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...î,t) + ACO(î,t) (2) ¢A(î,t) ) Adeoxy(î,t) - ACO/ (î,t) (3) ACO / (î,t) ) ACO(î) - ACO(î,t) (4) A(î) ) M2î[L(î)XG(î)XP(î)] (5) L(î) ) ∑ m1âââmNh ∞ [∏hNh Shmhe-Shmh! ] ¡ [î - î0(T) - ∑ h Nh mhîh]2 + ¡2 =-=(6)-=- G(î) ) 1 ó(T)(2ð)1/2 e -î2/2ó2(T) (7) P(î) ) exp[- (xî - î00 + Q0x b)22bä2 ] + exp[- (xî - î00 - Q0x b)22bä2 ] 2ä x2ðb(î - î00) (8) Kinetic Hole Burning in Hemoglobin Biochemistry, Vol. 42, No. 15, 2...

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...the deoxy-component and negative signals from the COcomponent. ¢A(î,t) ) A(î,t) - ACO(î) (1) A(î,t) ) Adeoxy(î,t) + ACO(î,t) (2) ¢A(î,t) ) Adeoxy(î,t) - ACO/ (î,t) (3) ACO / (î,t) ) ACO(î) - ACO(î,t) =-=(4)-=- A(î) ) M2î[L(î)XG(î)XP(î)] (5) L(î) ) ∑ m1âââmNh ∞ [∏hNh Shmhe-Shmh! ] ¡ [î - î0(T) - ∑ h Nh mhîh]2 + ¡2 (6) G(î) ) 1 ó(T)(2ð)1/2 e -î2/2ó2(T) (7) P(î) ) exp[- (xî - î00 + Q0x b)22bä2 ] + exp[- (xî -...

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...tical conformational substates have been highlighted essentially from studies on myoglobin (4-8). For hemoglobin (Hb), studies on dynamic properties are complicated by quaternary structure relaxation =-=(9)-=- and by R-â heterogeneity (10). Nevertheless, such kind of studies are important since the characterization of the dynamic properties of both equilibrium species (e.g., fully ligand free and fully lig...

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...,t) - ACO/ (î,t) (3) ACO / (î,t) ) ACO(î) - ACO(î,t) (4) A(î) ) M2î[L(î)XG(î)XP(î)] (5) L(î) ) ∑ m1âââmNh ∞ [∏hNh Shmhe-Shmh! ] ¡ [î - î0(T) - ∑ h Nh mhîh]2 + ¡2 (6) G(î) ) 1 ó(T)(2ð)1/2 e -î2/2ó2(T) =-=(7)-=- P(î) ) exp[- (xî - î00 + Q0x b)22bä2 ] + exp[- (xî - î00 - Q0x b)22bä2 ] 2ä x2ðb(î - î00) (8) Kinetic Hole Burning in Hemoglobin Biochemistry, Vol. 42, No. 15, 2003 4501 the deconvolution of the CO-c...

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...ain the final hemoglobin concentration (8 íM in heme) in 75% v/v glycerol and 0.03 M phosphate buffer pH 7. The silica sol was synthesized from TMOS (Merck) according to the method of Ellerby et al. =-=(23)-=- with some modifications. A mixture of 60% TMOS, 38% deionized water, and 2% HCl 0.04 M was sonicated for 20 min in an ice bath and then mixed in a 2:3 proportion with the Hb solution. The gel is form...

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...e signals from the COcomponent. ¢A(î,t) ) A(î,t) - ACO(î) (1) A(î,t) ) Adeoxy(î,t) + ACO(î,t) (2) ¢A(î,t) ) Adeoxy(î,t) - ACO/ (î,t) (3) ACO / (î,t) ) ACO(î) - ACO(î,t) (4) A(î) ) M2î[L(î)XG(î)XP(î)] =-=(5)-=- L(î) ) ∑ m1âââmNh ∞ [∏hNh Shmhe-Shmh! ] ¡ [î - î0(T) - ∑ h Nh mhîh]2 + ¡2 (6) G(î) ) 1 ó(T)(2ð)1/2 e -î2/2ó2(T) (7) P(î) ) exp[- (xî - î00 + Q0x b)22bä2 ] + exp[- (xî - î00 - Q0x b)22bä2 ] 2ä x2ðb(î ...

