Enhanced photostability of the anthracene chromophore in aqueous medium upon protein encapsulation.
In the present work, 9-anthraceneacetic acid (1) has been selected as a simple, water-compatible derivative of the anthracene chromophore to investigate the photophysical and photochemical behavior upon binding to human and bovine serum albumins (HSA and BSA) and alpha-acid glycoproteins (HAAG and BAAG). The UV-vis absorption spectrum of 1 exhibited the typical four maxima between 320 and 400 nm, which were slightly red-shifted in the presence of proteins. These minor changes suggested the formation of 1@protein complexes; their stoichiometry (1:1) was determined by means of the corresponding Job plots. As expected, the fluorescence spectrum of 1 in phosphate-buffered saline (PBS) consisted of a structured emission with maxima between 390 and 470 nm. The addition of increasing amounts of HSA resulted in a decrease in the emission intensity. In the presence of BSA, HAAG, or BAAG, the same trend was observed, although the changes were less pronounced. The determination of binding constants was achieved from fluorescence titration, considering one (AAGs) or two (SAs) binding sites. The binding constants (K(B)) were found to be 2.3 x 10(6) M(-1) (HAAG), 2.4 x 10(6) M(-1) (BAAG), 4.57 x 10(4)/1.45 x 10(6) M(-1) (HSA), and 1.44 x 10(4)/1.20 x 10(6) M(-1) (BSA). Binding within two different sites of SAs was confirmed by displacement experiments using warfarin and ibuprofen as site I and site II probes, respectively. Laser flash photolysis of 1 at lambda(exc) = 355 nm in PBS/air gave rise to several transient species; by contrast, in the presence of 1 equiv of proteins, only the triplet excited state was detected. Moreover, the triplet lifetime (tau(T)) monitored at 420 nm lengthened considerably (up to 50-fold) in the protein media. This can be attributed to a slower deactivation of the species inside the protein binding pockets, where an exceptional microenvironment provides protection from attack by a second molecule of 1, oxygen, or other reagents. In agreement with the results from fluorescence titration, the presence of two binding sites in SAs was revealed by two different triplet lifetimes; by contrast, only one tau(T) value was found for HAAG and BAAG. The major, longer-lived component under nonsaturating conditions was assigned to (1@SA)(II), while the minor component was assigned to (1@SA)(I). Irradiation of 1 at 350 nm in PBS/air led to anthraquinone as a major product. In the presence of proteins, the degree of conversion was markedly lower than in PBS, as revealed by the photodegradation kinetics monitored through the absorbance changes at 367 nm. Thus, a dramatic protection from photooxidation is provided within the protein microenvironment.
Alonso
Rafael
R
Departamento de Química/Instituto de Tecnología Química UPV-CSIC, Universidad Politécnica de Valencia, Camino de Vera s/n, E-46022 Valencia, Spain.
Yamaji
Minoru
M
Jiménez
M Consuelo
MC
Miranda
Miguel A
MA
D000085:Acetates / Q000737:chemistry*; D000818:Animals; D000873:Anthracenes / Q000737:chemistry*; D002417:Cattle; D006801:Humans; D009961:Orosomucoid / Q000737:chemistry*; D010782:Photolysis; D012709:Serum Albumin / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D014867:Water / Q000737:chemistry
C554279:9-anthraceneacetic acid; D000085:Acetates; D000873:Anthracenes; D009961:Orosomucoid; D012709:Serum Albumin; D014867:Water; D012710:Serum Albumin, Bovine