Fluorescent lifetime of near infrared dyes for structural analysis of serum albumin

Structural alteration of serum albumin, the major extracellular multifunctional protein in mammals, has been linked to a number of diseases. Herein we present a method based on fluorescence lifetime analysis of near-infrared (NIR) probes bound to albumin to interrogate its structural state without prior isolation of the protein. Molecular modeling study revealed that albumin binds polymethine dyes via two binding sites with different sizes and polarities. As a result, a NIR molecular probe typically exhibits two distinct lifetimes with corresponding fractional contributions. The distribution of fractional contributions along with individual fluorescence lifetimes represents unique parameters for characterizing albumin architecture. To evaluate the effect of size and polarity of binding sites on fluorescence lifetime we studied NIR probes in solutions with different viscosity and polarity. We established that viscosity has negligible effect on polymethine dyes lifetime while the change in polarity has a profound impact. We also established a correlation between fluorescence lifetime and solvent polarity function for a number of NIR dyes for quantitative description of binding sites polarity. After screening a library of dyes, we identified a probe with optimal fluorescence lifetime properties to assess structure-related differences of albumins. The results show that changes in the lifetime of NIR dyes reflect the perturbation of albumin's tertiary structures. Because of the reduced absorption of light by blood in the NIR region, the method developed can be used to determine structural changes of albumins in whole blood.

[1]  A. Gasbarrini,et al.  Oxygen free radical formation by rat hepatocytes during postanoxic reoxygenation: scavenging effect of albumin. , 1994, The American journal of physiology.

[2]  M. N. Komarova,et al.  Serum albumin binding sites properties in donors and in schizophrenia patients: the study of fluorescence decay of the probe K-35 using S-60 synchrotron pulse excitation , 2000 .

[3]  E. Sevick-Muraca,et al.  Fluorescence lifetime spectroscopy in multiply scattering media with dyes exhibiting multiexponential decay kinetics. , 2002, Biophysical journal.

[4]  Sharon Bloch,et al.  Targeting Beta-3 integrin using a linear hexapeptide labeled with a near-infrared fluorescent molecular probe. , 2006, Molecular pharmaceutics.

[5]  P. Brick,et al.  Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites , 1998, Nature Structural Biology.

[6]  J. Lakowicz,et al.  Fluorescence properties of rhodamine 800 in whole blood and plasma. , 2000, Analytical biochemistry.

[7]  Samuel Achilefu,et al.  Heptamethine cyanine dyes with a robust C-C bond at the central position of the chromophore. , 2006, The Journal of organic chemistry.

[8]  Bill X. Huang,et al.  Probing conformational changes of human serum albumin due to unsaturated fatty acid binding by chemical cross-linking and mass spectrometry. , 2005, The Biochemical journal.

[9]  Don W. Green,et al.  Perry's Chemical Engineers' Handbook , 2007 .

[10]  J. Levine,et al.  Albuminuria and renal injury--beware of proteins bearing gifts. , 2001, Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association.

[11]  W. Schneider,et al.  An alternative method of large scale plasma fractionation for the lsolation of serum albumin , 1975, Blut.

[12]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[13]  S. Achilefu,et al.  Novel receptor-targeted fluorescent contrast agents for in vivo tumor imaging. , 2000, Investigative radiology.

[14]  Jürgen Wolfrum,et al.  Inter- and intramolecular fluorescence quenching of organic dyes by tryptophan. , 2003, Bioconjugate chemistry.

[15]  F. Lesage,et al.  Whole-body fluorescence lifetime imaging of a tumor-targeted near-infrared molecular probe in mice. , 2005, Journal of biomedical optics.

[16]  I. Vakser.  Low-resolution docking: prediction of complexes for underdetermined structures. , 1998, Biopolymers.

[17]  S. Kazmierczak,et al.  Electron spin resonance spectroscopy of serum albumin: a novel new test for cancer diagnosis and monitoring. , 2006, Clinical chemistry.

[18]  Rafael Yuste,et al.  Fluorescence microscopy today , 2005, Nature Methods.

[19]  S. Achilefu,et al.  Near infrared dyes as lifetime solvatochromic probes for micropolarity measurements of biological systems. , 2007, Biophysical journal.

[20]  M. Hariharan,et al.  Site-selective binding and dual mode recognition of serum albumin by a squaraine dye. , 2006, Journal of the American Chemical Society.

[21]  S. P. Firsov,et al.  Chronic liver and renal diseases differently affect structure of human serum albumin. , 2002, Archives of biochemistry and biophysics.

[22]  José-Angel Conchello,et al.  Fluorescence microscopy , 2005, Nature Methods.

[23]  Feng Yang,et al.  Effect of human serum albumin on drug metabolism: structural evidence of esterase activity of human serum albumin. , 2007, Journal of structural biology.

[24]  H. Moriwaki,et al.  Problems in serum albumin measurement and clinical significance of albumin microheterogeneity in cirrhotics. , 2004, Nutrition.

[25]  S. Curry,et al.  Structural basis of the drug-binding specificity of human serum albumin. , 2005, Journal of molecular biology.

[26]  M. Haidekker,et al.  Effects of solvent polarity and solvent viscosity on the fluorescent properties of molecular rotors and related probes. , 2005, Bioorganic chemistry.

[27]  C. Harris,et al.  The Pile-Up Problem in Pulse Fluorometry , 1983 .

[28]  E J COHN,et al.  Preparation and properties of serum and plasma proteins; a system for the separation into fractions of the protein and lipoprotein components of biological tissues and fluids. , 1946, Journal of the American Chemical Society.

[29]  R. Weissleder,et al.  Charge-coupled-device based scanner for tomography of fluorescent near-infrared probes in turbid media. , 2002, Medical physics.

[30]  Carl Frieden,et al.  Steady‐state and time‐resolved fluorescence studies of the intestinal fatty acid binding protein , 2006, Proteins.

[31]  T. Peters,et al.  All About Albumin: Biochemistry, Genetics, and Medical Applications , 1995 .

[32]  W. Schneider,et al.  An Alternative method of large scale plasma fractionation for the isolation of serum albumin , 2005, Blut: Zeitschrift für die Gesamte Blutforschung.

[33]  C. Dobson.  Experimental investigation of protein folding and misfolding. , 2004, Methods.

[34]  C. Dash,et al.  Production of human albumin solution: a continually developing colloid. , 2000, British journal of anaesthesia.

[35]  T. Moon,et al.  Effect of Disulfide Bond Reduction on Bovine Serum Albumin-Stabilized Emulsion Gel Formed by Microbial Transglutaminase , 2003 .

[36]  G. Sudlow,et al.  Further characterization of specific drug binding sites on human serum albumin. , 1976, Molecular pharmacology.