. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. SEA CUCUMBER HEMOGLOBIN 117 mA/ organic phosphates: adenosine 5'-triphosphate (ATP), disodium salt; acid (2,3-DPG), pentasodium salt: and inositol hexaphosphoric acid (IHP), dodecasodium salt (Sigma Chem. Co.). When IHP was tested, the Hb sample was first dialyzed against a Tris- HC1 buffer containing 550 mA/ Cl and 500 mM Na+, because the addition of IHP to saline buffer resulted in precipitation. For structural studies, hemocytes were obtained as de- scribed above. After centrifugation, washing, an
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. SEA CUCUMBER HEMOGLOBIN 117 mA/ organic phosphates: adenosine 5'-triphosphate (ATP), disodium salt; acid (2,3-DPG), pentasodium salt: and inositol hexaphosphoric acid (IHP), dodecasodium salt (Sigma Chem. Co.). When IHP was tested, the Hb sample was first dialyzed against a Tris- HC1 buffer containing 550 mA/ Cl and 500 mM Na+, because the addition of IHP to saline buffer resulted in precipitation. For structural studies, hemocytes were obtained as de- scribed above. After centrifugation, washing, and lysing of the cells, the red supernatant was chromatographed on a Sephadex G-100 column (100 X cm) in equilibrium with a M sodium chloride, M sodium phosphate buffer, pH Spectra of elution fractions were measured with either a Zeiss PMQ-II or a Beckman DU-70 spec- trophotometer. Hemoglobins were analyzed by non-denaturing poly- acrylamide gel electrophoresis (PAGE) and sodium do- decyl sulfate polyacrylamide gel electrophoresis (SDS PAGE). PAGE was carried out on oxy-, carboxy-, cyan- met-, and met-Hb samples on mm slab gels, acrylamide. with a discontinuous buffer system, pH (Tris-glycine) as a cathodal buffer, and pH (Tris-HCl) as the anodal buffer (Davis, 1964). Hemoglobins were prepared for SDS PAGE by incubation in 2% SDS, 5% mercaptoethanol, and 1 mA/ phenylmethylsulfonyl flu- oride (PMSF) at 100°C for min. Samples were elec- trophoresed on mm slab gels, acrylamide, with a continuous buffer system of pH (Laemmli, 1970). Analyses of variance were performed to test the signif- icance of regressions of log P50 and nH \ersus pH. The slopes of none of the regressions were significantly different from zero (P = ). Therefore, for comparisons between treatments, oxygen binding data obtained at dif- ferent pH were pooled. Results are reported as mean ± standard deviation. Analyses of variance and Student's /-tests
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