• Bulletin of the Korean Chemical Society
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• 제30권4호
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• pp.913-916
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• 2009

Femtosecond vibrational spectroscopy was used to probe the dynamics of CO rebinding to hemoglobin (Hb), denatured by 4.0 M GdnHCl in $D_2O# at 283 K, after photolysis of HbCO. The stretching mode of $^{13}CO$ bound to the denatured $Hb^{13}CO$ showed a single band centered at 1922 $cm^{-1}$, indistinguishable from that of denatured $Mb^{13}CO$. Geminate rebinding of CO to the denatured Hb was accelerated more than 1000 times, suggesting that the native structure of the Hb is required to suppress efficient geminate rebinding of CO, as is the case in Mb. The geminate yield and rate for CO rebinding are almost the same in both the denatured Hb and Mb. Similarity in the equilibrium spectrum and rebinding dynamics of CO indicates that the state of the denatured Hb is very similar to that of the denatured Mb. In the denatured Hb, quaternary contact of the protein is likely severed, with the denatured protein existing as an independent subunit much like Mb.

• Bulletin of the Korean Chemical Society
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• 제30권1호
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• pp.177-182
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• 2009

We present the geminate rebinding kinetics measurements of CO to 2-methylimidazole (2-MeIm) bound ferrous protoporphyrin- IX (FePPIX) in alkaline glycerol/water mixtures at 293 K after photolysis. The kinetics was probed by monitoring the CO stretching mode using femtosecond vibrational spectroscopy. When 2-MeIm is used in excess, heme dimers that typically form in low viscosity solutions disappear as the viscosity of the solvent increases. Heme aggregates formed in low viscosity solutions turn monomeric as more 2-MeIm is added, suggesting that 6-coordinated heme, including a strong proximal histidine tends to be in the monomeric form. The vibrational band of CO in the 2-MeIM-FePPIX-CO is well described by a single Gaussian function centered at 1958 $cm^-1$ and 28 $cm^-1$ full width at half maximum. The efficiency and rate of the geminate rebinding of CO to the heme increase with viscosity of the solvent, suggesting that retention of the dissociated CO near the heme, for a longer period by the viscous solvent media, accelerates rebinding.

• Bulletin of the Korean Chemical Society
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• 제33권3호
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• pp.925-928
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• 2012

The accurate expression for the steady-state velocity of an irreversible enzyme-catalyzed reaction obtained by Shin and co-workers (J. Chem. Phys. 2001, 115, 1455) is generalized to allow for the rebinding of the product. The amplitude of the power-law ($t^{-1/2}$) relaxation of the free- and bound-enzyme concentrations to steady-state values is expressed in terms of the steady-state velocity and the intrinsic (chemical) rate constants. This result is conjectured to be exact, even though our expression for the steady-state velocity in terms of microscopic parameters is only approximate.

• Biotechnology and Bioprocess Engineering:BBE
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• 제3권2호
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• pp.82-86
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• 1998

The maltose binding protein (MBP) fusion protein system is a versatile tool to express and isolate recombinant proteins in E. coli. In this system, MBP fusion proteins are efficiently isolated from whole cell lysate using amylose conjugated agarose beads and then eluted by competition with free maltose. Since MBP is a rather large molecule (∼42 kDa), for further experiments, the MBP part is usually proteolytically cleaved from the fusion protein and subsequently removed by ion-exchange chromatography or rebinding to amylose columns after washing out excess and MBP-bound maltose. In the present study, we have developed an improved method for the removal of cleaved MBP, which is advantageous over conventional methods. In this method, factor Xa cleaved MBP fusion proteins were incubated with Sepharose beads conjugated with MBP specific monoclonal antibodies and then precipitated buy centrifugation, resulting in highly purified proteins in the supernatant.