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.