Reactions of 5,5\'-dithiobis(2-nitrobenzoate) with CysB5(23)β and CysF9(93)β Of Duck Minor Hemoglobin

The reactivities of the sulphydryl groups of duck minor hemoglobin have been studied for a comparison with those of duck major hemoglobin. Boyer titration with p-hydroxymercuri(II)benzoic acid (pMB) gave four reactive sulphydryl groups per (tetramer) molecule. Titration with 5,5'- dithiobis(2-nitrobenzoic acid) (DTNB) also gave four reactive sulphydryl groups per molecule. The kinetics of the reaction of DTNB with the oxy-, carbonmonoxy- and aquomet derivatives was studied at 200C as a function of pH (7.0 9.0) under pseudo- first-order conditions. Biphasic time courses were obtained under all experimental conditions and the two kinetic phases were well separated along the time axis. The apparent second-order rate constants, kapp, were calculated from the observed pseudo-first-order rate constant, kobs. The dependence of kapp on pH for each of the two kinetic phases is simple, resembling the titration curve of a diprotic acid. For the fast kinetic phase, quantitative analyses of the simple pH dependence profiles gave pKas of 5.5 ± 0.5 and 8.4 ± 0.3. The pKa of 5.5 is assigned to HisHC3(146) β; the pKa of 8.4 is assigned to CysF9(93)β. For the slow kinetic phase, quantitative analyses of the simple pH dependence profiles gave pKas of 5.3 ± 0.3 and 8.7 ± 0.1. The pKa of 5.3 is assigned to HisG19(117) β; the pKa of 8.7 is assigned to CysB5(23) β. CysF9(93) β is responsible for the fast phase while CysB5(23) β is responsible for the slow phase. The result obtained showed that the reactivity of the CysF9(93) β sulphydryl of the minor hemoglobin differs from that of the major hemoglobin. It was observed that for the CysF9(93) β reaction, the oxy and aquomet derivatives of the major hemoglobin reacted about 3-fold faster than the corresponding derivatives of the minor hemoglobin. The carbonmonoxy derivative of the major hemoglobin reacted about 20-fold faster than the corresponding derivative of the minor hemoglobin. For the CysB5(23) β reaction, the oxy, carbonmonoxy and aquomet derivatives of the major hemoglobin reacted at about the same rate as the corresponding derivatives of the minor hemoglobin. KEY WORDS: Hemoglobin, sulphydryl groups, duck, p-hydroxymercuri (II) benzoate, 5,5'-dithiobis(2-nitrobenzoic acid). Global Journal of Pure and Applied Sciences Vol.11(2) 2005: 225-23

Reactions Of 5,5\'-Dithiobis(2 -Nitrobenzoate) with CysB5(23)β and CysF9(93)β 0f Duck Major Hemoglobin

Duck major hemoglobin contains ten sulphydryl groups per tetramer molecule as revealed by its amino acid sequence. Boyer titration of this hemoglobin with p-hydroxymercuri(II)benzoic acid (pMB) indicates that there are only six reactive sulphydryl groups per (tetramer) molecule, while titration with 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) indicates only four. The kinetics of the reaction of DTNB with the oxy-, carbonmonoxy-, and aquomet derivatives of this hemoglobin were studied at 20oC as a function of pH (pH 7.0 9.0) under pseudo-first order conditions. Biphasic time courses were obtained under all experimental conditions and the two kinetic phases were well separated along the time axis. The apparent second-order rate constants, kapp, were determined from the observed pseudo- first-order rate constants, kobs. The dependence of kapp on pH for each of the two kinetic phases is simple, resembling the titration curve of a diprotic acid. For the fast kinetic phase, quantitative analyses of the simple pH dependence profiles gave pKas of 5.5 ± 0.5 and 8.6 ± 0.2. The pKa of 5.5 ± 0.5 is assigned to HisHC3(146)β, the pKa of 8.6 0.2 is assigned to CysF9(93)β. For the slow kinetic phase, quantitative analyses of the simple pH dependence profiles gave pKas of 6.2 ± 0.6 and 8.8 ± 0.1. The pKa of 6.2 ± 0.6 is assigned to HisG19(117) β; the pKa of 8.8 is assigned to CysB5(23) β. It is significant to note that the rates of both CysF9(93) β and CysB5(23) β of the carbonmonoxy derivative were found to be much greater than those of oxy and aquomet derivatives. KEY WORDS:- Hemoglobin, sulphydryl groups, duck, 5,5'-dithiobis(2-nitrobenzoic acid). Global Journal of Pure and Applied Sciences Vol.11(2) 2005: 209-21

β-Catenin safeguards the ground state of mousepluripotency by strengthening the robustness of the transcriptional apparatus

Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that β-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by β-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts