Biochemical and spectroscopic characterization of morning glory peroxidase from an invasive and hallucinogenic plant weed Ipomoea carnea.
A novel heme peroxidase MGP from the latex of Ipomoea carnea subsp. fistulosa (morning glory) belonging to the Convolvulaceae family was purified to homogeneity using ammonium sulfate precipitation, anion exchange, hydrophobic interaction, and gel filtration chromatography. The enzyme is glycosylated and has a molecular mass of 42.06 kDa (MALDI-TOF) and an isoelectric point of pH 4.3. The enzyme has high yield, broad substrate specificity, and a high stability toward pH, temperature, chaotrophs, and organic solvents. The extinction coefficient (epsilon 280 (1%)) of the enzyme was estimated as 20.56 and it consists of 13 tryptophan, 9 tyrosine, and 8 cysteine residues forming 4 disulfide bridges. There is significant effect of inhibitors targeting S-S bridges (mercaptoethanol, l-cysteine, glutathione), as well as of inhibitors targeting heme (sodium azide and hydroxylamine) on peroxidase activity, whereas inhibition was not observed with ethylmaleinimide due to the absence of reduced cysteine in the enzyme. Polyclonal antibodies against the enzyme have been raised in rabbit, and immunodiffusion suggests that the antigenic determinants of MGP are unique. The N-terminal sequence of MGP (D-E-A-C-I-F-S-A-V-K-E-V-V-D-A) exhibited considerable similarity to the sequence of other known plant peroxidases. Spectroscopic studies (absorbance, fluorescence, and circular dichroism) reveal that MGP has secondary structural features with alpha/beta type with approximately 20% alpha-helicity.
Patel
Ashok Kumar
AK
Molecular Biology Unit, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005, India.
Singh
V K
VK
Moir
A J
AJ
Jagannadham
Medicherla V
MV
D000595:Amino Acid Sequence; D004795:Enzyme Stability; D006213:Hallucinogens; D027741:Ipomoea / Q000201:enzymology*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D009195:Peroxidase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
D006213:Hallucinogens; D010446:Peptide Fragments; D009195:Peroxidase