Impact of low- and high-molecular-mass components of human serum on NAMI-A binding to transferrin

Imidazolium trans-tetrachloridodimethylsulfoxideimidazolruthenate(III), NAMI-A, a novel antimetastatic ruthenium complex was investigated towards affinity to transferrin (Tf), whether Tf–Ru adducts might be formed after its intravenous injection. Studies were focused on the holotransferrin due to its preferential binding to transferrin receptor. Here, we showed that holotransferrin is able to bind NAMI-A as readily as apotransferrin. The simulation of biological conditions of human serum performed by application of simplified serum models allowed to analyse ruthenium distribution between transferrin and albumin. The presence of physiological concentration of albumin (ca. 18-fold excess over Tf) resulted in a twofold decrease of ruthenium binding to Tf. Interestingly, the introducing of low-molecular-mass components of serum dramatically increased the ruthenation of Tf. Intermolecular competition binding studies between transferrin and albumin showed that both proteins bound similar amount of ruthenium species. Investigation of NAMI-A binding to Tf in human serum showed that this protein was not the major binding partner for Ru complex. However, in spite of many competing proteins still the ruthenation of Tf was observed. The lack of free Ru species (protein unbounded) after incubation with human serum allowed to make an assumption of high affinity of NAMI-A towards serum proteins

Temperature- and pressure-dependent stopped-flow kinetic studies of jack bean urease : implications for the catalytic mechanism

Urease, a Ni-containing metalloenzyme, fea- tures an activity that has profound medical and agricultural implications. The mechanism of this activity, however, has not been as yet thoroughly established. Accordingly, to improve its understanding, in this study we analyzed the steady-state kinetic parameters of the enzyme (jack bean), K M and k cat , measured at different temperatures and pres- sures. Such an analysis is useful as it provides information on the molecular nature of the intermediate and transition states of the catalytic reaction. We measured the parame- ters in a noninteracting buffer using a stopped-flow tech- nique in the temperature range 15–35 ° C and in the pressure range 5–132 MPa, the pressure-dependent mea- surements being the first of their kind performed for urease. While temperature enhanced the activity of urease, pres- sure inhibited the enzyme; the inhibition was biphasic. Analyzing K M provided the characteristics of the formation of the ES complex, and analyzing k cat , the characteristics of the activation of ES. From the temperature-dependent measurements, the energetic parameters were derived, i.e. thermodynamic D H o and D S o for ES formation, and kinetic D H = and D S = for ES activation, while from the pressure- dependent measurements, the binding D V b and activation D V 6 ¼ cat volumes were determined. The thermodynamic and activation parameters obtained are discussed in terms of the current proposals for the mechanism of the urease reaction, and they are found to support the mechanism proposed by Benini et al. ( Structure 7:205–216; 1999), in which the Ni–Ni bridging hydroxide—not the terminal hydroxide—is the nucleophile in the catalytic reaction

Encapsulation of iron-saturated lactoferrin for proteolysis protection with preserving iron coordination and sustained release

Lactoferrin (Lf) is a globular glycoprotein found mainly in milk. It has a very high affinity for iron(III) ions, and its fully saturated form is called holoLf. The antimicrobial, antiviral, anticancer, and immunomodulatory properties of Lf have been studied extensively for the past two decades. However, to demonstrate therapeutic benefits, Lf has to be efficiently delivered to the intestinal tract in its structurally intact form. This work aimed to optimize the encapsulation of holoLf in a system based on the versatile Eudragit® RS polymer to protect Lf against the proteolytic environment of the stomach. Microparticles (MPs) with entrapped holoLf were obtained with satisfactory entrapment efficiency (90–95%), high loading capacity (9.7%), and suitable morphology (spherical without cracks or pores). Detailed studies of the Lf release from the MPs under conditions that included simulated gastric or intestinal fluids, prepared according to the 10th edition of the European Pharmacopeia, showed that MPs partially protected holoLf against enzymatic digestion and ionic iron release. The preincubation of MPs loaded with holoLf under conditions simulating the stomach environment resulted in the release of 40% of Lf from the MPs. The protein released was saturated with iron ions at 33%, was structurally intact, and its iron scavenging properties were preserved

Inhibition of matrix metalloproteinases and cancer cell detachment by Ru(II) polypyridyl complexes containing 4,7-diphenyl-1,10-phenanthroline ligands : new candidates for antimetastatic agents

Primary tumor targeting is the dominant approach in drug development, while metastasis is the leading cause of cancer death. Therefore, in addition to the cytotoxic activity of a series of Ru(II) polypyridyl complexes of the type [Ru(dip)(2)L](2+) (dip: 4,7-diphenyl-1,10-phenanthroline while L = dip; bpy: 2,2′-bipyridine; bpy-SC: bipyridine derivative bearing a semicarbazone 2-formylopyridine moiety; dpq, dpq(CH(3))(2), dpb: quinoxaline derivatives) their ability to inhibit cell detachment was investigated. In vitro studies performed on lung cancer A549 cells showed that they accumulate in cells very well and exhibit moderate cytotoxicity with IC(50) ranging from 4 to 13 µM. Three of the studied compounds that have dip, bpy-SC, or dpb ligands after treatment of the cells with a non-toxic dose (<(1)/(2)IC(50)) enhanced their adhesion properties demonstrated by lower detachment in the trypsin resistance assay. The same complexes inhibited both MMP-2 and MMP-9 enzyme activities with IC(50) ranging from 2 to 12 µM; however, the MMP-9 inhibition was stronger. More detailed studies for [Ru(dip)(2)(bpy-SC)](2+), which induced the greatest increase in cell adhesion, revealed that it is predominately accumulated in the cytoskeletal fraction of A549 cells. Moreover, cells treated with this compound showed the localization of MMP-9 to a greater extent also in the cytoskeleton. Taken together, our results indicate the possibility of a reduction of metastatic cells escaping from the primary lesion to the surrounding tissue by prevention of their detachment and by influencing the activity of MMP-2 and MMP-9