MnO 2 :Fe 2 O 3 are widely used in many applications such as supercapacitor [18]. MnO 2 and Fe 2 O 3 nanoparticles were prepared by a hydrothermal method, the hydrothermal method considered as a powerful for synthesizing numerous forms of MnO 2 because of the it is a simple method, cheap technique, can be control temperature, pH, choice of precursors, and reaction time [19].
The present work is a new improved method that uses a colorimetric method for determination of hydrogen peroxide (H 2 O 2 ) in acidic solution contain potassium permanganate.

Preparation of MnO 2 nanoparticles
The reaction was performed by the hydrothermal method in a Teflon-lined (100 ml) stainless steel autoclave. The synthesis in briefly, 4.115 g of KMnO 4 into deionized water (70 ml) was added with stirring about 20 min The solution should be filtered to remove any contaminates, after that added about 3.4 ml of HCl (concentrated) to the above solution (filtrated) with vigorous stirring to get the precursor solution. After that the solution transferred into an autoclave, sealed and treated with hydrothermally at 200°C for 9 h. After complete the reaction, taken out the autoclave and cooled to room temperature (naturally). The resulting precipitates (brownblack) were filtered by centrifugation and washed by deionized water several times, to remove other products, washed with ethanol, and finally the as-prepared dried at 100°C in air for 120 min The reaction between KMnO 4 and HCl took place as the following steps: The final above reactions can be write by the following equation: The MnO 2 precipitate (brown-black) annealed at (250°C, 400°C, 550°C and 700°C) temperatures for 120 min

Preparations of iron oxide (Fe 2 O 3 ) nanoparticles
Iron oxide nanoparticles were prepared by the hydrothermal method using an autoclave, by dissolved 6 g of FeCl 3 (anhydrous), in 20 ml of distilled water. The solution mixed in a beaker about 10 min at room temperature to ensure dissolute all the salt. In another beaker, 3.33 g of NH 2 CONH 2 were dissolved in 10 ml distilled water with stirring for 10 min at room temperature, until the solution became colorless. The first solution pours on the urea solution and stirred by using a magnetic stirrer for 20 min at room temperature. The mixed solutions were placed into an autoclave, then placed in the oven and heated at 200°C for 12 h. After he tend of time, the autoclave cooled at normal temperature slowly. The precipitate form in reddish-brown color was washed by water (several times) after that collected by centrifugation, then the precipitate washed several times with ethanol. Finally dried the precipitate in an oven at 100°C for 120 min The dark red color precipitate was obtained when annealed at 400°C.
The reaction between FeCl 3 and NH 2 CONH 2 write by the following equations: The final equation can be assumed as: Heat 2 3 4 2

Preparation of MnO 2: Fe 2 O 3 Composite
This composite was prepared by mixing (3:1) mole ratio of MnO 2: Fe 2 O 3 respectively, starting from the equivalent amount of these metal hydroxides. In a beaker containing distilled water the metal oxides were dispersed and mixed for 3 h by using a magnetic stirrer. then the mixture was placed in the autoclave and located in the oven at temperature 200°C for 6 h. After end of the time the dark powder is wished several time with distilled water and annealing at temperature 400°C for 120 min.

Catalytic activity procedure
The first time, the KMnO 4 concentration was measured by titration with sodium oxalate solution (known concentration). The second time, the unknown concentration of H 2 O 2 was measured by titration with the KMnO 4 (known concentration). The standard curve contained the following concentration of KMnO 4 (0, 1, 2, 3, 4 and 5) ×10 -5 M, was prepared to calculate the different color concentrations absorbed by KMnO 4 at the length wave 525 nm (as shown in figure 1). The mimic activity, was measured using the reaction with the solution of MnO 2 nanoparticle (2 mM), and solution of H 2 O 2 (750 μM), as shown in the reaction [10]: After five minutes, the solution of potassium permanganate (300 μM), acidity by some drops of sulfuric acid (H 2 SO 4 ) was added. The purple color of KMnO 4 solution, will be reacting with the H 2 O 2 (the excess residual), and converted to colorless produce (MnSO 4 ), as explain s in the following equation: The concentration of H 2 O 2 is directly comparative to the KMnO 4 concentration. The decreasing which happen in the KMnO 4 color (concentration), was determined calorimetrically at λ=525 nm using the equation get from figure 1. The following steps in table 1, explain the work of mimics activity.  same card (JCPDS Card No.44-0141). The intensity of diffraction peak (211) increased when annealing temperatures increase from 250 to 550°C. At 400°C and 550°C the sample showed orientation of regular grains shaped, that suggesting to the pure phase of α-MnO 2 nanoparticle was increased, these results are identical to the results of the XRD spectra of the reference [20].