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...and in the deoxy ligation state. The gel (still in the protective solution) was then exposed to CO by bubbling the protective solution for 4 h with opportunely humidified CO gas. As previously shown =-=(11, 15)-=-, this procedure leads to a gel in which Hb is still in the T quaternary conformation but in the CO ligation state. Experimental Apparatus. The hemoglobin gel was placed in a 4 mm thick sample holder,...

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...scape and suggests enhanced dynamics of ligation intermediate species such as T-state HbCO or R-state deoxyHb. Studies on protein dynamics are important since dynamics is crucial for protein function =-=(1)-=-. Myoglobin (Mb)1 is the protein most commonly used for such kind of studies; works on the structural dynamics of Mb (2, 3), besides contributing to the understanding of the structure-dynamics-functio...

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...d. Sol-gel encapsulation is therefore a promising approach toward the characterization of the dynamic properties and of the energy landscape of both equilibrium and intermediate species of hemoglobin =-=(15, 16)-=-. Ligand rebinding kinetics has been extensively used to probe protein dynamics. In a typical experiment, a fully CO saturated protein sample is photodissociated with a short light pulse, and CO rebin...

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...d. Sol-gel encapsulation is therefore a promising approach toward the characterization of the dynamic properties and of the energy landscape of both equilibrium and intermediate species of hemoglobin =-=(15, 16)-=-. Ligand rebinding kinetics has been extensively used to probe protein dynamics. In a typical experiment, a fully CO saturated protein sample is photodissociated with a short light pulse, and CO rebin...

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...ded CO molecule) starts within the time range of our experiment. As a final comment, we would like to discuss our data in comparison with analogous results from the pioneering work of Friedman et al. =-=(14, 15, 32)-=-. In these works, a sol-gel technique is used to encapsulate T-state and R-state HbCO, and kinetic properties are investigated by following the CO rebinding after flash photolysis at room (from -10 up...

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...n protein dynamics are important since dynamics is crucial for protein function (1). Myoglobin (Mb)1 is the protein most commonly used for such kind of studies; works on the structural dynamics of Mb =-=(2, 3)-=-, besides contributing to the understanding of the structure-dynamics-function relationships in this protein, have provided some general concepts in protein dynamics. In fact, concepts such as energy ...

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... ∑ m1âââmNh ∞ [∏hNh Shmhe-Shmh! ] ¡ [î - î0(T) - ∑ h Nh mhîh]2 + ¡2 (6) G(î) ) 1 ó(T)(2ð)1/2 e -î2/2ó2(T) (7) P(î) ) exp[- (xî - î00 + Q0x b)22bä2 ] + exp[- (xî - î00 - Q0x b)22bä2 ] 2ä x2ðb(î - î00) =-=(8)-=- Kinetic Hole Burning in Hemoglobin Biochemistry, Vol. 42, No. 15, 2003 4501 the deconvolution of the CO-component, ACO / (î,t), of the difference spectra. ACO / (î,t) is, actually, the difference of ...

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...s have been highlighted essentially from studies on myoglobin (4-8). For hemoglobin (Hb), studies on dynamic properties are complicated by quaternary structure relaxation (9) and by R-â heterogeneity =-=(10)-=-. Nevertheless, such kind of studies are important since the characterization of the dynamic properties of both equilibrium species (e.g., fully ligand free and fully liganded Hb) and nonequilibrium i...

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...ded CO molecule) starts within the time range of our experiment. As a final comment, we would like to discuss our data in comparison with analogous results from the pioneering work of Friedman et al. =-=(14, 15, 32)-=-. In these works, a sol-gel technique is used to encapsulate T-state and R-state HbCO, and kinetic properties are investigated by following the CO rebinding after flash photolysis at room (from -10 up...

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... frequency î0, it has been shown (25, 26) that, for heme proteins in unliganded state, the spectral heterogeneity can be well-approximated by an expression first introduced by Champion and co-workers =-=(19, 25)-=-: This is the expression that we have assumed in eq 5 to analyze the deoxy-component, Adeoxy(î,t), of the difference spectra. For heme proteins in liganded state, instead, the P(î) term is a further G...

