Spectroscopically and Kinetically Distinct Conformational Populations of Sol-Gel-encapsulated Carbonmonoxy Myoglobin
2002; Elsevier BV; Volume: 277; Issue: 28 Linguagem: Inglês
10.1074/jbc.m200301200
ISSN1083-351X
AutoresUri Samuni, David Dantsker, Imran H. Khan, Adam Friedman, Eric S. Peterson, Joel M. Friedman,
Tópico(s)Neonatal Health and Biochemistry
ResumoWe have used sol-gel encapsulation protocols to trap kinetically and spectroscopically distinct conformational populations of native horse carbonmonoxy myoglobin. The method allows for direct comparison of functional and spectroscopic properties of equilibrium and non-equilibrium populations under the same temperature and viscosity conditions. The results implicate tertiary structure changes that include the proximal heme environment in the mechanism for population-specific differences in the observed rebinding kinetics. Differences in the resonance Raman frequency of ν(Fe-His), the iron-proximal histidine stretching mode, are attributed to differences in the positioning of the F helix. For myoglobin, the degree of separation between the F helix and the heme is assigned as the conformational coordinate that modulates both this frequency and the innermost barrier controlling CO rebinding. A comparison with the behavior of encapsulated derivatives of human adult hemoglobin indicates that these CO binding-induced conformational changes are qualitatively similar to the tertiary changes that occur within both the R and T quaternary states. Protein-specific differences in the time scale for the proposed F helix relaxation are attributed to variations in the intra-helical hydrogen bonding patterns that help stabilize the position of the F helix. We have used sol-gel encapsulation protocols to trap kinetically and spectroscopically distinct conformational populations of native horse carbonmonoxy myoglobin. The method allows for direct comparison of functional and spectroscopic properties of equilibrium and non-equilibrium populations under the same temperature and viscosity conditions. The results implicate tertiary structure changes that include the proximal heme environment in the mechanism for population-specific differences in the observed rebinding kinetics. Differences in the resonance Raman frequency of ν(Fe-His), the iron-proximal histidine stretching mode, are attributed to differences in the positioning of the F helix. For myoglobin, the degree of separation between the F helix and the heme is assigned as the conformational coordinate that modulates both this frequency and the innermost barrier controlling CO rebinding. A comparison with the behavior of encapsulated derivatives of human adult hemoglobin indicates that these CO binding-induced conformational changes are qualitatively similar to the tertiary changes that occur within both the R and T quaternary states. Protein-specific differences in the time scale for the proposed F helix relaxation are attributed to variations in the intra-helical hydrogen bonding patterns that help stabilize the position of the F helix. myoglobin carbonmonoxy myoglobin 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)- propane-1,3-diol geminate yield Myoglobin (Mb)1continues to be used as a model protein system for investigations into how structure, dynamics, and reactivity interconnect to give rise to functionality. Recently there has been a renewed interest in myoglobin based on several new developments. From a functional point of view there are indications and suggestions that, apart from its physiological importance in facilitating oxygen diffusion, myoglobin has functions arising from its ability to participate in catalytic multisubstrate reactions involving such molecules as NO, O2, and H2O2 (1Brunori M. Trends Biochem. 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Kotani M. Yonetani T. Biochim. Biophys. Acta. 1974; 371: 126-139Crossref PubMed Scopus (88) Google Scholar, 25Huang J. Ridsdale A. Wang J. Friedman J.M. Biochemistry. 1997; 36: 14353-14365Crossref PubMed Scopus (41) Google Scholar, 26Frauenfelder H. Sligar S.G. Wolynes P.G. Science. 1991; 254: 1598-1603Crossref PubMed Google Scholar, 27Nienhaus G.U. Mourant J.R. Frauenfelder H. