Skip to main content
Biophysical Journal logoLink to Biophysical Journal
. 2002 Dec;83(6):3637–3651. doi: 10.1016/S0006-3495(02)75364-2

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 1, Emil A Pastrana 1, Yancy Ferrer 1, Qing Huang 1, Reinhard Schweitzer-Stenner 1, Kai Griebenow 1
PMCID: PMC1302439  PMID: 12496131

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.

Full Text

The Full Text of this article is available as a PDF (460.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Affleck R., Xu Z. F., Suzawa V., Focht K., Clark D. S., Dordick J. S. Enzymatic catalysis and dynamics in low-water environments. Proc Natl Acad Sci U S A. 1992 Feb 1;89(3):1100–1104. doi: 10.1073/pnas.89.3.1100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allison S. D., Chang B., Randolph T. W., Carpenter J. F. Hydrogen bonding between sugar and protein is responsible for inhibition of dehydration-induced protein unfolding. Arch Biochem Biophys. 1999 May 15;365(2):289–298. doi: 10.1006/abbi.1999.1175. [DOI] [PubMed] [Google Scholar]
  3. Allison S. D., Dong A., Carpenter J. F. Counteracting effects of thiocyanate and sucrose on chymotrypsinogen secondary structure and aggregation during freezing, drying, and rehydration. Biophys J. 1996 Oct;71(4):2022–2032. doi: 10.1016/S0006-3495(96)79400-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Baker L. J., Hansen A. M., Rao P. B., Bryan W. P. Effects of the presence of water on lysozyme conformation. Biopolymers. 1983 Jul;22(7):1637–1640. doi: 10.1002/bip.360220703. [DOI] [PubMed] [Google Scholar]
  5. Belton P. S., Gil A. M. IR and Raman spectroscopic studies of the interaction of trehalose with hen egg white lysozyme. Biopolymers. 1994 Jul;34(7):957–961. doi: 10.1002/bip.360340713. [DOI] [PubMed] [Google Scholar]
  6. Berman H. M., Westbrook J., Feng Z., Gilliland G., Bhat T. N., Weissig H., Shindyalov I. N., Bourne P. E. The Protein Data Bank. Nucleic Acids Res. 2000 Jan 1;28(1):235–242. doi: 10.1093/nar/28.1.235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burke P. A., Griffin R. G., Klibanov A. M. Solid-state NMR assessment of enzyme active center structure under nonaqueous conditions. J Biol Chem. 1992 Oct 5;267(28):20057–20064. [PubMed] [Google Scholar]
  8. Careri G., Giansanti A., Gratton E. Lysozyme film hydration events: an ir and gravimetric study. Biopolymers. 1979 May;18(5):1187–1203. doi: 10.1002/bip.1979.360180512. [DOI] [PubMed] [Google Scholar]
  9. Carpenter J. F., Prestrelski S. J., Dong A. Application of infrared spectroscopy to development of stable lyophilized protein formulations. Eur J Pharm Biopharm. 1998 May;45(3):231–238. doi: 10.1016/s0939-6411(98)00005-8. [DOI] [PubMed] [Google Scholar]
  10. Carrasquillo K. G., Carro J. C., Alejandro A., Toro D. D., Griebenow K. Reduction of structural perturbations in bovine serum albumin by non-aqueous microencapsulation. J Pharm Pharmacol. 2001 Jan;53(1):115–120. doi: 10.1211/0022357011775091. [DOI] [PubMed] [Google Scholar]
  11. Carrasquillo K. G., Costantino H. R., Cordero R. A., Hsu C. C., Griebenow K. On the structural preservation of recombinant human growth hormone in a dried film of a synthetic biodegradable polymer. J Pharm Sci. 1999 Feb;88(2):166–173. doi: 10.1021/js980272o. [DOI] [PubMed] [Google Scholar]
