A new technique with controlled interface generation allows separation and quantitation of enzyme inactivation by both solvent/aqueous interface and dissolved solvent. This has now been used in n-butanol, isopropylether, 2-octanone, n-hexane, n-butylbenzene, and n-tridecane. Ribonuclease was stable with all the solvent/aqueous interfaces studied. Chymotrypsin was mainly inactivated by the more hydrophobic solvent/aqueous interfaces, whereas lipase was only inactivated by the less hydrophobic solvent/aqueous interfaces. Urease was inactivated by some interfaces, but not all, without an obvious trend. Thus, the commonly expected simple relationship with solvent polarity (e.g., log P) does not apply when interfacial inactivation is determined specifically. Greater dissolved solvent inactivation occurred with the more polar solvents, though only a general trend was apparent with log P. A better correlation was noted with the Hilde-brand solubility parameter. Interfacial effects are discussed with reference to enzyme molecular weight, denaturation temperature, hydrophobicity, and adiabatic compressibility.
A liquid-liquid bubble column apparatus allows exposure of enzyme solutions to water-immiscible organic solvents with a known total interfacial area and welldefined time scales and flow. It allows clear distinction of the different classes of inactivation mechanism. With urease as a model enzyme, octan-2-one and butylbenzene act only through the effects of solvent molecules dissolved in the aqueous phase, giving first-order inactivation at 0.34 and 0.21 h(-1), respectively. Hexane and tridecane act only through exposure to the interface. The amount of urease inactivated is proportional to the total area of interface exposed, rather than to elapsed time, and may be characterized by a rate of about 0.5 mukat m(-2). This is consistent with the formation and (partial) inactivation of a complete adsorbed monolayer of protein. With butan-1-ol, both mechanisms contribute significantly to the observed inactivation. The presence of O(2) increases the rate of interfacial inactivation, but not that by dissolved solvent. (c) 1994 John Wiley & Sons, Inc.
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