Spectral analysis of t-α1m after incubation with hemoglobin.
(A) Methemoglobin-Sepharose (75 μM) and t-α1m (3 μM) were incubated in PBS at 37°C for 1 hour. The sample was centrifuged and the absorbance spectrum of the supernatant measured at the indicated time intervals. The spectrum of a control sample (methemoglobin-Sepharose only) was subtracted. (B) Oxyhemoglobin (7 μM) and t-α1m (5 μM) were incubated in PBS at 37°C for 1 hour. The t-α1m was purified by affinity chromatography on a column of monoclonal anti-α1m Affigel, concentrated, and the absorbance spectrum read after 0 and 5 hours. (C) Lysed erythrocytes (original cell volume diluted 1:1) and plasma-α1m (40 μM) were mixed and incubated at 37°C for 3 hours. The t-α1m formed was purified by affinity chromatography on a column of monoclonal anti-α1m Affigel and gel chromatography on Sephacryl S-300, concentrated, and the absorbance spectrum read after the indicated time intervals. (D) Methemoglobin-Sepharose (75 μM) and plasma-α1m (10 μM) were incubated for 1 hour at 37°C. The sample was centrifuged and the absorbance spectrum of the supernatant measured at the indicated time intervals.
