The conformational stability of aponeocarzinostatin, an all-beta-sheet protein with 113 amino-acid residues, is investigated by thermal-induced equilibrium unfolding between pH 2.0 and 10.0 with and without urea. At room temperature, the protein is stable in a pH range of 4.0-10.0, whereas the stability of the protein drastically decreases below pH 4.0. The thermal unfolding of aponeocarzinostatin is reversible and follows a two-state mechanism. By two-dimensional unfolding studies, the enthalpy change, heat capacity change, and free energy change for unfolding of the protein are estimated. Circular dichroism profiles suggest that this protein undergoes both heat- and cold-induced unfolding. The ellipticity changes at far- and near-UV circular dichroism suggest that the tertiary structure is disrupted but the secondary structure remains folded at low temperatures. Interestingly, the labile enediyne chromophore, which is highly stabilized by the protein, is able to protect the protein against cold-induced unfolding, but not the heat-induced unfolding.

Most conjugate proteins undergo both conformational and stability changes on ligand removal. When architecture remains unchanged in the protein holo and apo forms, it is uncertain whether the protein stability also remains unaltered in both of the forms. Neocarzinostatin (NCS), a chromoprotein possessing a potent enediyne chromophore stands for such an instance. Protein-chromophore interaction has not been thoroughly explored previously due to a lack of strategies to independently and simultaneously monitor changes in the NCS conjugates. Here we report a method by which one can detect the signal exclusively from only one of the NCS conjugates without the spectral interference from the other. Stability of the NCS protein is significantly correlated to the proteinbound chromophore, irrespective of denaturation by heat, pH, urea, or ethanol. Despite the similarity in protein backbone conformation, protein stability of the NCS holo form diminishes and equalizes to that of the apo form when the chromophore is released and degraded. Although the enediyne chromophore is highly unstable, it intriguingly protects the protein by which it is protected. Significant mutual reliance between the carrier protein and its naturally associated ligand unveils important information on the NCS drug stability. KeywordsEnediyne; Neocarzinostatin; Chromophore; Ligand binding; Holoprotein; Protein stability Recently, much attention has been focused on the protein-ligand conjugation. Most conjugate proteins undergo changes in their conformation and subsequently, in stability on ligand removal [1][2][3]. For example, binding of heme to cytochrome changes the protein conformation and consequently, increases the protein stability [4]. It is rather uncommon for conjugate proteins to adopt a like conformation with or without the ligand. This includes azurin (β protein with a copper ion), flavodoxin (α/β protein with a flavin mononucleotide) [5], neocarzinostatin (NCS, 1 β protein with an enediyne chromophore) [6,7] and so on. In spite of structural similarity, the holo form of azurin is more stable than its apo form [8]. The flavodoxin holo form, in contrast, has little changes in either conformation or stability on its cofactor removal *Corresponding author. Fax: +886 4 22862547. E-mail address: chdhchin@dragon.nchu.edu.tw (D.-H. Chin). 1 Abbreviations used: NCS, neocarzinostatin; holoNCS, neocarzinostatin chromoprotein conjugate complex; apoNCS, apoprotein of neocarzinostatin; NCS-C, neocarzinostatin chromophore; NCS-C(b), protein-bound neocarzinostatin chromophore; CD, circular dichroism; HPLC, high-performance liquid chromatography; EtBr, ethidium bromide; NOESY, nuclear Overhauser effect spectroscopy; NMR, nuclear magnetic resonance; T m , temperature at which half of the protein is unfolded; T r , temperature at which half of the NCS-C(b) is released from NCS complex; C m , concentration of denaturant at which half of the protein is unfolded; C r , concentration of denaturant at which half of the NCS-C(b) is released from NCS com...

Electrocatalytic oxidative cyclization of dithiothreitol (DTT(SH)2) to a disulfide product was demonstrated on a Nafion/lead-ruthenium oxide pyrochlore chemically modified electrode (NPyCME). The process at the NPyCME with DTT(SH)2 is similar to the behaviour of protein in a disulfide linkage, which can be demonstrated by product analysis using HPLC coupled with UV spectroscopy. A possible electrocatalytic mechanism for DTT(SH)2 oxidation to dihydroxydithiane [i.e. cyclized DTT(S-S)] on the NPyCME was proposed in terms of Py-Ru(IV)/Py-Ru(VI) redox active sites. This physical aspect was further utilized for high precision analytical assays using flow injection analysis (FIA), with a linearity up to 50 microM and a detection limit (S/N = 3) of 28 nM (8.64 pg) in a 20 microL sample loop. This is the most sensitive method ever reported for DTT(SH)2 detection assays. The interference from dissolved oxygen, disulfide and glucose is almost negligible. The present method offers an easy route for extension to redox-related protein studies.