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2902-2906Crossref PubMed Google Scholar, 28Ansari A. Jones C.M. Henry E.R. Hofrichter J. Eaton W.A. Biochemistry. 1994; 33: 5128-5145Crossref PubMed Google Scholar, 29Steinbach P.J. Ansari A. Berendzen J. Braunstein D. Chu K. Cowen B.R. Ehrenstein D. Frauenfelder H. Johnson J.B. Lamb D.C. Biochemistry. 1991; 30: 3988-4001Crossref PubMed Google Scholar, 30Ansari A. Berendzen J. Bowne S.F. Frauenfelder H. Iben I.E. Sauke T.B. Shyamsunder E. Young R.D. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 5000-5004Crossref PubMed Google Scholar), in which the equilibrium deoxy derivative is compared with the non-equilibrium deoxy derivative derived from the photodissociation of COMb under conditions where there is either no or slowed relaxation of the photoproduct, indicate that the proximal heme environment is different in the two cases. How and through what mechanism these different tertiary conformations impact ligand reactivity are uncertain. A complication in addressing the role of conformational relaxation arises from the difficulty in trapping COMb derivatives having different tertiary structure distributions at the same temperature under ambient conditions. In this study, we describe the use of sol-gel encapsulation as a method of trapping different tertiary conformations of COMb that allows for both spectroscopic and functional characterization at the same ambient conditions. Relatively inert and transparent amorphous sol-gel matrices, composed of a porous network of silica-oxygen-silica bonds have been prepared under conditions that allow for the non-destructive encapsulation of proteins (31Avnir D. Braun S. Lev O. Ottolenghi M. Chem. Mater. 1994; 6: 1605-1614Crossref Google Scholar, 32Ellerby L.M. Nishida C.R. Nishida F. Yamanaka S.A. Dunn B. Valentine J.S. Zink J.I. Science. 1992; 255: 1113-1115Crossref PubMed Google Scholar, 33Braun S. Shtelzer S. Avnir D. Ottolenghi M. J. Non-Cryst. Solids. 1992; 148: 739-743Crossref Scopus (145) Google Scholar). In most instances the trapped protein molecules are isolated and immobile, preventing any possible complications due to aggregation. Advantageously, the porous matrix does allow water and small molecules to diffuse in and out. Thus, when the matrix is placed in a bathing buffer, the encapsulated proteins are solvated and their function can be examined and compared with solution phase work. Studies have revealed that the encapsulated proteins remain intact and functional. For example encapsulated hemeproteins undergo ligand binding and dissociation as well as redox reactions (32Ellerby L.M. Nishida C.R. Nishida F. Yamanaka S.A. Dunn B. Valentine J.S. Zink J.I. Science. 1992; 255: 1113-1115Crossref PubMed Google Scholar, 34Dave B.C. Miller J.M. Dunn B. Valentine J.S. Zink J.I. J. Sol Gel Sci. Technol. 1997; 8: 629-634Google Scholar). Many encapsulated enzymes have been shown to retain enzymatic activity within the sol-gel matrix (35Gill I. Ballesteros A. TIBTECH. 2000; 18: 282-296Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar). The sol-gel matrix also seems to confer enhanced structural stability (36Das T.K. Khan I. Rousseau D.L. Friedman J.M. J. Am. Chem. Soc. 1998; 120: 10268-10269Crossref Scopus (70) Google Scholar, 37Eggers D.K. Valentine J.S. Protein Sci. 2001; 10: 250-261Crossref PubMed Scopus (342) Google Scholar, 38Samuni U. Navati M.S. Juszczak L.J. Dantsker D. Yang M. Friedman J.M. J. Phys. Chem. B. 2000; 104: 10802-10813Crossref Google Scholar). Most significantly, with respect to the present project, sol-gel encapsulation has been shown to limit conformational change in hemoglobin. Oxygen binding (39Shibayama N. Saigo S. J. Mol. Biol. 1995; 251: 203-209Crossref PubMed Scopus (147) Google Scholar, 40Shibayama N. J. Mol. Biol. 1999; 285: 1383-1388Crossref PubMed Scopus (34) Google Scholar, 41Bettati S. Mozarrelli A. J. Biol. Chem. 1997; 272: 32050-32055Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 42Bruno S. Bonaccio M. Bettati S. Rivetti C. Viappiani C. Abbruzzetti S. Mozzarelli A. Protein Sci. 2001; 10: 2401-2407Crossref PubMed Scopus (86) Google Scholar), kinetic (43Khan I. Shannon C.F. Dantsker D. Friedman A.J. Perez-Gonzalez-de-Apodaca J. Friedman J.M. Biochemistry. 