Comprehensive product characterization was performed for the photodegradation of protein disulfides, representatively of human growth hormone (somatotropin; hGH), in order to provide a product database, which will be useful for the general analysis of protein stability.

Industry experience suggests that g-forces sustained when vials containing protein formulations are accidentally dropped can cause aggregation and particle formation. To study this phenomenon, a shock tower was used to apply controlled g-forces to glass vials containing formulations of two monoclonal antibodies and recombinant human growth hormone (rhGH). High-speed video analysis showed cavitation bubbles forming within 30μs and subsequently collapsing in the formulations. As a result of echoing shock waves, bubbles collapsed and reappeared periodically over a millisecond time course. Fluid mechanics simulations showed low-pressure regions within the fluid where cavitation would be favored. A hydroxyphenylfluorescein assay determined that cavitation produced hydroxyl radicals. When mechanical shock was applied to vials containing protein formulations, gelatinous particles appeared on the vial walls. Size-exclusion chromatographic analysis of the formulations after shock did not detect changes in monomer or soluble aggregate concentrations. However, subvisible particle counts determined by microflow image analysis increased. The mass of protein attached to the vial walls increased with increasing drop height. Both protein in bulk solution and protein that became attached to the vial walls after shock were analyzed by mass spectrometry. rhGH recovered from the vial walls in some samples revealed oxidation of Met and/or Trp residues.

Thioredoxin reductases, important biological redox mediators for two-electron transfers, contain either 2 cysteines or a cysteine (Cys) and a selenocysteine (Sec) at the active site. The incorporation of Sec is metabolically costly, and therefore surprising. We provide here a rationale: in the case of an accidental one-electron transfer to a S-S or a S-Se bond during catalysis, a thiyl or a selanyl radical, respectively would be formed. The thiyl radical can abstract a hydrogen from the protein backbone, which subsequently leads to the inactivation of the protein. In contrast, a selanyl radical will not abstract a hydrogen. Therefore, formation of Sec radicals in a GlyCysSecGly active site will less likely result in the destruction of a protein compared to a GlyCysCysGly active site.

The reaction of singlet oxygen with water to form hydrogen peroxide was catalyzed by antibodies and has been termed as the antibody catalyzed water oxidation pathway (ACWOP) (Nieva and Wentworth, Trends Biochem. Sci. 2004, 29, 274-278; Nieva et al. Immunol. Lett. 2006, 103, 33-38). While conserved and buried tryptophans in the antibody are thought to play a major role in this pathway, our studies with a monoclonal antibody, mAb-1 and its mutant W53A, clearly demonstrate the role of surface-exposed tryptophans in production of hydrogen peroxide, via the photo-oxidation pathway. Reactive oxygen species (ROS) such as singlet oxygen and superoxide were detected and site-specific tryptophan (Trp53) oxidation was observed under these conditions using RP-HPLC and mass spectrometry. The single mutant of the surface exposed Trp53 to Ala53 (W53A) results in a 50% reduction in hydrogen peroxide generated under these conditions, indicating that surface exposed tryptophans are highly efficient in transferring light energy to oxygen and contribute significantly to ROS generation. ACWOP potentially leads to the chemical instability of mAb-1 via the generation of ROS and is important to consider during clinical and pharmaceutical development of mAbs.

The exposure of protein pharmaceuticals to light can cause loss of potency, oxidation, structural changes and aggregation. To elucidate the chemical pathways of photodegradation, we irradiated human growth hormone (hGH) at λ = 254 nm, λ ≈ 265-340 nm, and λ ≈ 295-340 nm (using the spectral cutoff of borosilicate glass) and analyzed the products by mass spectrometry. By means of LC-MS/MS analysis, we observed an unusual peptide backbone cleavage between Glu-88 and Pro-89. The crystal structure of hGH indicates that these residues are in proximity to Trp-86, which likely mediates this backbone cleavage. The two cleavage fragments observed by MS/MS analysis indicate the loss of CO from the amide bond and replacement of the Glu-C(═ O)Pro bond with a Glu-H bond, accompanied by double bond formation on proline. The reaction is oxygen-independent and likely involves hydrogen transfer to the Cα of Glu-88. To probe the influence of the protein fold, we irradiated hGH in its unfolded state, in 1:1 (v/v) acetonitrile/water, and also the isolated tryptic peptide Ile-78-Arg-90, which contains the Glu-88-Pro-89 sequence. In both cases, the cleavage between Glu-88 and Pro-89 was largely suppressed, while other cleavage pathways became dominant, notably between Gln-84 and Ser-85, as well as Ser-85 and Trp-86.

Selenocysteine is present in a variety of proteins and catalyzes the oxidation of thiols to disulfides and the reduction of disulfides to thiols. Here, we compare the kinetic and thermodynamic properties of cysteine with its selenium-containing analogon, selenocysteine. Reactions of simple selenols at pH 7 are up to four orders of magnitude faster than their sulfur analogs, depending on reaction type. In redox-related proteins, the use of selenium instead of sulfur can be used to tune electrode, or redox, potentials. Selenocysteine could also have a protective effect in proteins because its one-electron oxidized product, the selanyl radical, is not oxidizing enough to modify or destroy proteins, whereas the cysteine-thiyl radical can do this very rapidly.

