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SERUMDAKİ PROTEİN HİDRASYONUNUN NMR T1 ÖLÇÜMLERİ İLE İNCELENMESİ

Yıl 2007, Sayı: 9, 115 - 122, 01.12.2007

Öz

Bu çalışmada bir serum örneğinin spin-örgü durulma zamanı(T1) ölçümleri, bu örneğe kademe kademe albümin eklenerek ve inversion-recovery(IR) puls adımı kullanılarak, 60 MHz’ de çalışan bir FT-NMR ile ölçüldü. Her albümin eklemeden sonra saptanan 1/T1 oranları, o andaki Ma/Ms(albümin kütlesi/su kütlesi) oranına karşı grafiklendi. Böyle bir grafik, ucuca ekli dört doğru parçası vermektedir. Bir protein molekülünü saran su bölümlerinin, eklenen albümin vasıtası ile, dıştan içe doğru tükendiği varsayılmıştır. Bulunan doğru parçaları: serbest su, engellenmiş su, dönerek ve dönmeyerek bağlı su tabakalarına atfedildi. Bu doğrulara ait verilerden, bir gram proteine düşen su miktarı: 4,71 g (serbest), 1.5 g (engellenmiş), 0.38g(dönerek bağlı) ve 0.05 g (dönmeden bağlı) olarak bulundu. Bu bölümlere ait durulma oranları ise sırası ile, 0.59s-1 (serbest), 0.95s-1 (engellenmiş),1.83 s-1 (dönerek bağlı) ve 3.82 s-1 (dönmeden bağlı) olarak saptandı. Sonuçlar serbest ve hidrasyon suyu arasında su moleküllerinin hızlı kimyasal değiş-tokuş mekanizmasına dayanan durulma modeli ile tutarlı bulundu. Bloembergen, Purcell ve Pound tarafından ortaya konan durulma bağıntıları kullanılarak, serbest, engellenmiş, dönerek bağlı ve dönmesiz bağlı tabakaların ilgi zamanları sırası ile 10-12s, 5*10-10s, 1.2*10-9s ve 10-7s olarak saptandı. Literatürle uyumlu bulunan bu sonuçlar, bir çözeltideki hidrasyon suyunun çeşitli bölümlerine ait durulma zamanı ve su miktarlarının, protein ilavesine karşılık yapılan T1 ölçümleri ile saptanabileceğini önermektedir.

