Journal article
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
Biophysical journal, v 83(6), pp 3637-3651
2002
PMID: 12496131
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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
α-helices and
β-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.
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Details
- Title
- 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
- Creators
- Wasfi Al-Azzam - University of Puerto Rico SystemEmil A. Pastrana - University of Puerto Rico SystemYancy Ferrer - University of Puerto Rico SystemQing Huang - University of Puerto Rico SystemReinhard Schweitzer-Stenner - University of Puerto Rico SystemKai Griebenow - University of Puerto Rico System
- Publication Details
- Biophysical journal, v 83(6), pp 3637-3651
- Publisher
- Elsevier
- Resource Type
- Journal article
- Language
- English
- Academic Unit
- Chemistry
- Web of Science ID
- WOS:000180256300067
- Scopus ID
- 2-s2.0-0036929348
- Other Identifier
- 991019196515904721
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- Web of Science research areas
- Biophysics