Result and discussion
The new phase as cubic Mn 2 O 3 was identified at annealing temperature 700°C (JCPDS Card No.41 −1442) as in figure 2(d). The average crystallite size (D) for the peak alone was estimated using the Debye-Scherer formula: Where D is the crystallite size, k is the constant (0.9), β is the full width at half maximum (FWHM) intensity of the diffraction peak, λ is the wavelength of the x-ray radiation, and θ is the diffraction angle [21]. Table 2 shows the diffraction patterns of x-ray nanoparticles product at annealing temperatures (250, 400, 550 and 700°C) for 120 min The hydroxide phase (Mn(OH) 4 ) decrease when increase annealing temperature from 250°C to 400°C and convert from hydroxide (Mn(OH) 4 ) to only oxide phase (MnO 2 ), whereas the increasing temperature to 550°C cause formed mixture phases from MnO 2 and Mn 2 O 3 , the increased in both of the lattice constant and the diffraction peaks ( intensity), are agreement with the results of the reference [22]. The lattice constant is decreases when annealing temperature becomes 700°C, due to the formation of one phase called Mn 2 O 3 [23].  Figures 3(a) to (d) show atomic force microscope (AFM) images 3D and 2D of MnO 2 nanopowders annealing at several temperatures (250-700)°C, and the chart of granularity accumulation distribution. It is clear that at an average diameter of prepared nanoparticles and its composite are change with different annealing temperatures, as in table 3. the surface morphologies of the MnO 2 are different. We found the average diameter of the nanoparticles were (66.27-81.65 nm). According to the results of AFM the average diameter increasing by increase annealing temperature this result maybe relate to improvements in the crystalline of the nanoparticles and the agglomeration of small grains form larger grains [24]. But when the annealing temperature is 700°C the average diameter decreases due to the formation of a new phase (Mn 2 O 3 ) and these results correspond to XRD analysis.   Figure 4 shows the FE-SEM images of α-MnO 2 nanostructures prepared by hydrothermal method and annealed at temperatures 400°C, for 120 min, at two magnifications (1 μm and 500 nm). The top-view FE-SEM image presented in figure 4 shows that the sample is composed of nanorods and nanorods bundle with average diameters about 65 nm.

The morphology surface for MnO 2 :Fe 2 O 3 by (FE-SEM)
The high magnification FE-SEM images of the Fe 2 O 3 powder and MnO 2 -Fe 2 O 3 composite powders prepared by hydrothermal method with annealing temperature 400°C for 120 min are shown in figures 4(b) and (c)) with magnifications 500 nm, Nanoparticles were observed as individual clusters with few conglomerates over the surface. The expected shapes are hexagonal micro pyramid which belongs to α-Fe 2 O 3 was a match the XRD patterns results. The FE-SEM images of MnO 2 -Fe 2 O 3 shows that there are nanorods and the bundle of nanorods with different diameters and lengths, the faces of nanorods have quadrilateral shapes which belonged to tetragonal crystalline structure for α-MnO 2 nano-powder. The figure also shows that there are aggregations of granules appear around the nanorods which belonged to hexagonal crystalline structure for α-Fe 2 O 3 .

Fourier transform infrared spectrum (FTIR) study
FT-IR spectra of manganese dioxide nanostructures synthesized by the hydrothermal method. FTIR spectroscopy was carried out in order to ascertain the purity and nature of manganese dioxide nanostructures.     We used a new and simple colorimetric method to determine catalase mimic activity of MnO 2, Fe 2 O 3 nanoparticles and MnO 2 :Fe 2 O 3 composite. This method is simple, cheap, fast and easy in the application. In order to estimate the catalase mimic activity (rate of reaction) of MnO 2 nanoparticles as-prepared and when the annealing of the samples at different temperatures (250°C-700°C) for 120 min, we used the following equation: Where: t mean the reaction time (in seconds); C o and C are the entire concentration of H 2 O 2 in the test tube before and after reaction respectively. We found that there is increase in rate of reactions (K) with increase annealing temperatures and become maximum at annealing temperature is 400°C (K = 2.59×10 -2 Ssc −1 ), as shown in figure 6 and Table 4, this may be related to conversion of manganese compound from metal hydroxide (Mn(OH) 4 ) to metal oxide (MnO 2 ), after that the rate of reactions decreases with increasing the temperatures from 550°C to 700°C. This may be due to a decrease in the surface area of particles when increase annealing and accumulate particles. At the same annealing temperature (400°C) the catalase mimic activity of Fe 2 O 3 (1.44×10 -2 s −1 ) is lower than MnO 2 (2.59×10 -2 s −1 ). Therefore, we suggested to use the mole ratio (3:1), (of the composite MnO 2 : Fe 2 O 3 respectively. while its composite with MnO 2 shows a decrease in activity compared with MnO 2 only, and an increase in activity when compared with Fe 2 O 3 alone. These changes in activities maybe relate to the difference in ionic potential (charge/radius) between these ions (for Fe 3+ =4.138 and for Mn 4+ =5.970). This differences in ionic potential make the strong one (Mn 4+ ) effect on oxygen atoms of Fe 2 O 3 , therefore, the bond strength between Mn 4+ ion and oxygen relate to Fe 2 O 3 will increase and cause a decrease in bond strength (elongation of bond length) between Mn-O relate to MnO 2 molecules compared with MnO 2 alone. These results

Conclusion
In summary, we successfully used a new and simple colorimetric method to determine catalase mimic activity of MnO 2 , Fe 2 O 3 nanoparticles and its composite (MnO 2 : Fe 2 O 3 ), against low concentration (750 μM) of H 2 O 2 solution as a substrate. The MnO 2 and Fe 2 O 3 nanoparticles were successfully prepared by the hydrothermal method (autoclave), then MnO 2 annealed at different temperatures (250°C-700°C). By using our new colorimetric method the results show the annealing temperature at 400°C of MnO 2 nanoparticles, had the greatest catalase mimic activity (2.59×10 -2 s −1 ) among all other annealing temperatures. Therefore this annealing temperature (400°C), was used to calculate the catalase mimic activity of Fe 2 O 3 and MnO 2 : Fe 2 O 3 composite. The results show these are decreasing in the catalase mimic activities of both Fe 2 O 3 nanoparticles and the composite (MnO 2 : Fe 2 O 3 ) which were (1.44×10 -2 s −1, 1.78×10 -2 s −1 ) respectively, compared with MnO 2 alone. The catalase mimic activity it is a special specification for each material, but the decrease in the activity of composite may be due to the interference between them may be effected on the activity.