Kaynakça

  • Balasubramanian, S., Bandyopadhyay, S. Pal, S. & Bagchi, B. (2003). Dynamics of water at the interface of a small protein, enterotoxin, Current Science, 85(11), 1571-1578.
  • Bertini, I., Fragai, M., Luchinat, C. & Parigi, G. (2000). 1H NMRD profiles of diamagnetic proteins: a model-free analysis. Magn. Reson. Chem, 38, 543-550.
  • Bertini, I., Luchinat, C., Nerinovski, K., Parigi, G., Cross, M., Xiao, Z. & Wedd, A. G. (2003). Application of NMRD to hydration of rubredoxin and a variant containing a (Cys- S)3Feııı(OH) site, Biophys. J. 84, 545-551.
  • Bloembergen, N., Purcell, E. M. & Pound, R. F. (1948). Relaxation Effects in Nuclear Magnetic Resonance Absorption Phys.Rev, 73, 679-712.
  • Brooks, R. A. Battocletti, J. H. Sances, JR Larson, S. J. Bowman, L.R. & Kudravcev,V. (1975). Nuclear magnetic relaxation in blood. IEEE Transac.Biomed, Engin.BME-22(1), 12- 16.
  • Cameron, I. L, Ord, V. A & Fullerton, G. D. (1988). Water of hydration in the intra and extra cellular environment of human erythrocytes. Biochem.Cell.Biol, 66, 1186-1199.
  • Daskiewicz, O. K., Hennel, J. W. & Lubas, B. (1963). Proton magnetic relaxation and protein hydration, Nature, 200, 1006-1007.
  • Fullerton, G.D., Ord, V. A. & Cameron,I.L. (1986). An evaluation of the hydration of lysozyme by a NMR titration method. Biochim.Biophys.Acta, 869, 230-246.
  • Fullerton, G. D. (1988). Physiologic Basic of Magnetic Relaxation in Magnetic Resonance Imaging, David D. Stark & W. G. Bradley (Ed.)(ss. 36). St. Louis: The C.V. Mosby Company.
  • Gallier, J., Rivet, P. & Certaınes, J.de. (1987). 1H- and 2H-NMR study of bovine serum albumin solutions. Biochimica et Biophysica Acta, 915, 1-18.
  • Grösch, L. & Noack, F. (1976). NMR relaxation investigation of water mobility in aquueous bovine serum albumin solutions. Biochimica et Biophysica Acta, 453, 218-232.
  • Halle, B. (2004). Protein hydration dynamics in solution: a critical survey, Phil. Trans. R. Soc. Lond. B. 359, 1207-1224.
  • Hennel, J. W. & Klinowsky, J. (1996). Çekirdek Magnetik Rezonansın Temelleri. (Çev: S. Bahçeli). Ankara:Bizim Büro Basımevi (Özgün kitap 1993’da yayımlandı).
  • Kavak, G. (2000). “Serum Proteinlerinin Spin-Örgü Durulma Mekanizmalarının NMR ile incelenmesi”. Yayımlanmış doktora tezi, Dicle Üniversitesi Fen Bilimleri Enstitüsü, Diyarbakır.
  • Koenig, S. H. & Schillinger, W. E. (1969). Nuclear magnetic relaxation dispersion in protein solutions, J. Biol. Chem. 244(23), 6520-6526.
  • Koivula, A., Suominen, K., Timonen,T. & Kiviniitty, K. (1982). The spin-lattice relaxation time in the blood of healthy subjects and patients with malignant blood disease. Phys.Med.Biol., 27(7), 937-947.
  • Marshall, A. G., Schmidt, P. G. & Sykes, B.D. (1972). Effect of Internal Rotation on Nuclear Magnetic Relaxation Times for Macromolecules. Biochemistry, 11(21), 3875-3879.
  • Pouliquen,D. & Gallois, Y. (2001). Physicochemical properties of structured water in human albumin and gammaglobulin solutions. Biochimie, 83, 891-898.
  • Raeymaekers, H. H., Borghys, D. & Eisendrath, H. (1988). Determinants of water proton T1 in blood serum. Magn.Reson.Med., 6(2), 212-216.
  • Tarek, M. & Tobias D. J. (2000). The dynamics of protein hydration water: A quantitative comparison of molecular dynamics simulations and neutron-scattering experiments. Biophysical Journal, 79, 3244-3257.
  • Yılmaz,A., Ulak F. Ş. & Batun, M. S. 2004. Proton T1 and T2 relaxivities of serum proteins, Magnetic Resonance İmaging, 22(7), 683-688.
  • Yılmaz, A. Chu, S. C. & Osmanoğlu, S. (1988). Dependence of the solvent proton 1 Ton the 1 iron content in normal human serum. Magn.Reson.Med., 7(6), 337-339.
  • Yılmaz, A., Otludil, B., Batun, M. S., Ensari,Y., Longo, R. & Palma, L.D. (1992). Determination of ion and iron binding capacity by NMR. Phys.Med.Biol., 37, 1589- 1596.
  • Yılmaz, A., Budak, H. & Longo, R. (1998). Paramagnetic contribution of serum iron to the spin- lattice relaxation rate(1 T) determined by MRI. Applied Magnetic Resonance, 1 14(1), 51-58.
  • Zimmerman, J. R, & Brittin, W. E. (1957). Nuclear magnetic resonance studies in multiple phase systems: lifetime of a water molecule in an adsorbing phase on silica gel. J.Phys. Chem, 61, 1328-1333.