2000; 39: 16099-16109Crossref PubMed Scopus (96) Google Scholar, 44Khan I. Dantsker D. Samuni U. Friedman A.J. Bonaventura C. Manjula B. Acharya S.A. Friedman J.M. Biochemistry. 2001; 40: 7581-7592Crossref PubMed Google Scholar), and spectroscopic (45Juszczak L. Friedman J. J. Biol. Chem. 1999; 274: 30357-30360Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 46Das T. Khan I. Rousseau D. Friedman J. Biospectroscopy. 1999; 5: S64-S70Crossref PubMed Google Scholar) studies all indicate that the sol-gel can be used to stabilize the quaternary state conformation of the initially encapsulated derivative to the extent that subsequent addition or removal of ligand does not induce the usual quaternary state transition that occurs in solution. As a result non-equilibrium species associated with the liganded T state and the deoxy R state can be generated and studied. Furthermore, the sol-gel-induced slowing of the conformational dynamics is dramatically dependent on temperature (40Shibayama N. J. Mol. Biol. 1999; 285: 1383-1388Crossref PubMed Scopus (34) Google Scholar, 45Juszczak L. Friedman J. J. Biol. Chem. 1999; 274: 30357-30360Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 46Das T. Khan I. Rousseau D. Friedman J. Biospectroscopy. 1999; 5: S64-S70Crossref PubMed Google Scholar). The tertiary and quaternary relaxation times can be tuned from essentially days or weeks at 4 °C to hours and minutes as the temperature is progressively raised (46Das T. Khan I. Rousseau D. Friedman J. Biospectroscopy. 1999; 5: S64-S70Crossref PubMed Google Scholar). Potentially, this sol-gel property opens the way to overcome the "diffusional barrier" that exists in rapid mix experiments, where the diffusion time of ligands/reactants is often longer than the time it takes for conformational relaxation. Encapsulation can extend the relaxation time beyond the diffusion time for small substrate molecules. Thus, encapsulation of proteins in sol-gel may become a new method both for trapping non-equilibrium species and for studying the evolution of short-lived non-equilibrium structures. In this work we show that the sol-gel-based approach of "locking in" of the quaternary structure of Hb can be applied to the much smaller amplitude motions associated with the ligation-dependent tertiary structure of myoglobin. As a result we are able to expose the ligation-induced conformational and functional changes in myoglobin at a temperature well above the glass transition. The Soret band-enhanced resonance Raman spectrum is used to characterize the proximal environment of the 8-ns photoproduct as a function of trapped conformation (47Friedman J.M. Methods Enzymol. 1994; 232: 205-231Crossref PubMed Scopus (53) Google Scholar, 48Rousseau D.L. Friedman J.M. Spiro T.G. Biological Applications of Raman Spectroscopy. III. John Wiley & Sons, New York1988: 133-215Google Scholar), whereas the geminate and solvent phase rebinding of CO is used to expose the functional consequences of conformation (11Scott E.E. Gibson Q.H. Biochemistry. 1997; 36: 11909-11917Crossref PubMed Scopus (147) Google Scholar, 12Scott E.E. Gibson Q.H. Olson J.S. J. Biol. Chem. 2001; 276: 5177-5188Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). Buffered solutions of filtered horse Mb (Sigma Chemical Co.) were prepared in either BisTris acetate, pH 6.5, or the same with 25% or 50% glycerol by volume. All chemicals were from Aldrich. To improve the degree of locking in, the original sol-gel preparation protocol (32Ellerby L.M. Nishida C.R. Nishida F. Yamanaka S.A. Dunn B. Valentine J.S. Zink J.I. Science. 1992; 255: 1113-1115Crossref PubMed Google Scholar) was modified as previously described (43Khan I. Shannon C.F. Dantsker D. Friedman A.J. Perez-Gonzalez-de-Apodaca J. Friedman J.M. Biochemistry. 