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...endence on quaternary conformation. The novelty of the approach lies on the combined use of the solgel encapsulation technique and of the Soret band spectral deconvolution developed at our laboratory =-=(21)-=-: this enables us to study spectral shifts, KHB, and relaxations of both the deoxy and CO components in the same experiment. Therefore, information is obtained not only on the equilibrium deoxyHb and ...

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...th improved time resolution are needed to definitely discriminate between the above two alternative hypotheses. To evaluate the presence of KHB, we plot the peak frequency shift as a function of N(t) =-=(29, 30)-=-. Peak frequency shifts are defined as ¢î(t) ) î(t) - î(∞) (i.e., as the difference between the peak frequency observed at time t after photolysis and that observed at a time after photolysis sufficie...

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...havior observed for ¢îCO of the R-state HbCO, in turn, argues against the possibility that the observed red shift is due to dynamic hole filling: indeed, substates interconversion responsible for DHF =-=(20)-=- could hardly generate a universal behavior. For Hb in R quaternary conformation, no ¢îdeoxy has been observed, within our experimental error, between 100 and FIGURE 4: Time evolution at T ) 100 K of ...

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...ccessible intermediate species R-state deoxyHb and T-state HbCO. MATERIALS AND METHODS Sample Preparation. Human hemoglobin was prepared and stored following a standard procedure previously described =-=(22)-=-. HbO2 from concentrated stocks (12 wt %) was diluted in suitable water-glycerol-buffer solutions to obtain the final hemoglobin concentration (8 íM in heme) in 75% v/v glycerol and 0.03 M phosphate...

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...he coupling of the electronic transition with a bath of soft modes (hî of the same order of magnitude as kT, or smaller than kT). It can be shown that G(î), in the so-called short times approximation =-=(24)-=-, is a Gaussian The third term, P(î), describes the spectral heterogeneity of the system: molecules in different conformational substates may correspond to different frequencies of the considered elec...

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...rom CO docking sites in which the interaction of the heme group with photolyzed CO molecule is minimal, thus resulting in weakened KHB, much like as it happens in Mb at temperatures higher than 180 K =-=(31)-=-; (2) interconversion between conformational substates responsible for KHB in the deoxycomponent (substates likely corresponding to different orientations of the photolyzed CO molecule in the B site a...

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...n protein dynamics are important since dynamics is crucial for protein function (1). Myoglobin (Mb)1 is the protein most commonly used for such kind of studies; works on the structural dynamics of Mb =-=(2, 3)-=-, besides contributing to the understanding of the structure-dynamics-function relationships in this protein, have provided some general concepts in protein dynamics. In fact, concepts such as energy ...

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...erent frequencies of the considered electronic transition. In the hypothesis of a quadratic coupling between the iron out-of-plane coordinate Q and the Soret band peak frequency î0, it has been shown =-=(25, 26)-=- that, for heme proteins in unliganded state, the spectral heterogeneity can be well-approximated by an expression first introduced by Champion and co-workers (19, 25): This is the expression that we ...

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...erent frequencies of the considered electronic transition. In the hypothesis of a quadratic coupling between the iron out-of-plane coordinate Q and the Soret band peak frequency î0, it has been shown =-=(25, 26)-=- that, for heme proteins in unliganded state, the spectral heterogeneity can be well-approximated by an expression first introduced by Champion and co-workers (19, 25): This is the expression that we ...

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...îCO(t) sGaussian width: ódeoxy(t), óCO(t) Values of the other parameters (i.e., ¡, Sh, îh, Q0b1/2, and äb1/2) are reported in Table 1: they have been determined from equilibrium spectral measurements =-=(27)-=- and have been maintained constant in the fittings. This is justified by the fact that spectral variations observed involve essentially intensity variations, peak shifts, and band broadenings; moreove...

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...th improved time resolution are needed to definitely discriminate between the above two alternative hypotheses. To evaluate the presence of KHB, we plot the peak frequency shift as a function of N(t) =-=(29, 30)-=-. Peak frequency shifts are defined as ¢î(t) ) î(t) - î(∞) (i.e., as the difference between the peak frequency observed at time t after photolysis and that observed at a time after photolysis sufficie...