Light-induced aggregation of type I soluble tumor necrosis factor receptor.
We evaluated the effect of UV-B light at 302 nm on a model therapeutic protein, 2.6 D type I soluble tumor necrosis factor receptor (sTNF-RI). This protein contains a single Trp at position 97 and seven native disulfide bonds along its interior from the N to the C-terminus. At a protein concentration of 0.1 mg/mL photoirradiation was found to induce the formation of soluble disulfide cross-linked dimers with greater levels of these species formed at pH 8 than at pH 5. Intermolecular disulfide formation was also directly correlated with the photoinduced unfolding of the protein as measured by changes in secondary structure by CD spectroscopy. Trp was implicated as the initiator of the observed photoreactions by the detection of the Trp oxidation products and the absence of dimer formation when Trp97 was replaced with Gln. Reactive oxygen species or triplet state species of Trp were not involved in the reaction suggesting that disulfides were cleaved through one-electron reduction by either hydrated or peptide bound electrons produced by the photoirradiated Trp resulting in thiyl radical formation with disruption of the protein structure and intermolecular cross-linking. Photodegradation was not prevented by deoxygenation, methionine or sucrose commonly used for formulation of biopharmaceuticals. To our knowledge this is the first report directly documenting disulfide mediated aggregation through thiyl radical formation initiated by photoirradiation of Trp.
Roy
Shouvik
S
Department of Pharmaceutical Sciences, Center for Pharmaceutical Biotechnology, University of Colorado Health Sciences Center, University of Colorado, Denver, Colorado 80262, USA.
Mason
Bruce D
BD
Schöneich
Christian S
CS
Carpenter
John F
JF
Boone
Thomas C
TC
Kerwin
Bruce A
BA
D020106:Acrylamide / Q000737:chemistry; D005079:Excipients / Q000737:chemistry; D010084:Oxidation-Reduction; D011487:Protein Conformation / Q000528:radiation effects*; D055550:Protein Stability / Q000528:radiation effects*; D047888:Receptors, Tumor Necrosis Factor, Type I / Q000737:chemistry*; D019810:Sodium Azide / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays
D005079:Excipients; D047888:Receptors, Tumor Necrosis Factor, Type I; D013438:Sulfhydryl Compounds; D020106:Acrylamide; D014364:Tryptophan; D019810:Sodium Azide