An efficient protocol for the extraction of pigment-free active polyphenol oxidase and soluble proteins from plant cells

Abstract

The elimination of brownish pigments from plant protein extracts has been a challenge in plant biochemistry studies. Although numerous approaches have been developed to reduce pigments for enzyme assays, none has been able to completely remove pigments from plant protein extracts for biochemical studies. A simple and effective protocol was developed to completely remove pigments from plant protein extracts. Proteins were extracted from red anthocyanin-rich transgenic and greenish wild-type tobacco cells cultured on agar-solidified Murashige and Skoog medium. Protein extracts from these cells were brownish or dark due to the pigments. Four approaches were comparatively tested to show that the diethylaminoethyl (DEAE)-Sephadex anion exchange gel column was effective in completely removing pigments to obtain transparent pigment-free protein extracts. A Millipore Amicon^® Ultra 10K cut-off filter unit was used to effectively desalt proteins. Moreover, the removal of pigments significantly improved the measurement accuracy of total soluble proteins. Furthermore, enzymatic assays using catechol as a substrate coupled with high-performance liquid chromatography analysis demonstrated that the pigment-free proteins not only showed polyphenol oxidase (PPO) activity but also enhanced the catalytic activity of PPO. Taken together, this protocol is effective for extracting pigment-free plant proteins for plant biochemistry studies. A simple and effective protocol was successfully developed to not only completely and effectively remove anthocyanin and polyphenolics-derived quinone pigments from plant protein extracts but also to decrease the effects of pigments on the measurement accuracy of total soluble proteins. This robust protocol will enhance plant biochemical studies using pigment-free native proteins, which in turn increase their reliability and sensitivity.

Keywords: anthocyanin; diethylaminoethyl (DEAE)-sephadex anion exchange gel column; pigments; plant protein extraction; polyphenolics; quinones.

Graphical Abstract

Figure 1.

Different calli cultured on agar-solidified medium and colors of crude protein extracts. (a, b) transgenic red calli cultured for 15 days (a) and crude protein extract with dark color (b). (c, d) Greenish wild-type calli cultured for 15 days (c) and crude protein extract with light brownish color (d)

Figure 2.

Four methods and steps used to remove pigments from crude protein extracts. (a) Step of methods I, II, and III reported in literatures for removal of pigments using PVP and PVPP; (b) steps of our new method (IV) for removal of pigments

Figure 3.

Comparison of pigment removal results obtained with different methods. (a–c) Colors of crude protein extracts from P3 and 6R calli after treated with PVP (method I) (a), PVPP (method II) (b), PVPP and (NH4)₂SO4 (method III) (c); (d) colors of crude protein extracts from P3 and 6R calli after pigment removal using the new method IV; (e) pigment-free protein extracts were obtained from crude protein extracts in (a–c) resulted from treatment of PVP, PVPP, and PVPP and (NH4)₂SO₄ after re-treated using the new method IV

Figure 4.

An SDS-PAGE image shows band profiles from protein extracts using four different methods. All crude proteins were extracted from red 6R cells. Protein extracts from methods I, II, and III were retreated with the new method IV. Protein extracts prior to and after treatment using the method IV were loaded gels side by side

Figure 5.

Polyphenol oxidase assay using catechol as substrate. (a) HPLC profiles showing catalytic products from incubations of catechol and pigment-free crude protein extracts from the new method IV. Catechol (1.0 mM) was incubated with pigment-free crude protein extracts (PE) and BSA for 45 min, respectively. Catechol (substrate, S) and pigment-free crude protein extract (PE) alone were used as controls, respectively. Absorbance values and chromatographs were recorded at 280 nm. Three new products 1, 2, and 3 are formed from catechol catalyzed by PPO. 6R PE+S: 6R protein extract from method IV was incubated with substrate catechol, 6R PE: 6R protein extract from method IV without substrate, S: substrate alone without protein extract, BSA+S: BSA was incubated with substrate, and MeOH: methanol solvent used as control. (b) Comparison of peak area values of catechol before and after incubation (“*” and “**” mean significant difference, P-value ≤.05, t-value: 2.07). (c) Production comparison of reaction products from crude enzyme extracts before and after the pigment removal using the new Method IV. Reaction using catechol was carried out in twenty minutes and absorbance values for products were recorded for each 30 s at 420 nm on a UV spectrophotometry. Four bars labeled “*” or “**” alone mean no significant difference. “Non-treatment” in panel c means that after extraction, crude protein extracts were immediately used to test PPO activity. ANOVA and Tukey’s test were performed to evaluate significant differences between non-treatment, methods I, II, and III, and method IV (Supplementary Result 4). In Tukey’s test, for non-treatment (n) vs. method IV (IV), M_IV-n (0.0190) > q₁ (0.0154); for method I (I) vs. method IV, M_IV-I (0.0267) > q₂ (0.0217), for method II (II) vs. method IV (IV), M_IV-II (0.0246) > q₃ (0.0200); for method III vs. method IV, M_IV-III (0.0177) > q₄ (0.0144). Bars between labels with “*” and “**” mean significant difference, while bars labeled by the same “*” or “**” mean insignificant difference