Design of Heteronuclear Metalloenzymes.

Heteronuclear metalloenzymes catalyze some of the most fundamentally interesting and practically useful reactions in nature. However, the presence of two or more metal ions in close proximity in these enzymes makes them more difficult to prepare and study than homonuclear metalloenzymes. To meet these challenges, heteronuclear metal centers have been designed into small and stable proteins with rigid scaffolds to understand how these heteronuclear centers are constructed and the mechanism of their function. This chapter describes methods for designing heterobinuclear metal centers in a protein scaffold by giving specific examples of a few heme-nonheme bimetallic centers engineered in myoglobin and cytochrome c peroxidase. We provide step-by-step procedures on how to choose the protein scaffold, design a heterobinuclear metal center in the protein scaffold computationally, incorporate metal ions into the protein, and characterize the resulting metalloproteins, both structurally and functionally. Finally, we discuss how an initial design can be further improved by rationally tuning its secondary coordination sphere, electron/proton transfer rates, and the substrate affinity.

Quinine- and quinidine-dependent antiplatelet antibodies. Requirement of factor VIII-related antigen for platelet damage and for in vitro transformation of lymphocytes from patients with drug-induced thrombocytopenia.

The requirement of Factor VIII-related antigen (VIIIR:Ag) for platelet damage by quinine-and quinidine-dependent antibodies was studied in platelet-rich plasma (PRP) of four patients with severe von Willebrand's disease (vWd) (Factor VIII deficiency). Platelet factor 3 availability, platelet aggregation, and release of [(14)C]serotonin from labeled vWd-PRP by drug-dependent antibodies were significantly reduced in comparison with PRP from normal controls. Addition of purified VIIIR:Ag restored levels of platelet damage to that of normal PRP. In vWd-PRP, platelet damage by two human antiplatelet sera, not dependent on drugs, and by a rabbit antiplatelet serum did not differ from that in normal PRP. PRP from patients deficient in Factor VIII coagulant activity, Factor IX, or Factors II, VII, IX, and X behaved like normal PRP. The role of VIIIR:Ag in forming antigen able to transform lymphocytes of patients who had recovered from drug-induced thrombocytopenia was investigated by measuring incorporation of [methyl-(3)H]thymidine into DNA. When lymphocytes were cultured for 7 d, significantly less transformation occurred in response to platelets and the drug in the presence of vWd sera than in normal sera or sera deficient only in Factor VIII coagulant activity or Factor IX. Addition of purified VIIIR:Ag to vWd sera restored transformation to that obtained in normal sera. Nonspecific lymphocyte transformation by pokeweed mitogen was not affected by VIIIR:Ag. Thus VIIIR:Ag is involved both in platelet damage by drug-dependent antibodies and in the interaction between platelet and drug which produces an antigen able to transform sensitized lymphocytes.

Study of the factors that cause specific transformation in cultures of lymphocytes from patients with quinine- and quinidine-induced immune thrombocytopenia.

Quinine- or quinidine-induced thrombocytopenic purpura is caused by synthesis of an immunoglobulin (Ig)G antibody, which caused platelet damage in the presence of the offending drug. The nature of the antigenic stimulus has been examined by measuring incorporation of [3H]thymidine into DNA during lymphocyte transformation to blast cells in the presence of the drug. Although patients' lymphocytes responded normally to the nonspecific mitogen, phytohemagglutinin P, they did not respond to either drug or platelets alone. However, significant transformation occurred when patients' lymphocytes were cultured for 7 d with homologous or autologous platelets in the presence of therapeutic concentrations of the drugs (0.39-39 microM). Platelet membranes were more active than intact platelets on the basis of protein content, whereas platelets from a patient with Bernard-Soulier syndrome were inactive. Washed platelets pretreated with the drugs were inactive when cultured with lymphocytes in the absence of the drugs, whereas platelets pretreated similarly in plasma caused transformation. Control lymphocytes from 20 normal patients and 6 patients with nondrug-induced thrombocytopenia were not transformed by drugs and platelets in the presence of normal serum or serum containing drug-dependent antibody, showing that the observed response was specific for presensitized lymphocytes. Thus lymphocytes of patients with drug-induced thrombocytopenia are transformed by an antigen that forms after interaction of plasma, specific platelet membrane components and the drug.