Human growth hormone (hGH) was exposed to oxygen-centered radicals generated through the thermolysis of AAPH in the presence of dioxygen. Such conditions mimic oxidative processes which protein pharmaceuticals can encounter during formulation in the presence of polysorbates. We detected the oxidation of Met to Met sulfoxide, the formation of protein carbonyls, the oxidation of Tyr to dityrosine and several additional Tyr oxidation products, the conformation-dependent oxidation of Trp, and the site-specific formation of protein hydroperoxides. The sensitivity of Met oxidation correlates with their solvent accessible surface, i.e. the yields of MetSO decreased in the order Met-14 > Met-125 > Met-170. Trp oxidation in native hGH was negligible, but was enhanced through denaturation. Dityrosine formed predominantly intramolecularly but did not contribute significantly to protein cross-linking. Hydroperoxides formed selectively on Leu-101 and were generated specifically by alkoxyl radicals, generated through the decomposition of peroxyl radicals. Tyr-103 was converted into a series of oxidation products characterized by mass shifts of Tyr + 14 Da and Tyr + 16 Da.

Cysteamine reduces selenocystamine to form hemiselenocystamine and then cystamine. The rate constants are k(1) = 1.3 × 10(5) M(-1) s(-1); k(-1) = 2.6 × 10(7) M(-1) s(-1); k(2) = 11 M(-1) s(-1); and k(-2) = 1.4 × 10(3) M(-1) s(-1), respectively. Rate constants for reactions of cysteine/selenocystine are similar. Reaction rates of selenium as a nucleophile and as an electrophile are 2-3 and 4 orders of magnitude higher, respectively, than those of sulfur. Sulfides and selenides are comparable as leaving groups.

The fast reaction of (•)NO and O(2)(•-) to give ONOO(-) has been extensively studied at irreversible conditions, but the reasons for the wide variations in observed forward rate constants (3.8 ≤ k(f) ≤ 20 × 10(9) M(-1) s(-1)) remain unexplained. We characterized the diffusion-dependent aqueous (pH > 12) chemical equilibrium of the form (•)NO + O(2)(•-) = ONOO(-) with respect to its dependence on temperature, viscosity, and [ONOO(-)](eq) by determining [ONOO(-)](eq) and [(•)NO](eq). The equilibrium forward reaction rate constant (k(f)(eq)) has negative activation energy, in contrast to that found under irreversible conditions. In contradiction to the law of mass action, we demonstrate that the equilibrium constant depends on ONOO(-) concentration. Therefore, a wide range of k(f)(eq) values could be derived (7.5-21 × 10(9) M(-1) s(-1)). Of general interest, the variations in k(f) can thus be explained by its dependence on the distance between ONOO(-) particles (sites of generation of (•)NO and O(2)(•-)).

The oxidation of proteins and other macromolecules by radical species under conditions of oxidative stress can be modulated by antioxidant compounds. Decreased levels of the antioxidants glutathione and ascorbate have been documented in oxidative stress-related diseases. A radical generated on the surface of a protein can: (1) be immediately and fully repaired by direct reaction with an antioxidant; (2) react with dioxygen to form the corresponding peroxyl radical; or (3) undergo intramolecular long range electron transfer to relocate the free electron to another amino acid residue. In pulse radiolysis studies, in vitro production of the initial radical on a protein is conveniently made at a tryptophan residue, and electron transfer often leads ultimately to residence of the unpaired electron on a tyrosine residue. We review here the kinetics data for reactions of the antioxidants glutathione, selenocysteine, and ascorbate with tryptophanyl and tyrosyl radicals as free amino acids in model compounds and proteins. Glutathione repairs a tryptophanyl radical in lysozyme with a rate constant of (1.05±0.05)×10(5) M(-1) s(-1), while ascorbate repairs tryptophanyl and tyrosyl radicals ca. 3 orders of magnitude faster. The in vitro reaction of glutathione with these radicals is too slow to prevent formation of peroxyl radicals, which become reduced by glutathione to hydroperoxides; the resulting glutathione thiyl radical is capable of further radical generation by hydrogen abstraction. Although physiologically not significant, selenoglutathione reduces tyrosyl radicals as fast as ascorbate. The reaction of protein radicals formed on insulin, β-lactoglobulin, pepsin, chymotrypsin and bovine serum albumin with ascorbate is relatively rapid, competes with the reaction with dioxygen, and the relatively innocuous ascorbyl radical is formed. On the basis of these kinetics data, we suggest that reductive repair of protein radicals may contribute to the well-documented depletion of ascorbate in living organisms subjected to oxidative stress.

The rate constant for the reduction of the tyrosyl radical with selenocysteine has been measured to investigate whether selenocysteine is capable of repair of protein radicals. Tyrosyl radicals, both free in solution and in insulin, were generated by means of pulse radiolysis and laser flash photolysis in aqueous solution. The rate constant for the reaction of free N-acetyl-tyrosyl-amine radicals with selenocysteine is (8 +/- 2) x 10 (8) M (-1) s (-1), and that for tyrosyl radicals in insulin is (1.6 +/- 0.4) x 10 (8) M (-1) s (-1). The rate constant for the reaction of selenoglutathione with the N-acetyl-tyrosyl-amine radical is (5 +/- 2) x 10 (8) M (-1) s (-1). In contrast, cysteine and glutathione react more slowly than their selenium analogues with the tyrosyl radical: the reactions of N-acetyl-tyrosyl-amine radicals with cysteine and glutathione are 3 and 5 orders of magnitude slower, respectively, than those with selenocysteine and selenoglutathione, while those of tyrosyl radicals in insulin are 3 and 2 orders of magnitude slower, respectively.