2000; 39: 16099-16109Crossref PubMed Scopus (96) Google Scholar) by avoiding sonication and by changing the buffer to 50 mm BisTris acetate, pH 6.5, diluted 3:1 with glycerol to yield 25% glycerol by volume. The gels were cast as thin films in 10-mm NMR tubes. Equal volumes of tetramethylorthosilicate, buffer, and Mb were combined and added to the NMR tubes (New Era and Willmad). The tubes were then spun using a high speed spinner (Princeton Photonics Inc., Princeton, NJ) to generate the thin film that eventually gelled after approximately 1 h of spinning. The samples were flushed and covered with buffer and then allowed to age a minimum of 1 day at 4 °C. The final concentration of Mb was 0.5 mm. DeoxyMb samples were prepared from nitrogen-purged solutions of metMb to which a slight excess of sodium dithionite (solution) was added. All solutions and gels were prepared in and stored under anaerobic conditions. The spinner was contained and operated within a nitrogen-purged glove box. The preparations for both visible resonance Raman and transient absorption were as described earlier (38Samuni U. Navati M.S. Juszczak L.J. Dantsker D. Yang M. Friedman J.M. J. Phys. Chem. B. 2000; 104: 10802-10813Crossref Google Scholar). In addition to the visible resonance Raman measurements described below, absorption, front-face fluorescence, and UV resonance Raman measurements on samples of Mb encapsulated using this protocol all indicate that the native structure is maintained. Scheme FS1 shows the two types of encapsulated samples that were prepared. In one case COMb is directly encapsulated in sol-gel. This protocol or sample type is denoted [COMb]. For the other protocol, deoxyMb is encapsulated in the sol-gel, and is denoted as [deoxyMb]. The Mb encapsulated in sol-gels was found to be intact and functional by exhibiting the unperturbed absorption spectra of the corresponding solution phase samples (not shown). The samples were aged at ∼4 °C, to allow the evolving gel to "template" around the equilibrium structure of the encapsulated Mb derivative to maximize the likelihood of locking in the initial distribution of conformations. After the encapsulated deoxyMb sample was allowed to age (1–5 days) and its spectra were recorded it was then exposed to CO. Rapid (within 5 min or less) ligation with CO occurred as determined by the characteristic changes in the visible absorption spectrum. Such samples are denoted [deoxyMb]+CO to indicate that the CO is added after encapsulation. A similar two-protocol approach was used to prepare the COHbA locked in either the R or the T quaternary state (43Khan I. Shannon C.F. Dantsker D. Friedman A.J. Perez-Gonzalez-de-Apodaca J. Friedman J.M. Biochemistry. 2000; 39: 16099-16109Crossref PubMed Scopus (96) Google Scholar). Fig. 1contains segments of Soret-enhanced resonance Raman spectra for both myoglobin and hemoglobin samples. The resonance Raman spectra from the sol-gel-encapsulated samples are very similar to those of the corresponding solution phase samples (as shown in the insetof Fig. 1) indicating that the protein conformation is not altered by the sol-gel matrix to any significant degree. Nonetheless, for some of the sol-gel-encapsulated samples there are differences in the frequency of the iron-proximal histidine mode, ν(Fe-His). TableI contains a summary of the relevant peak frequencies for ν(Fe-His). Fig. 1 displays a section of the visible resonance Raman spectrum, comparing the Raman band arising from ν(Fe-His) for [deoxyMb] (A), the 8-ns photoproduct of [deoxyMb]+CO (B), and the 8-ns photoproduct of [COMb] (C). Also shown are the solution phase spectra for deoxyMb (D) and the 8-ns photoproduct of COMb (E). The absolute peak positions are accurate to ∼0.5 cm−1 based on repeated spectral acquisitions. Peak position differences for ν(Fe-His) obtained when comparing spectra from different samples are accurate to within 0.2 cm−1 on average when the spectra are generated under similar conditions on the same day.Table Iν(Fe-His) frequency for Mb and Hb generated from either equilibrium deoxy derivatives or the 8-ns photoproduct of the CO-saturated derivativeSampleFe-HisTrace label in Fig. 1 cm −1[DeoxyMb]219.5 A blue[DeoxyMb]+CO220.8 B red[COMb]222.1 C greenDeoxyMb219.8 D blueCOMb220 E green[DeoxyMb]+CO+gly221.3 F red[COMb]+gly223.4 G green[DeoxyHb]214.0 H blue[DeoxyHb]+CO221.8 I red[COHb]230 J green[O2Hb]+dithionite223 Not shownCOHb230 Not shownDeoxyHb214 Not shownSamples are: in solution, in a buffer-bathed sol-gel (indicated bybrackets), or in glycerol-bathed sol-gel (indicated bybrackets and "+gly"). Open table in a new tab Samples are: in solution, in a buffer-bathed sol-gel (indicated