Immobilization of myoglobin from horse skeletal muscle in hydrophilic polymer networks

Abstract

This work examines the immobilization of myoglobin from horse skeletal muscle in hydrophilic polymer networks. Due to specific changes in the spectroscopic properties of hemoproteins during ligand binding, they could be employed in optical sensing devices. Two immobilization techniques were considered: imbibition and entrapment. Anionic hydrogels composed of methacrylic acid (MAA), cationic hydrogels composed of dimethylamino ethyl methacrylate (DMAEM), and neutral hydrogels composed of poly(ethylene glycol) monomethyl ether monomethacrylate (PEGMA; molecular weight = 200, 400, or 1000), all crosslinked with poly(ethylene glycol) dimethacrylate (PEGDMA) (molecular weight = 200, 600, or 1000), were synthesized by free-radical solution polymerization. By the imbibition method, MAA-based hydrogels incorporated the highest amount of myoglobin in comparison with PEGMA or DMAEM polymers. The evaluation of the correlation length of the networks revealed that MAA hydrogels had the highest correlation length in comparison with PEGMA-containing matrices or DMAEM hydrogels. Release experiments from MAA hydrogels at pHs 5.8 and 7.0 showed that the solute-transport mechanism was a combination of Fickian and chain relaxation diffusion. Myoglobin-loaded MAA hydrogels retained their heme reactivity after the immobilization process. The release of myoglobin incorporated by entrapment in MAA-PEGDMA hydrogels was highly influenced by the chain relaxation process. The diffusion coefficients of myoglobin incorporated by entrapment into anionic hydrogels were 2 orders of magnitude smaller (~10-13) than those for myoglobin incorporated by imbibition (10-11), both evaluated at pH 7.0. Substrate binding studies indicated that the protein biological activity was not compromised in those hydrogels loaded by the imbibition method, whereas prepolymeric solutions showed detrimental effects on protein stability.

Protein-responsive protein release of supramolecular/polymer hydrogel composite integrating enzyme activation systems

“…41). The molar absorption coefficients of Mb (18,800 70 ) and Alexa Flour 647 (290,000 69 ) were used.…”

Peroxidase versus Peroxygenase Activity: Substrate Substituent Effects as Modulators of Enzyme Function in the Multifunctional Catalytic Globin Dehaloperoxidase

“…The enzyme concentration was determined spectrophotometrically using an ε Soret of 116400 M –1 cm –1 . Horse skeletal muscle (HSM) myoglobin and horseradish peroxidase (HRP) were purchased from Sigma-Aldrich, and enzyme concentrations were determined using ε Soret values of 188000 M –1 cm –1 and 102000 M –1 cm –1 , respectively. Solutions of H 2 O 2 were prepared fresh daily in 100 mM KP i (pH 7) and kept on ice until they were needed.…”

Monitoring methanol-induced protein unfolding by fluorescence anisotropy measurements of covalently labelled rhodamine probe

“…Fluorescence measurements are performed after a 1:10 dilution. The final D/P ratio was determined using the molar extinction coefficients of Mb at 408 nm (188000 M -1 cm -1 ) 13 versus RhB one at 555 nm (106000 M -1 cm -1 ). 14…”