Alteration of tryptophan fluorescence properties upon dissociation of Lumbricus terrestris hemoglobin.
Fluorescence analysis has been used to study dissociation of the dodecameric 3.8 kDa Lumbricus terrestris hemoglobin. Since tryptophan intrinsic fluorescence has been used as a reporter group to study Lumbricus hemoglobin, it is of interest to study dissociation perturbed properties of the tryptophan residues. Shifts in the fluorescence emission maximum to longer wavelengths upon dissociation at pH 9.2 suggest that tryptophans buried at the subunit interface(s) become more exposed. Fluorescence lifetime and quenching studies are employed in this present investigation as a means to confirm the location of tryptophan residues at the subunit interfaces. Acrylamide titration (to 2.5 M) indicate only a fraction of the residues can be quenched at either pH. At pH 7.0, the Stern-Volmer plot has downward curvature, while at pH 9.2 there is slight upward curvature, again indicating a change in environment. The intrinsic fluorescence decay requires at least four exponentials at both pHs. The mean fluorescence lifetime of CO Lumbricus hemoglobin increases from 1.1 ns at pH 7 to 3.3 ns at pH 9.2. The lifetime data can be further interpreted as a decrease in the quenching of residues with a approximately 30 ps lifetime, and a concomitant increase in the longer lifetime components. This is consistent with interface tryptophans becoming exposed to solvent upon dissociation, and loss of quenching by intersubunit hemes. The overall results suggest that in the dodecamer, most of the tryptophans are located in a hydrophobic environment, not all of which are located at the subunit interface.
Hirsch
R E
RE
Department of Medicine (U-921), Albert Einstein College of Medicine, Bronx, New York, NY 10461.
Vidugiris
G J
GJ
Friedman
J M
JM
Harrington
J P
JP
D020106:Acrylamide; D000178:Acrylamides / Q000494:pharmacology; D000818:Animals; D002263:Carboxyhemoglobin / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D006454:Hemoglobins / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D009835:Oligochaeta / Q000737:chemistry*; D010108:Oxyhemoglobins / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D000178:Acrylamides; D006454:Hemoglobins; D010108:Oxyhemoglobins; D020106:Acrylamide; D014364:Tryptophan; C100003:deoxyhemoglobin; D002263:Carboxyhemoglobin