Screening And Mechanism Of Action Of Horseradish Peroxidase Thermal Stabilizer

Horseradish peroxidase (HRP, EC 1.11.1.7) is the most important member of the plant peroxidase family. It has been studied extensively and been used widely in scientific research and industry including the aspects of organic synthesis, biotransformation, chemiluminescent assays, immunoassays, biomedicine as well as environmental engineering. Keeping an enzyme in its native form and intrinsic activity is crucial for its roles in biological catalysis. However, an enzyme is usually separated from its native matrix prior to its pratical uses, which can result in the enzyme become unstable and extremely easy to lose activity. Many strategies have been developed for stabilizing labile enzymes, including physical entrapments, covalent and non-covalent immobilization, chemical derivation, genic engineering and crystallization. In the present study, a simple, low-cost and effective thermal stabilizer of HRP has been screened from metal salts and native macromolecules. The resultant ingredient, constituting of magnesium sulfate and gelatin, demonstrated strong protective effect on the intrinsic activity of HRP both under thermal stress and room temperature. In the presence of the stabilizer, the enzymatic activity of HRP remained 89% after stored for 80 h at 50℃and 57% for 90 days at room temperature. In the absence of the stabilizer, the enzymatic activity of HRP was very low and the percentage were 30% and 1% respectively. Thermal alterations of HRP structure in the absence and presence of the stabilizers have been explored by using UV absorption spectra at 402 nm (Soret band), intrinsic fluorescence and ANS fluorescence. The results suggested that the ingredient of magnesium sulphate and gelatin attenuated the extent of unfolding of HRP and therefore the native conformation was stabilized. The HRP stabilizer reported in this study may extend its use to other enzymes, particularly to the enzymes immobilized on solid supports.Part I Experimental methods1. Measurements of enzymatic activityThe HRP- H₂O₂- TMB (3,3',5,5'-tetramethylbenzidine) system has been used for measurement of enzymatic activity of HRP. TMB is oxidized during the enzymatic degradation of H₂O₂ by HRP. The oxidized product of TMB has a deep blue color with a maximum absorbance at 700 nm. Experimental conditions in terms of ratio between reactants, buffer, reactive time and concentration of substrate have been explored and optimized. HRP activity assay was performed by using substrate concentration of 1.2 mM for TMB and 1.5μM for H₂O₂ respectively. Under such a condition, the absorbance of the oxidative product of TMB at 700 nm had a linear relationship with the intrinsic activity of HRP. 2. Screening of thermal stabilizers for HRPHRP powder was dissolved in distilled water and mixed with additives. Aliquots of enzyme solution were added to a microplate and incubated in the air under thermal stress or room temperature. For enzymatic assay, samples were dissolved in distilled water and aliquots of solution were mixed with TMB and H₂O₂. Kinetics of enzymatic reaction was recorded at 700 nm and HRP activity was calculated in respect to the kinetic curve using fresh-prepared HRP as a control.3. Soret Band spectra of HRPThe Soret Band spectra of HRP were measured by scanning the absorbance curve of HRP samples between 360-450 nm in the presence and in the absence of additives.4. Intrinsic fluorescence of HRPFluorescence measurements were performed on a Perkin Elmer LS-55 spectrofluorimeter. Excitation was carried out at 295 nm in order to avoid the contribution of tyrosine to the intrinsic fluorescence spectrum of HRP. Spectra were recorded in the range of 310-490 nm and a emission peak was found at 330 nm.5. ANS fluorescence upon binding to HRPANS (8-anilinonaphthalene-1-sulfonic acid) is a fluorescent dye for probing the surface hydrophobicity of a protein. A Perkin Elmer LS-55 spectrofluorimeter was used for fluorescence measurements with an excitation at 350 nm and the emission spectra were recored and analyzed between 400-600 nm.Part II Results1. Thermal stabilizers for HRPA variety of compounds including metal salts and natural macromolecules have been screened for stabilizing HRP under thermal stress and room temperature. Magnesium sulfate and gelatin were found to be able to stabilize HRP. A synergical effect of magnesium sulphate and gelatin was observed. An ingredient constituting magnesium sulphate and gelatin was used as a thermal stabilizer for HRP and its protective efficiency was explored and evaluated. In the presence of the stabilizer, the enzymatic activity of HRP remained 89% after stored for 80 h at 50℃and 57% for 90 days at room temperature.2. Soret Band spectra of HRP under thermal stressA porphyrin is located at the active site of HRP, which afford the protein with a characteristic absorbance at 402 nm (Soret Band). Soret Band reflects the change in the redox state of the heme iron of porphyrin structure and provides information about possible denaturation or unfolding of HRP. The results indicated that gelatin and the ingredient stabilizer had protective effect on HRP against thermal denaturation. Under thermal stress, the Soret absorbance of HRP decreased significantly. While in the presence of magnesium sulphate and gelatin, the spectrum was kept unchanged for 24 hr under incubation at 60℃.3. Tryptophan fluorescenceTryptophan fluorescence has the maximal absorb spectra when excitation wavelength is 295 nm. Change of absorb spectrum is related with structure of protein. The intrinsic fluorescence of the enzyme is highly dependent on the fluorescence energy transfer from tryptophan to heme. Thus, changes in the structure of the heme cavity affecting the distance/orientation between the heme and the tryptophan would affect the intrinsic fluorescence of the enzyme. Denaturation of heme proteins is generally accompanied by an increase in the fluorescence intensity and a red shift of the fluorescence maximum as a result of a change in the tryptophan microenvironment. The native HRP has a maximal fluorescent emission at 340 nm. This emission peak was red-shifed and transformed into an M-form as a consequence of HRP unfolding. In contrast, the alteration in tryptophan fluorescence of HRP was attenuated by the ingredient of magnesium sulfate and gelatin. The result suggests that the conformation of HRP become more stable upon the addition of thermal stabilizer.4. ANS fluorescenceThe results of ANS fluorescence showed that the surface hydrophobicity of HRP increased upon thermal denaturation. In the presence of the ingredient of magnesium sulfate and gelatin, the blue-shift of ANS fluorescence was inhibited, indicating the stabilizer was capable to maintain HRP in native conformation under thermal stress.Part III ConclusionsKeeping an enzyme in its native form with high catalytic activity is of great significance. In the present study, thermal stabilizers of HRP were screened and their mechanisms of action were explored. The results indicated that thermal stability of HRP was enhanced by magnesium sulphate, gelatin and their ingredients. A synergic effect of magnesium sulphate and gelatin was observed. In the presence of the stabilizer, the life span of HRP was extended and the enzymatic activity was maintained in a period much longer than that of the naked enzyme. Thermal alterations of HRP structure in the absence and presence of the stabilizers were explored by using UV absorption spectra at 402 nm (Soret band), intrinsic fluorescence and ANS fluorescence. The results suggested that the ingredient of magnesium sulphate and gelatin attenuated the extent of unfolding of HRP under thermal stress and therefore the native conformation was stabilized.