You can try to measure the PO2 (Partial Pressure of Oxygen) reflects the amount of oxygen gas dissolved in the blood. It primarily measures the effectiveness of the lungs in pulling oxygen into the blood stream from the atmosphere and Oxygen Saturation (SO2) which measures the percent of hemoglobin which is fully combined with oxygen with AGB (Arterial Blood Gas) device.

You have to measure the oxygen affinity of the red blood cells through common gas blood analyzers, which measure the hemoglobin fractional saturation with oxygen and provide you an oxygen dissociation curve (ODC). From the ODC you can then calculate the p50 (the pressure at which half of hemoglobin is saturated with oxygen) and the Hill coefficient, or n (a measure of the cooperativity of the oxygen binding to hemoglobin).

As a first screening you could simply measure the oxygen saturation

A link you might find useful (it is free - but you need to register) http://acutecaretesting.org/en/articles/oxygen-saturation-better-measured-than-calculated. It is a site for testing in humans - but have many papers on the principles of oxygen - measures.

As mentioned before, as a rough measure you can analyze the oxygen saturation of the hemoglobin (after oxygenation of the sample). However, you should be careful when using a simple blood gas analyzer. If you are working with "toxic" agents you should be aware of Hb species (methhemoglon or HbS) which could interfere with a simple photometric measurement. For this reason you should perform your measurements on a hemoximeter which can determine all Hb spezies.

Just to point out that there a several analyzers on the market that is doing just that and some have even more features like metHb, HbS HbCO, FHb, calculates 2.3 DPG as pointed out by Prof Thews. An working with mice it is important that the equipment can handle micro-samples.

Using a hemoximeter (often incorporated in blood gas analysers) you can measure O2Hb, COHb, MetHb, SulfHb. Some apparatus (probably the analysers of Radiometer) contain equations for different animals. But most informative is oxygen capacity (measurement of O2 bound after equilibration with high O2 pressure) used since the time of Van Slyke (for methods search in pubmed). This shows you how much of Hb still functions as oxygen transporter. When registering oxygen dissociation curves, be aware that you get no Information about the amount of non-oxygen-binding Hb fractions. This fractions usually shift the curve leftward.

I would also use a Hemox analyzer, however, instead of their conventional buffer we use our own containing (mM) 140 NaCl, 4 KCl, 0.75 MgSO4, 10 glucose, 0.015 ZnCl2, 0.2 Glycine, 0.2 Glutamate, 0.2 Alanine, 0.1 Arginine, 0.6 Glutamine, 20 HEPES and 1.8 CaCl2. Very important is to control the pH of 7.40 exactly at the temperature you use for your measurements (I assume you shall be using 37C).

For buffer conditions, the main factors affecting the oxygen affinity of RBCs are:

- protons (as already Anna pointed out): lower oxygen affinity

- chloride ions: lower oxygen affinity

- glucose: you need to feed glycolysis to maintain constant concentration of ATP and 2,3-DPG, the main allosteric effector for hemoglobin.

- CO2: lowers oxygen affinity forming carbamates with hemoglobin.

You need to be very careful in replicating physiological conditions for these molecules.

We found that extracellular calcium is also an important issue for regulation of oxygen affinity of Hb in human red cells: Makhro et al Am J Physiol Cell Physiol. 2013 Dec 1;305(11):C1123-38

Is the effect of Calcium you found bound to its effect on intracellular pH, or did you observe any direct interaction with hemoglobin?

We currently think that intracellular calcium affects Hb protonation. This effect may be reproduced for the dissolved Hb. So, yes, the effect can be observed witg Hb alone and yes, it is related to the changes in protein protonation

You can use Blood Gas Analyzer to analyze the blood indexes including PCO2, PO2, HCO3-, pH, Ca2+(?).....It's easy and not expensive, much better than test by yourself.

Thank You All

You should measure oxygen capacity (oxygen content of a sample after equilibration with high oxygen pressure). If you want to know oxygen binding under the conditions of your experiment, you should determine at least O2 content or O2 saturation, together with PO2 and pH. The whole oxygen dissociation curve can be measured after mixing various volumes of saturated and unsaturated blood (described e. g. in Böning et al. PLoS One 9(6): e97932(2014). doi.10.1371/journal.pone.0097932).