  12. Carrasquillo K. G., Sanchez C., Griebenow K. Relationship between conformational stability and lyophilization-induced structural changes in chymotrypsin. Biotechnol Appl Biochem. 2000 Feb;31(Pt 1):41–53. doi: 10.1042/ba19990087. [DOI] [PubMed] [Google Scholar]
  13. Carrasquillo K. G., Stanley A. M., Aponte-Carro J. C., De Jésus P., Costantino H. R., Bosques C. J., Griebenow K. Non-aqueous encapsulation of excipient-stabilized spray-freeze dried BSA into poly(lactide-co-glycolide) microspheres results in release of native protein. J Control Release. 2001 Oct 19;76(3):199–208. doi: 10.1016/s0168-3659(01)00430-8. [DOI] [PubMed] [Google Scholar]
  14. Castellanos I. J., Cuadrado W. O., Griebenow K. Prevention of structural perturbations and aggregation upon encapsulation of bovine serum albumin into poly(lactide-co-glycolide) micropheres using the solid-in-oil-in water technique. J Pharm Pharmacol. 2001 Aug;53(8):1099–1107. doi: 10.1211/0022357011776487. [DOI] [PubMed] [Google Scholar]
  15. Castellanos Ingrid J., Cruz Gloydian, Crespo Rubén, Griebenow Kai. Encapsulation-induced aggregation and loss in activity of gamma-chymotrypsin and their prevention. J Control Release. 2002 Jun 17;81(3):307–319. doi: 10.1016/s0168-3659(02)00073-1. [DOI] [PubMed] [Google Scholar]
  16. Costantino H. R., Carrasquillo K. G., Cordero R. A., Mumenthaler M., Hsu C. C., Griebenow K. Effect of excipients on the stability and structure of lyophilized recombinant human growth hormone. J Pharm Sci. 1998 Nov;87(11):1412–1420. doi: 10.1021/js980069t. [DOI] [PubMed] [Google Scholar]
  17. Dong A., Prestrelski S. J., Allison S. D., Carpenter J. F. Infrared spectroscopic studies of lyophilization- and temperature-induced protein aggregation. J Pharm Sci. 1995 Apr;84(4):415–424. doi: 10.1002/jps.2600840407. [DOI] [PubMed] [Google Scholar]
  18. Grdadolnik J., Maréchal Y. Bovine serum albumin observed by infrared spectrometry. I. Methodology, structural investigation, and water uptake. Biopolymers. 2001;62(1):40–53. doi: 10.1002/1097-0282(2001)62:1<40::AID-BIP60>3.0.CO;2-C. [DOI] [PubMed] [Google Scholar]
  19. Griebenow K., Klibanov A. M. Lyophilization-induced reversible changes in the secondary structure of proteins. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):10969–10976. doi: 10.1073/pnas.92.24.10969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Griebenow K., Vidal M., Baéz C., Santos A. M., Barletta G. Nativelike enzyme properties are important for optimum activity in neat organic solvents. J Am Chem Soc. 2001 Jun 6;123(22):5380–5381. doi: 10.1021/ja015889d. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Habeeb A. F. Determination of free amino groups in proteins by trinitrobenzenesulfonic acid. Anal Biochem. 1966 Mar;14(3):328–336. doi: 10.1016/0003-2697(66)90275-2. [DOI] [PubMed] [Google Scholar]
  22. Henriksen A., Smith A. T., Gajhede M. The structures of the horseradish peroxidase C-ferulic acid complex and the ternary complex with cyanide suggest how peroxidases oxidize small phenolic substrates. J Biol Chem. 1999 Dec 3;274(49):35005–35011. doi: 10.1074/jbc.274.49.35005. [DOI] [PubMed] [Google Scholar]
