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Active uniform films of horseradish peroxidase (HRP) have been prepared by covalent binding on Si/SiO(2) or glass supports previously activated by silanization and succinylation. Labeling by fluorescent or by Electron Spin Resonance (ESR) probes was used to quantify the surface density of active groups and of horseradish peroxidase. Atomic Force Microscopy (AFM) imaging was used to characterize the surface morphology. We observed that a non-uniform protein adsorption due to physical interactions was present when the supports were not activated for covalent binding and was, in large part, removed by washing. The enzyme deposited by covalent binding formed homogeneous layers with a height in the range 60-90 A. By using a fluorescent label, we calculated a protein density of 3.6 x 10(12) molecules cm(-2) on Si/SiO(2), corresponding to an estimated area per molecule of 2800 A(2) which is in agreement with the value expected on the basis of the crystallographic data considering the formation of a monomolecular layer. The protein density of the layer immobilized on glass was similar (1.9 x10(12) molecules cm(-2)). The enzyme immobilized on both supports showed a k(cat)/K(M) being of the order of 3-5x10(5) M(-1)s(-1) that is 1/20th of free HRP. The half-life time of the activity of the enzyme immobilized by covalent binding was longer than 40 days at 6 degrees C.

Active uniform films of horseradish peroxidase (HRP) have been prepared by covalent binding on Si/SiO2 or glass supports previously activated by silanization and succinylation. Labeling by fluorescent or by Electron Spin Resonance (ESR) probes was used to quantify the surface density of active groups and of horseradish peroxidase. Atomic Force Microscopy (AFM) imaging was used to characterize the surface morphology. We observed that a non‐uniform protein adsorption due to physical interactions was present when the supports were not activated for covalent binding and was, in large part, removed by washing. The enzyme deposited by covalent binding formed homogeneous layers with a height in the range 60–90 Å. By using a fluorescent label, we calculated a protein density of 3.6 × 1012 molecules cm−2 on Si/SiO2, corresponding to an estimated area per molecule of 2800 Å2 which is in agreement with the value expected on the basis of the crystallographic data considering the formation of a monomolecular layer. The protein density of the layer immobilized on glass was similar (1.9 ×1012 molecules cm−2). The enzyme immobilized on both supports showed a kcat/KM being of the order of 3–5×105 M−1s−1 that is 1/20th of free HRP. The half‐life time of the activity of the enzyme immobilized by covalent binding was longer than 40 days at 6°C. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 68: 488–495, 2000.

In the continuous effort to find new metal-based compounds as alternatives to platinum-related anticancer drugs, 11th group metal phosphane compounds have been thoroughly taken into consideration. Tris-arylphosphane metal derivatives have been extensively considered as heteroleptic metal compounds exhibiting remarkable cytotoxic activities. Functional groups in the aryl moieties modulate the activity reinforcing or eliminating it. Previous works have highlighted that the presence of hydrophilic groups in the phosphane ligands, such as COOH or OH, hampers the anticancer activity of gold azolate/PPh3 compounds. To increase the polarity of the triarylphosphane ligand without affecting the activity, we considered the preparation of esters starting from the 4-diphenylphosphane-benzoic acid. The resulting phosphanes are poorer donators than the PPh3, leading to poly-phosphane M(I) compounds, and they exhibit intense emissive properties. A homologous series of L3MX-type compounds (where M = Au and X = Cl, M = Cu and X = BF4, and M = Ag and X = PF6) were obtained with the 4-methoxy-diphenylphosphane benzoate. The homologous metal compounds have been characterized by analytical and spectroscopic methods and, remarkably, their formation was associated with high frequencies of 31P NMR chemical shift variations (5–35 ppm in CDCl3). The new complexes and the ligand were evaluated on sensitive and cisplatin-resistant human tumor cell lines. The ligand is ineffective on cells while the complexes exert a notable antiproliferative effect. The homologous series of the L3MX complexes were able to significantly reduce the cell viability of human triple-negative breast cancer cells (MDA-MB-231), representing the most aggressive subtype of breast cancer, and of ovarian carcinoma (A2780). Among these coinage metal compounds, L3AgPF6 results the most interesting, showing the lowest GI50 values in all cell lines. Interestingly, this silver complex is more cytotoxic than cisplatin, taken as reference drug. The investigation of the mechanism of action of L3AgPF6 in A2780 cells highlighted the induction of the apoptotic pathway, the depolarization of the mitochondrial inner membrane, and a significant accumulation in cells.