bybrackets), or in glycerol-bathed sol-gel (indicated bybrackets and "+gly"). The frequency of ν(Fe-His) for [deoxyMb] is very close to that of the corresponding solution phase sample. Whereas in solution, the frequency of ν(Fe-His) for the 8-ns photoproduct of COMb is only a fraction of a cm−1 higher than for the equilibrium deoxyMb sample as previously reported (23Sage J.T. Schomacker K.T. Champion P.M. J. Phys. Chem. 1995; 99: 3394-3405Crossref Google Scholar, 49Findsen E. Scott T. Chance M. Friedman J. Ondrias M. J. Am. Chem. Soc. 1985; 107: 3355-3357Crossref Google Scholar, 50Peterson E. Chien E. Sligar S. Friedman J. Biochemistry. 1998; 37: 12301-12319Crossref PubMed Scopus (89) Google Scholar), in the sol-gel it is higher by ∼2 cm−1. This increase is also seen for COMb samples in highly viscous media (23Sage J.T. Schomacker K.T. Champion P.M. J. Phys. Chem. 1995; 99: 3394-3405Crossref Google Scholar) and for the unrelaxed picosecond photoproduct of COMb in solution (51Mizutani Y. Kitagawa T. J. Phys. Chem. 2001; 105: 10992-10999Crossref Scopus (0) Google Scholar). The frequency of ν(Fe-His) for the 8-ns photoproduct of [deoxyMb]+CO is slightly higher than for the [deoxyMb] sample but is still clearly lower than for [COMb]. Traces F and G illustrate the consequence of replacing the bathing buffer with 100% CO-saturated glycerol for the 8-ns photoproduct Raman spectrum of [deoxyMb]+CO and [COMb], respectively. The added glycerol has the effect of increasing the frequency of ν(Fe-His) for both samples (see Table I); nevertheless, the frequency difference between the two photoproduct populations is still maintained. It is noted that traces Fand G are for samples that have been kept for about a year in a cold room, after CO exposure, demonstrating that trapping of non-equilibrium structures for extended periods of time can be achieved. Fig. 1 also shows the Fe-His bands of sol-gel-encapsulated HbA, prepared using similar protocols ([deoxyHbA] (H); [deoxyHbA]+CO (I); and [COHbA] (J)). Based on the kinetics and UV resonance Raman spectra from such samples (43Khan I. Shannon C.F. Dantsker D. Friedman A.J. Perez-Gonzalez-de-Apodaca J. Friedman J.M. Biochemistry. 2000; 39: 16099-16109Crossref PubMed Scopus (96) Google Scholar, 45Juszczak L. Friedman J. J. Biol. Chem. 1999; 274: 30357-30360Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar,46Das T. Khan I. Rousseau D. Friedman J. Biospectroscopy. 1999; 5: S64-S70Crossref PubMed Google Scholar), we have unambiguously established that, in the first two samples, HbA is locked in the T quaternary structure and in the last it is locked in the R quaternary structure. The spectra from the two equilibrium forms, [deoxyHbA] and the 8-ns photoproduct of [COHbA], are both identical to the corresponding spectra obtained in solution (see inset in Fig. 1 and Table I). It can be seen that the frequency of ν(Fe-His) for the photoproduct of the liganded T state derivative, [deoxyHbA]+CO, is clearly higher than that of the deoxy T state but substantially lower than that of the R state photoproduct. This frequency is similar to what has been reported for either mutant Hbs stabilized in the T state even when fully liganded (52Friedman J.M. Rousseau D.L. Ondrias M.R. Stepnoski R.A. Science. 1982; 218: 1244-1246Crossref PubMed Google Scholar) and for the early time photoproduct of T state NOHbA+IHP (53Friedman J.M. Scott T.W. Stepnoski R.A. Ikeda-Saito M. Yonetani T. J. Biol. Chem. 1983; 258: 10564-10572Abstract Full Text PDF PubMed Google Scholar). Not shown is the spectrum obtained from a sample of [oxyHbA], which had been deoxygenated through the addition of dithionite after gelation and aging. In this case the frequency of ν(Fe-His) is ∼223 cm−1, a value much lower than that of the R state photoproduct but at a value in the frequency range for mutant or modified ferrous Hbs and transient forms that are in the R state despite being ligand free (54Kitagawa T. Spiro T.G. Biological Application of Raman Spectroscopy. III. Wiley & Sons, New York1988: 97-131Google Scholar, 55Ondrias M.R. Rousseau D.L. Kitagawa T. Ikeda-Saito M. Inubushi T. Yonetani T. J. Biol. Chem. 1982; 257: 8766-8770Abstract Full Text PDF PubMed Google Scholar, 56Ondrias M.R. Rousseau D.L. Shelnutt J.A. Simon S.R. Biochemistry. 