Studies On The Purification Of Lotus Root PPO And Environmental Effects On Its Solution Conformations

Polyphenol oxidase (PPO) was extracted by phosphate buffer from Lotus roots, and the best extracting condition was determined. PPO was purified through ammonium sulfate precipitation, Sephadex DEAE A-50 and Sephadex G-75 column chromatography. The purity and molecular weight were determined by SDS-PAGE. The zymologic characteristics of PPO were studied. Environmental effects on PPO's solution conformation were studied by circular dichroism (CD), fluorescence spectrometry (FP) and Fourier transform infrared spectroscopy (FT-IR). The main results are as follows:1. Extraction and purification of PPO from lotus rootsPPO was extracted by disodium hydrogen phosphate - citric acid buffer (including 5% PVPP and 10% NaCl, pH 5.4) for 4 h at 4℃, the stuff liquid ratio being 1: 2.2. PPO was precipitated by 80% saturation of ammonium sulfate, purified by DEAE Sephadex A-50 and Sephadex G-75 gel column, and gained a single band by SDS-PAGE.2. The zymologic characteristics, molecular weight and structure characterization of PPO from Lotus rootThe PPO's substrate is pyrogallol, the optimum pH is 7.0, the optimum temperature is 50℃, Km value is 11 mmol / L. Comparing with the standard protein molecular weight, the SDS-PAGE showed that PPO's molecular weight is about 21 kD.The PPO's amino acid composition was analysised. The number of total amino acids is about 164. The content of non-polar amino acid (Ala, Val, Leu, Ile, Pro, Met, Phe, Trp), polar amino acids (Gly, Ser, Thr, Cys, Tyr, Asp, Glu, Lys, Arg, His), acidic amino acids (Asp, Glu), basic amino acids (Lys, Arg, His) are about 39.62 % , 57.92% , 18.32% and 10.39% respectively. The conformation of PPO was studied by circular dichroism, fluorescence and infrared spectroscopy, the results showed that with the concentration of 0.3 mg/ml, PPO'sα-helix conformation andβ-sheet conformation accounted for 43% and 57% respectively. Fluorescence spectroscopy showed that the largest emissive wavelength was at 329nm which indicated that the Trp residues were partially buried in hydrophobic environment. The Infrared absorption at 1658 cm-1 and 1544 cm-1 were for amide I and amide II respectively, the absorption at 1240 cm-1 for Amide III band was attribute to a-helix conformation.3. Micro-environment effects on the solution conformation of PPO from Lotus rootsSecondary structural changes of PPO at different pH value: As the pH value increased or decreased, PPO's activity and secondary structure changed significantly. When pH <5 and pH>10, PPO' activity reduced significantly,α-helix conformation decreased rapidly,β-sheet and random coil conformation increased distinctively; the largest emissive wavelength red shifted, which indicated that the Trp residueds were exposed to the hydrophilic environment; fluorescence intensity increased as pH value enhanced. pH value affect the PPO's activity by disturbing on the secondary structure of PPO, and PPO's active center may be located in the a-helix conformation, the rational allocation ofα-helix conformation andβ-sheet conformation of PPO may be the suitable conformation to maintain its activity.Secondary structural changes of PPO at different temperatures: PPO's activity increased as temperature increased, when the temperature rose to 60℃, PPO's activity started to decline,α-helix conformation decreased whileβ-sheet conformation and the random coil increased. The fluorescence intensity decreased as the temperature increased and the largest emissive wavelength blue-shifted 3 nm. Amide bands shifted and the infrared absorption decreased at 60℃. Amide III band at 1240 cm-1 which attribute toα-helix conformation moved to 1245 cm-1. From the above , PPO's degeneration temperature is 60℃. 4. The interaction between substrates and PPO from lotus rootsThe results showed that PPO's activity is highest using pyrogallol as substrate. Circular Dichroism results showed that when interacting with pyrogallol, catechol, gallic acid, the PPO'sα-helix conformation decline significantly,β-sheet conformation increase and random coil go up to 16.5%. When interacting with pyrogallol, PPO's fluorescence intensity decreased. When interacting with catechol, its fluorescence intensity increases significantly. When interacting with gallic acid, the Trp residues red-shifted most significantly, it may be caused by excessive exposure. From the changes of solution conformation, excessive exposure of Trp residues may be disadvantageous to maintain the active conformation of PPO.Secondary comformation changes of PPO's interaction with endogenous substrate were studied by circular dichroism and fluorescence spectrometry. The results showed thatβ-turn could be observed in the secondary structure of PPO, theα-helix conformation reduced from 37.1% to 12.3%,β-sheet conformation droped from 62.9% to 58.5%, random coil rose up to 18.9%. Fluorescence spectrometry showed a increase in Trp residues fluorescence intensity, the largest emissive wavelength red shifted to 348 nm, this indicated that Trp residues partly exposed to hydrophobic environment,β-turn may be an important middle conformation in the secondary structure of PPO when interact with endogenous substrates, Trp residue's exposure to the partly hydrophobic environment may play a role in the maintenance of the active conformation.5. The interaction between inhibitors and PPO from Lotus rootThe activity changes of PPO interacting with different inhibitor were determined. The inhibitory effects of several commonly used inhibitors as Inhibitor A, calcium chloride, homocysteine, citric acid, EDTA on PPO's activity were investigated. The results showed that Inhibitor A, homocysteine and calcium chloride had stronger effects on the inhibition of the activity; the inhibiting rates are 96.2%, 86.8% and 82.4% respectively. Citric acid and EDTA had lower inhibitory effects.Circular Dichroism results showed that the Inhibitor A seriously damaged theα-helix conformation. Calcium chloride, homocysteine, and citric acid EDTA could damage the α-helix partly. PPO'sβ-sheet and random coil conformation increased in general by inhibitors. Fluorescence spectrometry results showed that when calcium chloride, homocysteine, citric acid and EDTA combined with PPO, the emission spectra got a similar-type change; when Inhibitor A added, the emission spectra got a distinguish change, the largest emissive wavelength red-shifted most significantly, indicating that the Trp residues expose most in the hydrophilic environment.