  23. Holzbaur I. E., English A. M., Ismail A. A. FTIR study of the thermal denaturation of horseradish and cytochrome c peroxidases in D2O. Biochemistry. 1996 Apr 30;35(17):5488–5494. doi: 10.1021/bi952233m. [DOI] [PubMed] [Google Scholar]
  24. Howes B. D., Feis A., Raimondi L., Indiani C., Smulevich G. The critical role of the proximal calcium ion in the structural properties of horseradish peroxidase. J Biol Chem. 2001 Aug 23;276(44):40704–40711. doi: 10.1074/jbc.M107489200. [DOI] [PubMed] [Google Scholar]
  25. Howes B. D., Rodriguez-Lopez J. N., Smith A. T., Smulevich G. Mutation of distal residues of horseradish peroxidase: influence on substrate binding and cavity properties. Biochemistry. 1997 Feb 11;36(6):1532–1543. doi: 10.1021/bi962502o. [DOI] [PubMed] [Google Scholar]
  26. Kabsch W., Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. doi: 10.1002/bip.360221211. [DOI] [PubMed] [Google Scholar]
  27. Karr L. J., Donnelly D. L., Kozlowski A., Harris J. M. Use of poly(ethylene glycol)-modified antibody in cell extraction. Methods Enzymol. 1994;228:377–390. doi: 10.1016/0076-6879(94)28037-1. [DOI] [PubMed] [Google Scholar]
  28. Klibanov A. M. Enzyme memory. What is remembered and why? Nature. 1995 Apr 13;374(6523):596–596. doi: 10.1038/374596a0. [DOI] [PubMed] [Google Scholar]
  29. Krimm S., Bandekar J. Vibrational spectroscopy and conformation of peptides, polypeptides, and proteins. Adv Protein Chem. 1986;38:181–364. doi: 10.1016/s0065-3233(08)60528-8. [DOI] [PubMed] [Google Scholar]
  30. Kuntz I. D., Jr, Kauzmann W. Hydration of proteins and polypeptides. Adv Protein Chem. 1974;28:239–345. doi: 10.1016/s0065-3233(08)60232-6. [DOI] [PubMed] [Google Scholar]
  31. Poole P. L., Finney J. L. Sequential hydration of a dry globular protein. Biopolymers. 1983 Jan;22(1):255–260. doi: 10.1002/bip.360220135. [DOI] [PubMed] [Google Scholar]
  32. Poole P. L., Finney J. L. Sequential hydration of dry proteins: a direct difference IR investigation of sequence homologs lysozyme and alpha- lactalbumin. Biopolymers. 1984 Sep;23(9):1647–1666. doi: 10.1002/bip.360230904. [DOI] [PubMed] [Google Scholar]
  33. Prestrelski S. J., Arakawa T., Carpenter J. F. Separation of freezing- and drying-induced denaturation of lyophilized proteins using stress-specific stabilization. II. Structural studies using infrared spectroscopy. Arch Biochem Biophys. 1993 Jun;303(2):465–473. doi: 10.1006/abbi.1993.1310. [DOI] [PubMed] [Google Scholar]
  34. Prestrelski S. J., Tedeschi N., Arakawa T., Carpenter J. F. Dehydration-induced conformational transitions in proteins and their inhibition by stabilizers. Biophys J. 1993 Aug;65(2):661–671. doi: 10.1016/S0006-3495(93)81120-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Pérez Caroline, Castellanos Ingrid J., Costantino Henry R., Al-Azzam Wasfi, Griebenow Kai. Recent trends in stabilizing protein structure upon encapsulation and release from bioerodible polymers. J Pharm Pharmacol. 2002 Mar;54(3):301–313. doi: 10.1211/0022357021778448. [DOI] [PubMed] [Google Scholar]
  36. Rupley J. A., Careri G. Protein hydration and function. Adv Protein Chem. 1991;41:37–172. doi: 10.1016/s0065-3233(08)60197-7. [DOI] [PubMed] [Google Scholar]
  37. Santos A. M., Vidal M., Pacheco Y., Frontera J., Báez C., Ornellas O., Barletta G., Griebenow K. Effect of crown ethers on structure, stability, activity, and enantioselectivity of subtilisin Carlsberg in organic solvents. Biotechnol Bioeng. 2001 Aug 20;74(4):295–308. [PMC free article] [PubMed] [Google Scholar]