The Investigation of Protein Hydration in Serum by T1 Relaxation Measurement

Yıl 2007, Sayı: 9, 115 - 122, 01.12.2007

Öz

In this study; T1 relaxation times were measured for serum doped albumin with FT-NMR spectrometer operating at 60 MHz. and using inversion-recovery pulse sequences (IR). After each albumin addition, spin-lattice relaxation rates (1/T1) were measured and plotted against mass albumin/mass water. The plotting gives four successive line segments. The water layers surrounding the protein molecule are assumed to be consistently exhausted from outer shell to inner shell by adding albumin. The obtained line segments were atributed to free, hindered water, rotatioanlly bound and irrotatioanlly bound water layers. Using the line segments, the mass of water per 1 g of protein were obtained 4,71 g(free), 1.5 g(hindered), 0.38g(rotationally bound) and 0.05g (irrotationally bound). The relaxation rates of this layers were determined as follows; 0.59s-1(free), 0.95s-1 (hindered),1.83 s-1 (rotatioally bound) and 3.82 s-1 (irrotatioanlly bound). These results were consistent with relaxation model based on fast chemical exchange mechanism of water molecules between free and hydration water. Using the relaxation rate and the dipolar relaxation formula found by Bloembergen, Purcell and Pound, correlation times were determined for free, hindered, rotatioanlly bound and irrotatioanlly bound layers; 10-12s, 5*10-10s, 1.2*10-9s ve 10-7s, respectively. These results suggest that the relaxation times of water layers surrounding a protein and amount of water in each layer could be determined by T1 measured versus addition protein.

Kaynakça

  • Balasubramanian, S., Bandyopadhyay, S. Pal, S. & Bagchi, B. (2003). Dynamics of water at the interface of a small protein, enterotoxin, Current Science, 85(11), 1571-1578.
  • Bertini, I., Fragai, M., Luchinat, C. & Parigi, G. (2000). 1H NMRD profiles of diamagnetic proteins: a model-free analysis. Magn. Reson. Chem, 38, 543-550.
  • Bertini, I., Luchinat, C., Nerinovski, K., Parigi, G., Cross, M., Xiao, Z. & Wedd, A. G. (2003). Application of NMRD to hydration of rubredoxin and a variant containing a (Cys- S)3Feııı(OH) site, Biophys. J. 84, 545-551.
  • Bloembergen, N., Purcell, E. M. & Pound, R. F. (1948). Relaxation Effects in Nuclear Magnetic Resonance Absorption Phys.Rev, 73, 679-712.
  • Brooks, R. A. Battocletti, J. H. Sances, JR Larson, S. J. Bowman, L.R. & Kudravcev,V. (1975). Nuclear magnetic relaxation in blood. IEEE Transac.Biomed, Engin.BME-22(1), 12- 16.
  • Cameron, I. L, Ord, V. A & Fullerton, G. D. (1988). Water of hydration in the intra and extra cellular environment of human erythrocytes. Biochem.Cell.Biol, 66, 1186-1199.
  • Daskiewicz, O. K., Hennel, J. W. & Lubas, B. (1963). Proton magnetic relaxation and protein hydration, Nature, 200, 1006-1007.
  • Fullerton, G.D., Ord, V. A. & Cameron,I.L. (1986). An evaluation of the hydration of lysozyme by a NMR titration method. Biochim.Biophys.Acta, 869, 230-246.
  • Fullerton, G. D. (1988). Physiologic Basic of Magnetic Relaxation in Magnetic Resonance Imaging, David D. Stark & W. G. Bradley (Ed.)(ss. 36). St. Louis: The C.V. Mosby Company.
  • Gallier, J., Rivet, P. & Certaınes, J.de. (1987). 1H- and 2H-NMR study of bovine serum albumin solutions. Biochimica et Biophysica Acta, 915, 1-18.
  • Grösch, L. & Noack, F. (1976). NMR relaxation investigation of water mobility in aquueous bovine serum albumin solutions. Biochimica et Biophysica Acta, 453, 218-232.
  • Halle, B. (2004). Protein hydration dynamics in solution: a critical survey, Phil. Trans. R. Soc. Lond. B. 359, 1207-1224.
  • Hennel, J. W. & Klinowsky, J. (1996). Çekirdek Magnetik Rezonansın Temelleri. (Çev: S. Bahçeli). Ankara:Bizim Büro Basımevi (Özgün kitap 1993’da yayımlandı).
  • Kavak, G. (2000). “Serum Proteinlerinin Spin-Örgü Durulma Mekanizmalarının NMR ile incelenmesi”. Yayımlanmış doktora tezi, Dicle Üniversitesi Fen Bilimleri Enstitüsü, Diyarbakır.
  • Koenig, S. H. & Schillinger, W. E. (1969). Nuclear magnetic relaxation dispersion in protein solutions, J. Biol. Chem. 244(23), 6520-6526.
  • Koivula, A., Suominen, K., Timonen,T. & Kiviniitty, K. (1982). The spin-lattice relaxation time in the blood of healthy subjects and patients with malignant blood disease. Phys.Med.Biol., 27(7), 937-947.
  • Marshall, A. G., Schmidt, P. G. & Sykes, B.D. (1972). Effect of Internal Rotation on Nuclear Magnetic Relaxation Times for Macromolecules. Biochemistry, 11(21), 3875-3879.
  • Pouliquen,D. & Gallois, Y. (2001). Physicochemical properties of structured water in human albumin and gammaglobulin solutions. Biochimie, 83, 891-898.
  • Raeymaekers, H. H., Borghys, D. & Eisendrath, H. (1988). Determinants of water proton T1 in blood serum. Magn.Reson.Med., 6(2), 212-216.
  • Tarek, M. & Tobias D. J. (2000). The dynamics of protein hydration water: A quantitative comparison of molecular dynamics simulations and neutron-scattering experiments. Biophysical Journal, 79, 3244-3257.
  • Yılmaz,A., Ulak F. Ş. & Batun, M. S. 2004. Proton T1 and T2 relaxivities of serum proteins, Magnetic Resonance İmaging, 22(7), 683-688.
  • Yılmaz, A. Chu, S. C. & Osmanoğlu, S. (1988). Dependence of the solvent proton 1 Ton the 1 iron content in normal human serum. Magn.Reson.Med., 7(6), 337-339.
  • Yılmaz, A., Otludil, B., Batun, M. S., Ensari,Y., Longo, R. & Palma, L.D. (1992). Determination of ion and iron binding capacity by NMR. Phys.Med.Biol., 37, 1589- 1596.
  • Yılmaz, A., Budak, H. & Longo, R. (1998). Paramagnetic contribution of serum iron to the spin- lattice relaxation rate(1 T) determined by MRI. Applied Magnetic Resonance, 1 14(1), 51-58.
  • Zimmerman, J. R, & Brittin, W. E. (1957). Nuclear magnetic resonance studies in multiple phase systems: lifetime of a water molecule in an adsorbing phase on silica gel. J.Phys. Chem, 61, 1328-1333.
Toplam 25 adet kaynakça vardır.

Ayrıntılar

Birincil Dil Türkçe
Bölüm Research Article
Yazarlar

Gülten Kavak

Yayımlanma Tarihi 1 Aralık 2007
Yayımlandığı Sayı Yıl 2007 Sayı: 9

Kaynak Göster

APA Kavak, G. (2007). SERUMDAKİ PROTEİN HİDRASYONUNUN NMR T1 ÖLÇÜMLERİ İLE İNCELENMESİ. Dicle Üniversitesi Ziya Gökalp Eğitim Fakültesi Dergisi(9), 115-122.