Developing blood substitutes is in urgent demand for chronic blood shortage all over the world. In this connection, the oxygen binding behavior of hemoglobin-based oxygen carriers (HBOCs) is one of the most important characteristics. However, present methods available for estimating oxygen binding behavior have need of expensive apparatus, and also are not suitable for high-throughput and the time-course analysis. To overcome these problems, we proposed a simple analysis method for the time-course oxygen binding behavior of HBOCs, which employs a general UV-Vis microplate reader and a common reagent, sodium dithionite, as a reductant for HBOCs and an oxygen scavenger. Our method enabled time-course oxygen binding behavior analysis of HBOCs in a simple manner, and obtained data corresponding with those by the conventional method. Thus, our developed method will accelerate the development of HBOCs due to easy oxygen binding analysis.

A method to detect the L-proline- (L-Pro-) catalyzed Michael addition reaction in model biomembranes has been established, using N-[p(2-benzimidazolyl)phenyl]maleimide and acetone as reactants. The effect of liposome membranes on this reaction was kinetically analyzed using fluorescence spectroscopy. The kinetics of the reaction were different from those of the constituent lipids of the liposomes. Zwitterionic 1,2-dipalmitoyl-sn-glycero-3-phosphocholine liposome, which is in the solid-ordered phase, had a better value of reaction rate, suggesting that the reaction rate constants of this reaction in liposome membrane systems could be regulated by the characteristics of the liposome membrane (i.e., the phase state and surface charge). Based on the results obtained, a plausible model of the L-Pro-catalyzed Michael addition reaction was discussed. The obtained results provide us with an easily detectable method to assess the reactivity of L-Pro in biological systems.