  38. Sarciaux J. M., Hageman M. J. Effects of bovine somatotropin (rbSt) concentration at different moisture levels on the physical stability of sucrose in freeze-dried rbSt/sucrose mixtures. J Pharm Sci. 1997 Mar;86(3):365–371. doi: 10.1021/js960217k. [DOI] [PubMed] [Google Scholar]
  39. Schweitzer-Stenner R. Allosteric linkage-induced distortions of the prosthetic group in haem proteins as derived by the theoretical interpretation of the depolarization ratio in resonance Raman scattering. Q Rev Biophys. 1989 Nov;22(4):381–479. doi: 10.1017/s0033583500003164. [DOI] [PubMed] [Google Scholar]
  40. Sirotkin V. A., Zinatullin A. N., Solomonov B. N., Faizullin D. A., Fedotov V. D. Calorimetric and Fourier transform infrared spectroscopic study of solid proteins immersed in low water organic solvents. Biochim Biophys Acta. 2001 Jun 11;1547(2):359–369. doi: 10.1016/s0167-4838(01)00201-1. [DOI] [PubMed] [Google Scholar]
  41. Smulevich G., Paoli M., Burke J. F., Sanders S. A., Thorneley R. N., Smith A. T. Characterization of recombinant horseradish peroxidase C and three site-directed mutants, F41V, F41W, and R38K, by resonance Raman spectroscopy. Biochemistry. 1994 Jun 14;33(23):7398–7407. doi: 10.1021/bi00189a046. [DOI] [PubMed] [Google Scholar]
  42. Smulevich G., Paoli M., De Sanctis G., Mantini A. R., Ascoli F., Coletta M. Spectroscopic evidence for a conformational transition in horseradish peroxidase at very low pH. Biochemistry. 1997 Jan 21;36(3):640–649. doi: 10.1021/bi960427b. [DOI] [PubMed] [Google Scholar]
  43. Spiro T. G. Resonance Raman spectroscopy as a probe of heme protein structure and dynamics. Adv Protein Chem. 1985;37:111–159. doi: 10.1016/s0065-3233(08)60064-9. [DOI] [PubMed] [Google Scholar]
  44. Stocks S. J., Jones A. J., Ramey C. W., Brooks D. E. A fluorometric assay of the degree of modification of protein primary amines with polyethylene glycol. Anal Biochem. 1986 Apr;154(1):232–234. doi: 10.1016/0003-2697(86)90520-8. [DOI] [PubMed] [Google Scholar]
  45. Vecchio G, Zambianchi F, Zacchetti P, Secundo F, Carrea G. Fourier-transform infrared spectroscopy study of dehydrated lipases from candida antarctica B and pseudomonas cepacia . Biotechnol Bioeng. 1999 Sep 5;64(5):545–551. doi: 10.1002/(sici)1097-0290(19990905)64:5<545::aid-bit4>3.0.co;2-y. [DOI] [PubMed] [Google Scholar]
  46. Yu N. T. Comparison of protein structure in crystals, in lyophilized state, and in solution by laser Raman scattering. 3. Alpha-Lactalbumin. J Am Chem Soc. 1974 Jul 10;96(14):4664–4668. doi: 10.1021/ja00821a049. [DOI] [PubMed] [Google Scholar]
  47. Yu N. T., Jo B. H. Comparison of protein structure in crystals and in solution by laser raman scattering. I. Lysozyme. Arch Biochem Biophys. 1973 Jun;156(2):469–474. doi: 10.1016/0003-9861(73)90296-8. [DOI] [PubMed] [Google Scholar]
  48. van de Weert M., Haris P. I., Hennink W. E., Crommelin D. J. Fourier transform infrared spectrometric analysis of protein conformation: effect of sampling method and stress factors. Anal Biochem. 2001 Oct 15;297(2):160–169. doi: 10.1006/abio.2001.5337. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES