Class III Peroxidases

Abstract

Class III peroxidases are heme-containing proteins of the secretory pathway with an extremely high number of isoenzymes, indicating the tremendous and important functions of this protein family. This chapter describes fractionation of the cell in subproteomes, their separation by polyacrylamide gel electrophoresis (PAGE) and visualization of peroxidase isoenzymes by heme and specific in-gel staining procedures. Soluble and membrane-bound peroxidases were separated by differential centrifugation. Aqueous polymer two-phase partitioning and discontinuous sucrose density gradient were applied to resolve peroxidase profiles of plasma membranes and tonoplast. Peroxidase isoenzymes of subproteomes were further separated by PAGE techniques such as native isoelectric focussing (IEF), high resolution clear native electrophoresis (hrCNE), and modified sodium dodecyl sulfate (modSDS)-PAGE. These techniques were used as stand-alone method or in combination for two-dimensional PAGE.

Two-dimensional phos-tag zymograms for tracing phosphoproteins by activity in-gel staining

“…vroege, Lüneburg, Germany). Soluble proteins of corn and pea were separated by differential centrifugation from the microsomal fraction as described elsewhere (Meisrimler et al, 2011 ; Lüthje et al, 2014 ) and stored at −76°C until use. Total protein extracts from corn roots (12 days) were acquired by grinding with liquid nitrogen, followed by extraction in Tris-HCl buffer pH 7.6 (50 mM NaCl, 1 mM DTT, 1% Triton X-100) for 1 h at 4°C.…”

“…Before samples were applied, a pre-run of the gels was accomplished for 45 min at 30 V with no further restrictions. Electrophoresis conditions were described by Lüthje et al ( 2014 ). For NEPHGE, the polarity and the IEF buffer system was reversed (Figure 1 ).…”

“…Standard hrCNE was casted as a gradient gel of 4–16% as described by Lüthje et al ( 2014 ). The content of the phos-tag hrCNE was similar to the standard hrCNE containing additionally phos-tag (Wako Chemicals GmbH, Neuss, Germany) and Mn(II)Cl 2 .…”

“…The standard zymograms (non-reducing SDS-PAGE, without heating of the protein sample) are commonly used for proteolytic enzymes (Vandooren et al, 2013 ), but combination of different electrophoresis methods and various in-gel activity staining makes the method applicable for different enzyme activities (Manchenko, 2002 ). In the past, activity in-gel stainings after isoelectric focusing (IEF) slab gels were reported for different enzymes, e.g., malate dehydrogase, peroxidase, quinone reductase, Fe(III)-reductase, superoxide dismutase, catalase and others (Mika et al, 2010 ; Meisrimler et al, 2011 ; Kukavica et al, 2012 ; Lüthje et al, 2014 ). In the standard IEF-PAGE, protein separation is based on their pI and oriented from basic to acidic pH.…”

“…To date, native PAGE methods, as the described above, are well-established systems but none of them is usually named a standard method. Especially the combination of non-reducing IEF/NEPHGE with one or the other native PAGE in the second dimension has rarely been performed and is scarcely found in the literature, but has been important for two-dimensional zymograms (Lüthje et al, 2014 ). After multiple modifications and trials we now developed a protocol which we report in the present paper.…”