Structure of Poly(Ethylene Glycol)-Modified Horseradish Peroxidase in Organic Solvents: Infrared Amide I Spectral Changes upon Protein Dehydration Are Largely Caused by Protein Structural Changes and Not by Water Removal Per Se

Abstract

Fourier transform infrared (FTIR) spectroscopy has emerged as a powerful tool to guide the development of stable lyophilized protein formulations by providing information on the structure of proteins in amorphous solids. The underlying assumption is that IR spectral changes in the amide I and III region upon protein dehydration are caused by protein structural changes. However, it has been claimed that amide I IR spectral changes could be the result of water removal per se. Here, we investigated whether such claims hold true. The structure of horseradish peroxidase (HRP) and poly(ethylene glycol)-modified HRP (HRP-PEG) has been investigated under various conditions (in aqueous solution, the amorphous dehydrated state, and dissolved/suspended in toluene and benzene) by UV-visible (UV-Vis), FTIR, and resonance Raman spectroscopy. The resonance Raman and UV-Vis spectra of dehydrated HRP-PEG dissolved in neat toluene or benzene were very similar to that of HRP in aqueous buffer, and thus the heme environment (heme iron spin, coordination, and redox state) was essentially the same under both conditions. Therefore, the three-dimensional structure of HRP-PEG dissolved in benzene and toluene was similar to that in aqueous solution. The amide I IR spectra of HRP-PEG in aqueous buffer and of dehydrated HRP-PEG dissolved in neat benzene and toluene were also very similar, and the secondary structure compositions (percentages of alpha-helices and beta-sheets) were within the standard error the same. These results are irreconcilable with recent claims that water removal per se could cause substantial amide I IR spectral changes (M. van de Weert, P.I. Haris, W.E. Hennink, and D.J. Crommelin. 2001. Anal. Biochem. 297:160-169). On the contrary, amide I IR spectral changes upon protein dehydration are caused by perturbations in the secondary structure.

Effect of PEG modification on subtilisin Carlsberg activity, enantioselectivity, and structural dynamics in 1,4‐dioxane

“…This allowed for the quantitative assessment of dehydration-and modification-induced structural perturbations when compared to the enzyme in aqueous solution (Griebenow et al, 1999b). Transfer of the enzyme from aqueous buffer to the organic solvent led to a 10% decrease in the SCC value (see Table III) in accord with previously described dehydrationinduced structural changes for other enzymes (Al-Azzam et al, 2002;Carrasquillo et al, 2000;Griebenow and Klibanov, 1995;Griebenow et al, 1999b;Kendrick et al, 1997;Prestrelski et al, 1993a). Interestingly, PEGylation of the enzyme did not induce additional structural changes (SCC values of 0.9) within the solvent used in this study (see Table III).…”

On the relationship between the activity and structure of PEG‐α‐chymotrypsin conjugates in organic solvents

“…To extend our understanding of the factors that contribute to the drop in enzyme activity in organic solvents, we explored the covalent modification of a-chymotrypsin with poly(ethylene glycol) (PEG) (Al-Azzam et al, 2002;Inada and Matsushima, 1990;Inada et al, 1995;Koops et al, 1999). PEG has been widely employed in biomedical and biotechnological applications (Matsuchima et al, 1996).…”

“…PEG has been widely employed in biomedical and biotechnological applications (Matsuchima et al, 1996). Some research has already been performed to investigate the usefulness of enzyme modification with PEG to improve enzyme activity in organic solvents but no systematic investigation has been done on how the amount of PEG bound to an enzyme influences its activity (Al-Azzam et al, 2002;Inada and Matsushima, 1990;Inada et al, 1995;Koops et al, 1999). As mentioned previously, one explanation for diminished enzymatic activities in anhydrous solvents steams from reduced conformational mobility (Burke et al, 1993;Griebenow et al, 2001;Hutcheon et al, 2000;Rupley and Careeri, 1991;Tsou, 1998).…”

Dynamical properties of myoglobin in an ultraviscous water-glycerol solvent investigated with elastic neutron scattering and FTIR spectroscopy

“…In fact, as known, these bands are associated to vibrations involving the backbone atoms of the proteins, and are particularly sensitive, especially in the case of Amide I′, to the conformational changes of the protein, since different secondary structure elements give rise to different absorptions contributions [32][33][34][35].…”