RESUMEN
A CUA center engineered into Pseudomonas aeruginosa azurin was studied by metal substitution. Metal-binding properties were determined by electronic absorption (UV-vis) and electrospray ionization mass spectrometry (ESI-MS). The metal-binding site readily binds thiophilic metal ions, such as Hg(II), Ag(I), Cu(I), Cd(II), and Au(I). Harder metal ions, like Co(II), bind to apo-CuA-azurin only under basic conditions (pH 9.1-9.2). The results obtained from these studies indicate that two factors influence metal binding in CuA azurin: (1) the site favors metal combinations which produce an overall +3 charge, and (2) the site binds soft, thiophilic metal ions. The results demonstrate the remarkable ability of the CuA center to maintain valence delocalization of its native metal ions and to ensure redox accessibility of only one of the two redox couples (i.e., [Cu(1.5)...Cu(1.5)]<==> [Cu(I)...Cu(I)]) under physiological conditions. These findings may lead to the preparation of new metal ion derivatives and can serve as a basis for understanding this efficient electron transfer center.
Asunto(s)
Azurina/metabolismo , Cobre/química , Metales/metabolismo , Pseudomonas aeruginosa/química , Azurina/química , Modelos Moleculares , Unión Proteica , Ingeniería de Proteínas , Análisis EspectralRESUMEN
Microvascular anastomotic patency is the most important factor in determining a successful outcome in free-flap transfers. End-to-end and end-to-side techniques have been shown to provide equivalent arterial patency rates in clinical and basic science studies, and end-to-side anastomoses have been used extensively in microsurgical reconstruction. Nevertheless, the effect of venotomy shape on the patency of venous end-to-side anastomoses has not been previously reported. The purpose of this study was to compare the patency rates of end-to-side anastomoses using different techniques in both arteries and veins. In total, 104 Sprague-Dawley rats were subdivided into four groups. The rats were anesthetized, and anastomosis was performed on either the femoral artery or vein on the right with the left used as control. Vesselotomy was varied between an end-to-side hole and an end-to-side slit with patency measured immediately following surgery and at 2 weeks. No significant difference in patency or histology between these techniques was demonstrated in any group. We conclude there is no difference in patency rate between the two techniques in arterial or venous vesselotomies; however, in small vessels < 1.5 mm, the slit technique is technically easier, and clinical recommendations are given.
Asunto(s)
Anastomosis Quirúrgica/métodos , Microcirugia/métodos , Colgajos Quirúrgicos/irrigación sanguínea , Grado de Desobstrucción Vascular/fisiología , Animales , Arterias/cirugía , Femenino , Masculino , Ratas , Ratas Sprague-Dawley , Venas/cirugíaRESUMEN
The X-ray structure of an engineered purple CuA center in azurin from Pseudomonas aeruginosa has been determined and refined at 1.65 A resolution. Two independent purple CuA azurin molecules are in the asymmetric unit of a new P21 crystal, and they have nearly identical conformations (rmsd of 0.27 A for backbone atoms). The purple CuA azurin was produced by the loop-engineering strategy, and the resulting overall structure is unperturbed. The insertion of a slightly larger Cu-binding loop into azurin causes the two structural domains of azurin to move away from each other. The high-resolution structure reveals the detailed environment of the delocalized mixed-valence [Cu(1.5).Cu(1.5)] binuclear purple CuA center, which serves as a useful reference model for other native proteins, and provides a firm basis for understanding results from spectroscopic and functional studies of this class of copper center in biology. The two independent Cu-Cu distances of 2.42 and 2.35 A (with respective concomitant adjustments of ligand-Cu distances) are consistent with that (2.39 A) obtained from X-ray absorption spectroscopy with the same molecule, and are among the shortest Cu-Cu bonds observed to date in proteins or inorganic complexes. A comparison of the purple CuA azurin structure with those of other CuA centers reveals an important relationship between the angular position of the two His imidazole rings with respect to the Cu2S2(Cys) core plane and the distance between the Cu and the axial ligand. This relationship strongly suggests that the fine structural variation of different CuA centers can be correlated with the angular positions of the two histidine rings because, from these positions, one can predict the relative axial ligand interactions, which are responsible for modulating the Cu-Cu distance and the electron transfer properties of the CuA centers.