Photodegradation mechanism and reaction kinetics of recombinant human interferon-alpha2a.
The photodegradation mechanism of recombinant human interferon-alpha2a (IFNalpha2a) has been investigated using absorption, fluorescence, and circular dichroism (CD) spectroscopies, and fluorescence photobleaching kinetics measurements under various conditions. After photobleaching, the absorption profile of aromatic amino acid residues in IFNalpha2a was almost absent, and an absorption profile showing a monotonic increase toward short wavelengths was observed. According to the CD spectrum analysis, partial unfolding of IFNalpha2a was accompanied by a complete loss of fluorescence. This unfolding was attributed to tryptophan-mediated photoinduced disulfide bond cleavage. Photooxygenation and photoionization of tryptophan (Trp) residues followed by subsequent radical reactions were the main photodegradation pathways of IFNalpha2a. Photobleaching kinetics was faster in acidic solution (pH 2.5) than in neutral solution (pH 7.4). The variation of photobleaching kinetics seemed to be caused by the structural differences in IFNalpha2a according to the solution pH. The relationship between the protein conformation and photobleaching rate could be explained based on the competition between excited state energy transfer and the photoionization process in Trp residues.
Kim
Hyong-Ha
HH
Health Metrology Group, Division of Metrology for Quality Life, Korea Research Institute of Standards & Science, P.O. Box 102, Yuseong, Daejeon 305-600, Korea. kimhh@kriss.re.kr
Lee
Yun Mi
YM
Suh
Jung-Keun
JK
Song
Nam Woong
NW
D002942:Circular Dichroism; D004220:Disulfides / Q000737:chemistry; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D005978:Glutathione / Q000737:chemistry; D019791:Guanidine / Q000737:chemistry; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000528:radiation effects*; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D010100:Oxygen / Q000737:chemistry; D038761:Photobleaching; D010782:Photolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000528:radiation effects; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods; D013997:Time Factors; D014466:Ultraviolet Rays
D004220:Disulfides; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D004228:Dithionitrobenzoic Acid; D005978:Glutathione; D019791:Guanidine; D010100:Oxygen