Sol-gel trapping of functional intermediates of hemoglobin: geminate and bimolecular recombination studies.
The encapsulation of proteins in porous sol-gels is a promising technique for generating, trapping, and probing functionally significant nonequilibrium protein species. An essential step needed in the pursuit of that goal is establishing the degree to which the sol-gel limits conformational change upon adding or removing substrates. In the present study, geminate recombination and solvent phase bimolecular recombination of CO to human adult hemoglobin (HbA) are used as sensitive probes of the degree of conformational constraint within the sol-gel. Two forms of CO saturated encapsulated HbA are generated. In one case, designated [COHbA], the equilibrium form of COHbA is directly encapsulated. In the second case, designated as [deoxyHbA] + CO, the equilibrium form of deoxyHbA is encapsulated and only after the sample has aged is CO introduced to the HbA through the porous sol-gel matrix. Three different preparative protocols are used to generate the sol-gels for each of the two forms of encapsulated COHbA. The kinetic traces obtained from these encapsulated samples allow for an easy evaluation of the extent to which the sol-gel is locking in the initial tertiary/quaternary structure. The results show that the sol-gel encapsulated samples can be used with pulsed laser sources and that one of the tested encapsulation protocols is far superior with respect to conformational locking. This protocol is used to trap and probe nonequilibrium forms such as the liganded T state of HbA, a species whose properties are needed to fully explore allostery in HbA.
Khan
I
I
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Shannon
C F
CF
Dantsker
D
D
Friedman
A J
AJ
Perez-Gonzalez-de-Apodaca
J
J
Friedman
J M
JM
D002021:Buffers; D002248:Carbon Monoxide / Q000737:chemistry; D002263:Carboxyhemoglobin / Q000737:chemistry; D005782:Gels / Q000737:chemistry*; D005990:Glycerol / Q000737:chemistry; D006441:Hemoglobin A / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008024:Ligands; D017646:Organosilicon Compounds / Q000737:chemistry; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D012996:Solutions; D013053:Spectrophotometry
D002021:Buffers; D005782:Gels; D006454:Hemoglobins; D008024:Ligands; D017646:Organosilicon Compounds; D000644:Quaternary Ammonium Compounds; D012996:Solutions; C018408:3-(trimethoxysilyl)propyldimethyloctadecylammonium; D002248:Carbon Monoxide; C100003:deoxyhemoglobin; D006441:Hemoglobin A; D002263:Carboxyhemoglobin; D005990:Glycerol