| Abstract: | The fluorescence characteristics of 8-anilino-naphthalene-1-sulfonic acid (ANS) coupled to apohemoglobin and to apohemoglobin labeled with fluorescein iodoacetamide (FIA) at beta-93 have been compared. The quenching of emission of ANS produced by FIA was measured both with steady-state and with time-resolved techniques. In this system the emission of ANS in the beta-heme pockets was totally quenched by FIA at beta-93. Steady-state measurements indicated a 57% efficiency of energy transfer between ANS in the alpha-heme pockets and FIA at beta-93. Time resolution showed that the initial (unquenched) lifetime of ANS was 18.2 ns. In the presence of FIA two new components were generated with lifetimes of 2.0 and 6.6 ns. Assuming a random orientation of the probes, the distances inferred from these measurements were near 4.6 and 3.6 nm for the time-resolved and near 48 A for the steady-state measurements. In the tridimensional model of hemoglobin the distance between the iron atom of the alpha 1 chains and the SH group of the beta 1 chains at position 93 is 3.6 nm in oxyhemoglobin and 4.1 nm in deoxyhemoglobin. To these distances 0.5-1.0 nm may be added to allow for the dimensions of the probes. Thus it appears that removal of the heme fails to produce any important enlargement of the molecule. On the contrary, the data suggest a slight shrinking of apohemoglobin, which may be consistent with a collapse of the heme pocket when heme is removed. The rest of the molecule does not seem to be greatly affected.(ABSTRACT TRUNCATED AT 250 WORDS) |
