Reactions with Free Hemoglobin from Healthy Donors
Publications studying deoxygenated Hb reacting with oxygen and several biomolecules including spinach ferredoxin, horse metmyoglobin, horse heart ferricytochrome with dithionite report reactions occurring on the order of milliseconds (Coin & Lambeth, 1979). The timescale of reaction, not the rate constants, is confirmed using a basic spectrophotometer rather than a robust, airtight stopped-flow spectrophotometer. The spectra and relative oxygenation state over time for 13.4±2.7 SD μM free hemoglobin for three different reactions are shown in Figures 1A and 1B , respectively. Figure 1A is presented as a normalized absorption intensity. The spectra for dithionite-treated Hb has a notable shoulder at 630 nm, consistent with that of metHb. According to Di Iorio (1981), unstable dithionite produces H2O2, which can oxidize oxyHb into metHb (Winterbourn, 1900). The reaction with Oxyrase, however, shows no unwanted secondary peaks. As expected, the reaction inFigure 1B with dithionite is completed on the order of milliseconds. The reaction with sodium nitrite is on the order of 1 minute and the reaction with Oxyrase is completed on the order of 5-10 minutes. Overlaid spectra from deoxyHb treated with Oxyrase shows no significant change to the isosbestic point at 505nm reported in Tsao, Sethna, Sloan & Wyngarden (1955) (data not shown).
The manufacturer of Oxyrase defines 1 unit of EC-Oxyrase® as the “amount of Oxyrase that, under defined conditions, reduces dissolved oxygen at the rate of 1% per second” (“Assay of Oxyrase Activity”, 2019). In this study, we do not report Oxyrase in units defined by the manufacturer but rather the volume fraction of Oxyrase in the sample. In a reaction to eliminate dissolved oxygen, Oxyrase acts as the enzyme for dissolved oxygen reduction to water in the presence of an appropriate hydrogen donor. In the present case, Oxyrase and lactate are mixed beforehand to prepare the anaerobic solution before oxyHb is added.
Figures 2A, 2B presents the percent oxygenation of Hb over time with varying lactate molarities and Oxyrase concentrations. Interestingly, deoxygenation proceeds more slowly at higher concentrations of sodium lactate. It is suggested that this observation is a result of pH of the buffer and consistent with measured DO after an hour (Figures 3A, 3B). At high lactate concentration and acidic pH, DO remains in the mixture and therefore incomplete conversion of oxyHb to deoxyHb is achieved. However, at low lactate concentrations, near the reported optimal pH for Oxyrase at 8.4, DO measurements are near zero (Adler & Spady, 1997; “EC Oxyrase”, 2019). It should be emphasized that pH has a much stronger influence on DO than transport-limited kinetics due to increased viscosity from lactate, which is discussed in the next section.