RESUMO
The Rieske protein from Thermus thermophilus (TtRp) and a truncated version of the protein (truncTtRp), produced to achieve a low-pH crystallization condition, have been characterized using UV-visible and circular dichroism spectroscopies. TtRp and truncTtRp undergo a change in the UV-visible spectra with increasing pH. The LMCT band at 458 nm shifts to 436 nm and increases in intensity. The increase at 436 nm versus pH can be fit using the sum of two Henderson-Hasselbalch equations, yielding two pK(a) values for the oxidized protein. For TtRp, pK(ox1) = 7.48 +/- 0.12 and pK(ox2) = 10.07 +/- 0.17. For truncTtRp, pK(ox1) = 7.87 +/- 0.17 and pK(ox2) = 9.84 +/- 0.42. The shift to shorter wavelength and the increase in intensity for the LMCT band with increasing pH are consistent with deprotonation of the histidine ligands. A pH titration of truncTtRp monitored by circular dichroism also showed pH-dependent changes at 315 and 340 nm. At 340 nm, the fit gives pK(ox1) = 7.14 +/- 0.26 and pK(ox2) = 9.32 +/- 0.36. The change at 315 nm is best fit for a single deprotonation event, giving pK(ox1) = 7.82 +/- 0.10. The lower wavelength region of the CD spectra was unaffected by pH, indicating that the overall fold of the protein remains unchanged, which is consistent with crystallographic results of truncTtRp. The structure of truncTtRp crystallized at pH 6.2 is very similar to TtRp at pH 8.5 and contains only subtle changes localized at the [2Fe-2S] cluster. These titration and structural results further elucidate the histidine ligand characteristics and are consistent with important roles for these amino acids.
Assuntos
Proteínas de Bactérias/química , Complexo III da Cadeia de Transporte de Elétrons/química , Concentração de Íons de Hidrogênio , Thermus thermophilus/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Dicroísmo Circular , Cristalografia por Raios X , Transporte de Elétrons , Complexo III da Cadeia de Transporte de Elétrons/genética , Complexo III da Cadeia de Transporte de Elétrons/metabolismo , Cinética , Modelos Moleculares , Conformação Proteica , Espectrofotometria , Thermus thermophilus/genéticaRESUMO
Venous thromboembolism (VTE) is a major health problem that results in a significant burden on hospitals and patients. VTE screening and prophylaxis protocols in trauma patients vary significantly among hospitals and providers. In addition, many patients develop VTE even in the absence of "high-risk" categories. Therefore, more research is needed to better understand and prevent VTE in these patients. ABO blood group has long been recognised as a risk factor for VTE, but its contribution to VTE risk in the trauma setting is poorly studied. This paper reviews the literature describing the link between ABO blood group and VTE risk and the implications for VTE screening and prophylaxis in trauma patients. The effect of ABO blood groups are genotype-dependent - in most populations the A1 allele and the B allele increase risk while A2, O1, and O2 decrease risk of VTE. ABO group is a major determinant of plasma von Willebrand factor (vWF) and factor VIII levels, thereby (partially) mediating the effects of ABO blood group on VTE susceptibility. In addition, ABH antigens alter plasma levels of vWF via clearance mechanisms, which are in turn mediated by ADAMTS13. ABO blood group is a risk factor for VTE that warrants further investigation in trauma patients.