Five monomeric hemocyanin subunits from Portunus trituberculatus: purification, spectroscopic characterization, and quantitative evaluation of phenol monooxygenase activity.
Five kinds of monomeric subunits of arthropod hemocyanin have been isolated from swimming crab Portunus trituberculatus hemolymph. The copper centers holding a peroxo species, [(μ-η2:η2-peroxo)dicopper(II)], of these subunits exhibited almost the same UV-vis and visible region CD spectroscopic properties, indicating that they have a similar copper coordination geometry and an electronic structure. Under anaerobic conditions, the oxy-forms of the monomeric subunits were stable in 0.5 M borate buffer (pH 9.0) and reacted with 4-methylphenol (p-cresol) to show the phenolases (cresolase/phenol monooxygenase) activity in the presence of urea. To compare the phenolase (monooxygenase) reactivity, the reactivity of the isolated subunits has been examined quantitatively by using a simplified catalytic system, where the initial product catechol is trapped with borate anion of the buffer solution to prevent following catecholase reaction (Yamazaki and Itoh, 2003). The far-UV region CD spectra were measured in order to clarify the relationship between the content of the secondary structure and the phenolase reactivity. Even though the monomeric subunits exhibit a weak catalytic phenol monooxygenase activity, addition of urea (3 M) significantly enhances their catalytic activity. The differences of the phenolase activity among the monomeric subunits has been discussed on the basis of the spectroscopic analysis and reactivity studies in order to shed light on the enzymatic function of the arthropod hemocyanin in vivo.
Fujieda
Nobutaka
N
Department of Material and Life Science, Graduate School of Engineering, Osaka University, Osaka, Japan.
Yakiyama
Aki
A
Itoh
Shinobu
S
D000818:Animals; D002384:Catalysis; D002396:Catechols / Q000737:chemistry; D002942:Circular Dichroism; D003300:Copper / Q000737:chemistry; D003408:Cresols / Q000494:pharmacology; D003445:Crustacea / Q000378:metabolism*; D006433:Hemocyanins / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D002396:Catechols; D003408:Cresols; C032538:4-cresol; D003300:Copper; D006433:Hemocyanins; C034221:catechol