Determination Hypoiodous Acid (HIO) By Peroxidase System Using Peroxidase Enzyme

It has been understood that peroxidase enzyme including peroxidase serves as catalyzer to enzymatic reaction among hydrogen peroxide and halides, therefore this research was done for generating hypoiodous acid (HIO) from peroxidase system using peroxidase enzyme. Hydrogen peroxide, potassium iodide, and peroxidase enzyme were used to produce HIO. Determination the amount of formed HIO was done using 2,2'-azino-bis(3- ethylbenzothiazoline-6-sulphonic acid) or ABTS as substrate through the colorimetric measurement of hydrogen peroxide residue during reaction process using at 412 nm. The result indicated that residual hydrogen peroxide showed the minimum concentration after 60 minutes reaction time. Because the reaction started at the beginning time of mixing, hydrogen peroxide was unable to be eliminated totally to produce HIO. The reaction of peroxidase system was able to determine the beginning of mixing process but the reaction process could not eliminate the initial concentration of hydrogen peroxide indicating the maximum amount of production of HIO could be determined. In conclusion, the less of H2O2, higher HIO obtained and peroxidase enzymes can accelerate the formation of HIO.


Introduction
Enzymes are known as good catalyzer. One of important enzyme in food is peroxidase. Peroxidases are widely distributed in nature and can be extracted from most plant cells, some animal tissues and fungus [1]. Plant peroxidase is an enzyme that catalyzes the one electron oxidation of various organic and inorganic substrates in the presence of hydrogen peroxide [2]. Plant peroxidases have been isolated and purified from several plant sources, such as Leucaena leucocephala, Viscum angulatum, Vigna mungo, Solanum melongena, Beta vulgaris, Roystonea regia, Tamarix, Jatropha curcas [3]. Peroxidase enzyme is one of the enzymes that can catalyse reaction between substrate and oxygen molecule. Peroxidases are much less effective as catalysts of oxygen-transfer reactions than cytochromes P450 [4]. Iodine is a compound of anion halide or (X -), IO -3 , so application peroxidase can also catalyse the combination of hydrogen peroxide (H2O2) and potassium iodate (KIO3) to produce Hypoiodite (OI -) or Hypoiodous Acid (HIO). Peroxidase enzyme activity test was generally performed using UV-VIS spectrophotometer with 412 nm wavelength and using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) or ABTS as substrate [5]. ABTS was used as an indicator because it was easily transformed into a radical form. ABTS oxidized with H2O2, then would form a radical compound of metastable cations and blue or green chromophore [6]. This research was done to analyse concentration of hypoiodous from the reaction mixture of H2O2 and KIO3. Since hypoiodous has antibacterial properties, the minimum concentration of substrates H2O2 and KIO3 was very important in order to generate maximum HIO through peroxidase system.

Preparation of HIO
There were two methods for determination of HIO. A 450 μl of 0.15 mM H2O2 was mixed with 450 μl of 0.15 mM KI and 100 μl of enzyme peroxidase. The other one was 450 μl of 0.15 mM H2O2 was mixed with 450 μl of 0.15 mM KI and 100 μl of aquadest. Solution was reacted, then measured using ABTS method every 10 minutes for an hour. This method was adopted from previous researcher [5]. The mixture of H2O2 and KI was determined as HIO solution.

Residue H2O2 with ABTS method
A 450 μl of HIO solution was added to a reaction mixture containing 450 μl of 0.55 mM ABTS in Phosphate Buffer (0.1 M, pH 7) and 100 μl of horseradish peroxidase (4.5 U). ABTS as substrate through the measurement of hydrogen peroxide residue during reaction process using spectrophotometer at wavelength of 412 nm. This method was derived from previous researcher [7].

Residue of H2O2
Measurement of H2O2 residues is enabled to determine the acceleration of HIO formation using peroxide enzyme catalyst or not using a catalyst as shown at Figure 1 and 2. The residue of H2O2 was measured by the absorbance value of the HIO solution using ABTS as substrate. The addition was intended for produces a green colour of the reaction between the rest of H2O2 and ABTS. The thicker the green colour the higher of absorbance. The decrease of absorbance value could be interpreted as the acceleration of HIO acid formation. Based on Fig. 1 and 2, it can be seen that the reaction between H2O2 and KI without enzyme run slower than those of enzyme addition. The velocity reaction of both compounds could be seen at the interval of spectrophotometric absorbance. This is because enzymes accelerate the reaction process of the formation of new compounds and the new compound from that reaction was HIO. This is in accordance with the opinion from other researcher [8] which states that peroxidase enzyme as catalysts of the oxidation of iodide into HIO. The more enzymes were given, the faster the reaction changes may occur. The speed of reaction may also increase with increasing concentration enzyme. The concentration of enzymes was expressed in units of units (U) and this research used 4.5 U. This amount of concentration was determined as eligible concentration to analyse the rate of reaction. As time of reaction, the enzyme might stop working resulting the decrease in reaction speed. The initial reaction speed generated large value then followed by smaller concentration of substrate's reduction. This can be seen at Table 1 where at the absorbance of H2O2 residue with enzyme was 0.092 to 0.089 in ten minutes starting from 40 to 50, then the reaction started to run slowly. Based on Table 1, the higher decrease in residual value of H2O2 indicated the more HIO was formed, meanwhile the absorbance of residue H2O2 without enzyme showed that the high residual value of H2O2 and it indicated that the HIO formed was still less in concentration. This is in accordance with the opinion from other researcher [5] which states that as the decline in the value of hydrogen peroxide concentration and the halide ions used in the peroxidase system, the higher concentration in final product of peroxidase systems.  One of the factor that might affected the formation of HIO compound was the composition of the substrate. This was due to the peroxidase enzyme's ability to catalyse the oxidation reaction of KI with H2O2. According to other researcher [9], lactoperoxidase would be actively optimized when a state of sufficient ionic halide or pseudohalide. Meanwhile, the presence of an abundant substrate was also not providing the high concentration of HIO. According to previous researcher [10], the redox reactions existed in the peroxidase system could only occur if the reduction capability of the enzyme was weaker than the reduction capability of the substrate used. In addition to these factors, several things might also affected redox reactions in peroxidase systems such as anion size, anion access and anion ability to bind [11]. Besides these factors, the acidity level could also affect the reaction between H2O2 and KIO. According to other researcher [12], the acidity might hinder the action of hydrogen peroxide to oxidise iodine.

Conclusion
The peroxidase enzyme might accelerate the reaction process of peroxidase system for generating HIO and resulting minimum concentration of residual substrate. The maximum amount of production of HIO could be determined through the minimum rate of reaction that was reached at minute 40 th of reaction time.