help button home button Biophys. J.
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Al-Azzam, W.
Right arrow Articles by Griebenow, K.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Al-Azzam, W.
Right arrow Articles by Griebenow, K.

Biophys J, December 2002, p. 3637-3651, Vol. 83, No. 6

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

Wasfi Al-Azzam,* Emil A. Pastrana,dagger Yancy Ferrer,dagger Qing Huang,dagger Reinhard Schweitzer-Stenner,dagger and Kai Griebenowdagger

Departments of  *Biology and  dagger Chemistry, University of Puerto Rico, San Juan, Puerto Rico 00931 USA

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.

Biophys J, December 2002, p. 3637-3651, Vol. 83, No. 6
© 2002 by the Biophysical Society   0006-3495/02/12/3637/15  $2.00


This article has been cited by other articles:


Home page
 Biophys. JHome page
Q. Huang, W. Al-Azzam, K. Griebenow, and R. Schweitzer-Stenner
Heme Structural Perturbation of PEG-Modified Horseradish Peroxidase C in Aromatic Organic Solvents Probed by Optical Absorption and Resonance Raman Dispersion Spectroscopy
Biophys. J., May 1, 2003; 84(5): 3285 - 3298.
[Abstract] [Full Text] [PDF]


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Copyright © 2002 by the Biophysical Society.