Ratiometric analysis of fluorescence lifetime for probing binding sites in albumin with near-infrared fluorescent molecular probes.
A number of diseases have been linked to abnormal conformation of albumin, a major extracellular protein in blood. Current protein structural analysis requires pure isolated samples, thereby limiting their use for albumin analysis in blood. In this study, we report a new approach for high-throughput structure-related analysis of albumin by using the fluorescence lifetime properties of near-infrared (NIR) polymethine dyes. Based on molecular modeling, polymethine dyes are bound to two binding sites with different polarities on albumin. As a result, an NIR molecular probe exhibits two distinct lifetimes with two corresponding fluorescent fractional contributions. The distribution of fractional contributions along with individual fluorescence lifetimes represents unique parameters for characterizing albumin architecture by ratiometric analysis. After screening a small library of NIR polymethine dyes, we identified and used a polymethine dye with optimal fluorescence lifetime properties to assess structure-related differences in commercially available bovine serum albumin as model systems. The results show that changes in the lifetime of NIR dyes reflect the perturbation of the tertiary structures of albumin and that albumin prepared by different methods has slightly altered 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 in albumin in whole blood without prior isolation of the pure protein.
Berezin
Mikhail Y
MY
Department of Radiology, Washington University School of Medicine, St. Louis, MO, USA.
Lee
Hyeran
H
Akers
Walter
W
Nikiforovich
Gregory
G
Achilefu
Samuel
S
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry; D008958:Models, Molecular; D015394:Molecular Structure; D017510:Protein Folding; D012710:Serum Albumin, Bovine / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D019265:Spectroscopy, Near-Infrared; D013997:Time Factors
D005456:Fluorescent Dyes; D012710:Serum Albumin, Bovine