1982; 21: 3428-3437Crossref PubMed Google Scholar, 57Scott T.W. Friedman J.M. J. Am. Chem. Soc. 1984; 106: 5677-5687Crossref Google Scholar, 58Jayaraman V. Rodgers K.R. Mukerji I. Spiro T.G. Science. 1995; 269: 1843-1848Crossref PubMed Google Scholar). The question now arises as to whether the spectroscopically distinct encapsulated COMb samples are also functionally distinct. For Hb such a correlation exists. Earlier work on encapsulated HbA clearly shows a progression in the geminate recombination that parallels the changes in the quaternary state (43Khan I. Shannon C.F. Dantsker D. Friedman A.J. Perez-Gonzalez-de-Apodaca J. Friedman J.M. Biochemistry. 2000; 39: 16099-16109Crossref PubMed Scopus (96) Google Scholar, 44Khan I. Dantsker D. Samuni U. Friedman A.J. Bonaventura C. Manjula B. Acharya S.A. Friedman J.M. Biochemistry. 2001; 40: 7581-7592Crossref PubMed Google Scholar). The geminate yield for T state COHbA is substantially lower than that of R state COHbA. The kinetics for the solvent phase rebinding associated with the encapsulated CO derivatives of HbA show a direct correspondence to kinetic phases that are observed in solution (70Dasgupta S. Spiro T.G. Biochemistry. 1986; 25: 5941-5948Crossref PubMed Scopus (38) Google Scholar). Whereas in solution both the slow T state and fast R state solvent phases are observed, for the encapsulated HbA samples only one solvent phase is seen. The encapsulated T state COHbA derivative, [deoxyHbA]+CO, and the encapsulated R state COHbA derivative, [COHbA], exhibit the slow T state and fast R state solvent phase recombination phases, respectively. Fig. 2 shows a comparison of the kinetic traces of CO rebinding to photodissociated COMb under different conditions. In the top panel, there is a comparison of the kinetics at 3.5 °C for COMb in solution (trace a), [deoxyMb]+CO bathed in buffer (trace b), and [COMb] bathed in buffer (trace c) where the CO saturated buffer in all three cases contains 25% glycerol. It can be seen that the geminate yield increases in going from solution, to [deoxyMb]+CO, to [COMb]. It can also be seen that the solvent phase kinetics for the encapsulated samples are different both from each other and from the solution sample. The near vertical decay seen for the solution sample is characteristic of the expected exponential kinetics. Both encapsulated samples exhibit solvent phase kinetic traces that show a sloping decay suggestive of a distribution of rates. The distribution for [COMb] clearly consists of a faster rebinding kinetic population compared with that of [deoxyMb]. Again the pattern is similar to what is seen for the comparison of kinetics from [COHbA] and [deoxyHbA]+CO, but the magnitude of the protocol-specific differences is smaller for the Mb samples as might be anticipated from the Raman results. The Mb kinetic differences are similar in magnitude to the variation in the kinetic pattern within a given quaternary state of encapsulated Hb due to such factors such as the presence or absence of allosteric effectors or chemical modification (e.g.modification of β93-SH) (44Khan I. Dantsker D. Samuni U. Friedman A.J. Bonaventura C. Manjula B. Acharya S.A. Friedman J.M. Biochemistry. 2001; 40: 7581-7592Crossref PubMed Google Scholar). The second panel in Fig. 2 depicts the kinetic traces at 3.5 °C for the same three types of samples as above, but in each case the buffer used both for the solution phase sample and for the bathing solvent for the two sol-gel samples contains 50% glycerol. The pattern observed in the upper panel is further enhanced in this comparison. The geminate yield increases in going from solution (trace d) to [deoxyMb]+CO (trace e) to [COMb] (trace f). The geminate yield for both of the encapsulated samples is enhanced over that of the corresponding samples bathed in buffer containing 25% glycerol, but the protocol-specific difference remains. It is also apparent that the solvent phase rebinding is again faster for the [COMb] sample. In the bottom panel a kinetic comparison is shown for the two encapsulated samples, but in this case the bathing solvent is CO-saturated glyce
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