- A novel screening assay named the oxygen dissociation assay (ODA) was developed
- Assay is based on the spectral changes observed during hemoglobin deoxygenation
- SPECTROstar Nano captured spectral changes over time during N2 addition to microplate chamber
Hemoglobin (Hb) is an iron-containing globular protein present in the blood cells of all vertebrates. Its ability to perform transport of gases, primarily O2, make it critically important for maintaining aerobic metabolism.
Compounds which exogenously modify Hb affinity for O2 are desirable for treating multiple diseases. Modiﬁ ers that decrease Hb-O2 afﬁnity would be useful when improved delivery of O2 is needed, such as in wound healing. In addition, increased Hb-O2 afﬁnity would be useful in sickle cell anemia to delay the polymerization of deoxygenated sickle Hb.
Here we describe the Oxygen Dissociation Assay (ODA). Relatively high throughput was obtained using the SPECTROstar Nano to read 96-well plates. The reader was equipped with an optional gas-vent so that N2 could be directly added to the reader and spectral data collected over time to monitor the deoxygenation of Hb.
To determine the O2 afﬁnity of Hb the spectral differences between oxy- and deoxy-Hb were employed (Figure 1).
Differences between the 2 forms of Hb are primarily seen at the Soret band (400-500 nm) and the Q-band (500-600 nm) but the entire spectrum from 350 to 700 nm was monitored every 6 minutes for 2 hours while N2 was ﬂowing through the microplate reader. The spectral data were analyzed using Excel’s LINEST function and converted to % of oxygenated Hb.
Material & Methods
- 96-well, half-area microplate (µ-clear bottom, Greiner)
- SPECTROstar Nano equipped with gas vent
- For a complete description of reagents and sources see Patel et al.1
3 µM puriﬁed Hb was added to plates with or without various compounds. Plates were sealed and incubated at ambient air condition for 1 hour at 37°C. Plates were subsequently unsealed and placed in the SPECTROstar Nano to which gaseous dry N2 was applied via the gas vent. The plate was read in the following way:
|Optic settings||Absorbance, plate mode kinetic|
|Wavelength range:||350-770 nm|
|General settings||Number of flashes:||22|
|Settling time:||0.2 s|
|Kinetic settings||Number of cycles:||20|
|Cycle time:||360 s|
|Shaking settings||Shaking frequency:||300 rpm|
|Shaking mode:||Double orbital|
|Shaking time:||60 s - each cycle|
Results & Discussion
Figure 2 compares the spectral results for Hb at the beginning and end of the 2 hour exposure to N2. There is a clear rightward shift in the peak of the Soret band from 415 to 430 over the course of the experiment. Furthermore the 2 peaks at the Q band (541 and 577 nm) at the beginning of the exposure, resolve into one peak (555 nm) at the end of the 2 hours.
The 20 spectra that were collected over time were compared across the wavelength range from 380 to 700 nm and the results were expressed as %oxy Hb. The %oxy Hb results were plotted against time (Figure 3) showing the expected decrease in %oxy Hb over time.
The ODA was then tested in the presence of well characterized allosteric modiﬁers of Hb-O2 afﬁnity. Figure 4 shows a comparison of ODA results with Hb alone and in the presence of phytic acid and GBT1118.
Analyzing oxygen equilibrium curves (OEC) with the industry standard TCS Hemox Analyzer in presence of GBT1118 and phytic acid reveals differences in Hb oxygenation (data not shown). A more extensive ODA screen was compared to OEC results and it was found that the compounds that decrease oxygen affinity of hemoglobin, like phytic acid, are best evaluated at 30 minutes in the ODA. These compounds shift the ODA curve to the left due to low affinity and fast displacement of oxygen. Whereas, compounds that increase oxygen affinity of hemoglobin shift the ODA curve to the right as oxygen dissociation is delayed. These compounds such as GBT1118 are best evaluated at 108 min. These 2 detection times are indicated in figure 4. Regardless of the evaluation time used the results of ODA correlated well with OEC and exhibited acceptable robustness statistics (data not shown).
The ODA represents a major step toward HTS in analysis of Hb-afﬁnity modiﬁers. Compared to industry standards the ODA improves upon both the time required and volume required for the screening assay.
1. Patel, MP et al. Development and validation of an oxygen dissociation assay, a screening platform for discovering, and characterizing hemoglobin-oxygen afﬁ nity modiﬁers. Drug Des. Devel. Ther. (2018) 12: 1599-1607.