Effect of dioxane on the structure and hydration-dehydration of alpha-chymotrypsin as measured by FTIR spectroscopy.

Abstract

A new experimental approach based on FTIR spectroscopic measurements was proposed to study simultaneously the adsorption/desorption of water and organic solvent on solid enzyme and corresponding changes in the enzyme secondary structure in the water activity range from 0 to 1.0 at 25 degrees C. The effect of dioxane on the hydration/dehydration and structure of bovine pancreatic alpha-chymotrypsin (CT) was characterized by means of this approach. Dioxane sorption exhibits pronounced hysteresis. No sorbed dioxane was observed at low water activities (a(w)<0.5) during hydration. At a(w) about 0.5, a sharp increase in the amount of sorbed dioxane was observed. Dioxane sorption isotherm obtained during dehydration resembles a smooth curve. In this case, CT binds about 150 mol dioxane/mol enzyme at the lowest water activities. Three different effects of dioxane on the water binding by the initially dried CT were observed. At a(w)<0.5, water adsorption is similar in the presence and absence of dioxane. It was concluded that the presence of dioxane has little effect on the interaction between enzyme and tightly bound water at low a(w). At a(w)>0.5, dioxane increases the amount of water bound by CT during hydration. This behavior was interpreted as a dioxane-assisted effect on water binding. Upon dehydration at low water activities, dioxane decreases the water content at a given a(w). This behavior suggests that the suppression in the uptake of water during dehydration may be due to a competition for water-binding sites on chymotrypsin by dioxane. Changes in the secondary structure of CT were determined from infrared spectra by analyzing the structure of amide I band. Dioxane induced a strong band at 1628 cm(-1) that was assigned to the intermolecular beta-sheet aggregation. Changes in the intensity of the 1628 cm(-1) band agree well with changes in the dioxane sorption by CT. An explanation of the dioxane effect on the CT hydration and structure was provided on the basis of hypothesis on water-assisted disruption of polar contacts in the solid enzyme. The reported results demonstrate that the hydration and structure of alpha-chymotrypsin depend markedly on how enzyme has been hydrated - whether in the presence or in the absence of organic solvent. A qualitative model was proposed to classify the effect of hydration history on the enzyme activity-a(w) profiles.

• Authors: Vladimir A Sirotkin
• Journal: Biochimica et biophysica acta (Biochim Biophys Acta) Vol. 1750 Issue 1 Pg. 17-29 (Jun 15 2005) ISSN: 0006-3002 [Print] Netherlands
• PMID: 15882959 (Publication Type: Comparative Study, Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Dioxanes
• Water
• Chymotrypsin
• alpha-chymotrypsin
• 1,4-dioxane

Topics

• Animals
• Cattle
• Chymotrypsin (chemistry)
• Desiccation
• Dioxanes (chemistry)
• Models, Chemical
• Protein Conformation
• Spectroscopy, Fourier Transform Infrared
• Water