Kinetic study on oxygenation of Lingula unguis hemerythrin using the stopped-flow 0₂-jump method

Abstract

O₂-jump experiments with an improved stopped-flow apparatus have been used to study oxygenation and deoxygenation processes in Lingula unguis hemerythrin. With an O₂ electrode set in the observation cell, O₂ concentration conld be obtained directly. The reliability of this method has been compared with other conventional methods.

O₂-jump (up and down) experiments were carried out with L. unguis hemerythrin at pH 6.8 (non-cooperative pH) and at pH 7.6 (cooperative pH). At pH 6.8, both O₂-jump (up) and O₂-jump (down) experiments showed single exponential processes which were consistent with the following scheme: ${\text{Hr + O}}_{\text{2}} \mathop \rightleftharpoons \limits_{{\text{k}}_{{\text{off}}} }^{{\text{k}}_{{\text{on}}} } {\text{HrO}}_{\text{2}}$. The value of k _on was estimated to be (4.4 ± 0.5) × 10⁵ M⁻¹ s ⁻¹, and k _off was (15 ± 5) s⁻¹. These values are consistent with those obtained by the temperature-jump method (Zimmer et al. 1986). At pH 7.6, O₂-jump (up) experiments showed two relaxation processes, whereas O₂-jump (down) experiments showed a single exponential process. The faster process in the O₂-jump (up) experiments could be attributed to the same process as that seen in the temperature-jump experiments (Zimmer et al. 1986). The slower process in the O₂-jump (up) experiments corresponds to the process obtained in the O₂-jump (down) experiments. The results are discussed in terms of a state with intermediate affinity in O₂-binding and with the possible existence of a slow step in O₂-binding.