A STUDY OF THE MECHANISMS OF SLOW RELIGATION TO SICKLE-CELL HEMOGLOBIN POLYMERS FOLLOWING LASER PHOTOLYSIS

Time-resolved linear dichroism (TRLD) measurements are conducted on ge ls of sickle cell hemoglobin following laser photolysis of the carbony l adduct to monitor religation kinetics to hemoglobin S polymers. The return of the polymer phase to its equilibrium ligation state has been found to be about 1000 times slower than that of the solution phase h emoglobin tetramers. Several mechanisms describing this slow religatio n to the polymer were proposed: (1) religation occurs through a biomol ecular process involving all polymer hemes, (2) religation occurs thro ugh a bimolecular process in which only hemoglobin molecules at the po lymer ends can participate, and (3) religation occurs through the exch ange of ligated hemoglobin molecules in the monomer phase with unligat ed ones in the polymer phase. To test these mechanisms, measurements a re performed on gels having different domain sizes. The results show n o relation between domain size and religation kinetics. The independen ce of religation kinetics and domain size is most consistent with the first of the three mechanisms described above (bimolecular recombinati on involving all polymer hemes). This result is discussed in terms of a model in which diffusion of the ligand is inhibited in the polymer p hase. An understanding of the ligand binding kinetics of sickle hemogl obin polymers could have pathophysiological significance in its releva nce to polymer formation and melting during red blood cell circulation . (C) 1996 Academic Press Limited