A Novel Photoreactive Excipient to Probe Peptide-Matrix Interactions in Lyophilized Solids.

Excipients used in lyophilized protein drug products are often selected by a trial-and-error method, in part, because the analytical methods used to detect protein-excipient interactions in lyophilized solids are limited. In this study, photolytic labeling was used to probe interactions between salmon calcitonin (sCT) and excipients in lyophilized solids. Two diazirine-derived photo-excipients, photo-leucine (pLeu) and photo-glucosamine (pGlcN), were incorporated into lyophilized solids containing sCT, together with an unlabeled excipient (sucrose or histidine) at prelyophilization pH values from 6 to 9.9. Commercially available pLeu was selected as an ionizable photo-excipient and amino acid analog, while pGlcN was synthesized as an analog of sugar-based excipients. Photolytic labeling was induced by exposing the solids to UV light (365 nm, 30-60 min), and the resulting products were identified and quantified with liquid-chromatography mass spectrometry. The distribution of photo-reaction products was affected by the photoreactive reagent used, the type of unlabeled excipient, and the solution pH before lyophilization. When other components of the solid were identical, the extent and sites of labeling on sCT were different for pGlcN and pLeu. The results suggest that ionizable and nonionizable excipients interact differently with sCT in lyophilized solids and that photo-excipients can be used to map these interactions.

Removal of amino acid, peptide and protein hydroperoxides by reaction with peroxiredoxins 2 and 3.

Prxs (peroxiredoxins) are a ubiquitous family of cysteine-dependent peroxidases that react rapidly with H2O2 and alkyl hydroperoxides and provide defence against these reactive oxidants. Hydroperoxides are also formed on amino acids and proteins during oxidative stress, and they too are a potential cause of biological damage. We have investigated whether Prxs react with amino acid, peptide and protein hydroperoxides, and whether the reactions are sufficiently rapid for these enzymes to provide antioxidant protection against these oxidants. Isolated Prx2, which is a cytosolic protein, and Prx3, which resides within mitochondria, were reacted with a selection of hydroperoxides generated by γ-radiolysis or singlet oxygen, on free amino acids, peptides and proteins. Reactions were followed by measuring the accumulation of disulfide-linked Prx dimers, via non-reducing SDS/PAGE, or the loss of the corresponding hydroperoxide, using quench-flow and LC (liquid chromatography)/MS. All the hydroperoxides induced rapid oxidation, with little difference in reactivity between Prx2 and Prx3. N-acetyl leucine hydroperoxides reacted with Prx2 with a rate constant of 4 × 10(4) M-1 · s-1. Hydroperoxides present on leucine, isoleucine or tyrosine reacted at a comparable rate, whereas histidine hydroperoxides were ~10-fold less reactive. Hydroperoxides present on lysozyme and BSA reacted with rate constants of ~100 M-1 · s-1. Addition of an uncharged derivative of leucine hydroperoxide to intact erythrocytes caused Prx2 oxidation with no concomitant loss in GSH, as did BSA hydroperoxide when added to concentrated erythrocyte lysate. Prxs are therefore favoured intracellular targets for peptide/protein hydroperoxides and have the potential to detoxify these species in vivo.

Statistical vs. non-statistical deactivation pathways in the UV photo-fragmentation of protonated tryptophan-leucine dipeptide.

The excited state dynamics of protonated tryptophan-leucine ions WLH+, generated in an electrospray source, is investigated by photo-induced fragmentation in the gas phase, using femtosecond laser pulses. Two main features arise from the experiment. Firstly, the initially excited pipi* state decays very quickly with 2 time constants of 1 and 10 ps. Secondly, the transient signals recorded on different fragments are not the same which indicates two competing primary fragmentation processes. One involves a direct dissociation from the excited state that gives evidence for a non-statistical deactivation path. The other is attributed to a statistical decay following internal conversion to the ground electronic surface.

Photo-leucine and photo-methionine allow identification of protein-protein interactions in living cells.

Protein-protein interactions are the key to organizing cellular processes in space and time. The only direct way to identify such interactions in their cellular environment is by photo-cross-linking. Here we present a new strategy for photo-cross-linking proteins in living cells. We designed two new photoactivatable amino acids that we termed photo-methionine and photo-leucine based on their structures and properties closely resembling the natural amino acids methionine and leucine, respectively. This similarity allows them to escape the stringent identity control mechanisms during protein synthesis and be incorporated into proteins by the unmodified mammalian translation machinery. Activation by ultraviolet light induces covalent cross-linking of the interacting proteins, which can be detected with high specificity by simple western blotting. Applying this technology to membrane protein complexes, we discovered a previously unknown direct interaction of the progesterone-binding membrane protein PGRMC1 with Insig-1, a key regulator of cholesterol homeostasis.

Photofragmentation of C,N-protected alpha-amino acids: comparing tert-leucine with sulfur-containing amino acids methionine and cysteine.

The photochemical fingerprint for the N-acetyl methyl ester of the aliphatic amino acid tert-leucine 1 was investigated. This reaction path was compared with the electron transfer active amino acids methionine (N-acetyl methyl ester derivative 2a as well as the methyl ester derivative 2b) and the cysteine derivatives 3a and 3b (penicillamine derivative). Photofragmenation of the ester group dominated the photolysis of 1, whereas loss of methylmercaptane was observed for 2a and 3a. Vinylglycine derivatives 11 and 14 were formed to a minor extent. The gem-dimethylated compound 3b gave a solvent-characteristic product pattern with photoelimination products 18 and 19 in acetonitrile and loss of methylmercaptane to give 20 in methanol.

Mechanism of pH-dependent photolysis of aliphatic amino acids and enantiomeric enrichment of racemic leucine by circularly polarized light.

It has been proposed that the origin of biological homochirality may be the result of irradiation of a racemic sample of amino acids by circularly polarized light (CPL). To determine the mechanism of enantiomeric enrichment, the irradiation of aliphatic amino acids by CPL was undertaken. An enantiomerically enriched sample (e.g., L isomer enrichment from r-CPL) was found to result from the preferential excitation/decomposition of one enantiomer over another via a Norrish Type II mechanism (leucine, valine, and isoleucine), with the enantiomeric excess dependent on the degree of protonation of the amino/carboxylic acid moiety.

Asymmetric photolysis with elliptically polarized light.

Previous investigations have shown the efficacy of right-(RCPL) and left-(LCPL) circularly polarized light in promoting the asymmetric photolysis of racemic organic substrates and producing measurable enantiomeric excesses (e.e.s) when photolysis is incomplete. Synchrotron radiation, polychromatic and having out-of-plane components which are elliptically and ultimately circularly polarized, has been suggested as a universal source of RCPL and LCPL on a cosmic scale. The more prevalent right-(REPL) and left-(LEPL) elliptically polarized components have never been investigated for similar capabilities. The present study, using a 212.8 nm laser beam to mimic the synchtrotron radiation, explores the potential of REPL and LEPL in this context and finds a qualitative trend indicating that each induces asymmetric photolysis in the same sense as RCPL and LCPL, but to a lesser degree.

The radiolysis and radioracemization of poly-L-leucines.

The brief history of the discovery of radioracemization, the racemization of an optically active substance induced by ionizing radiation, is reviewed. Our early studies involving the radiolysis and radioracemization of D- and L-leucine using gamma radiation from a 111-TBq 60Co gamma-ray source are described briefly, as are later experiments involving other protein amino acids and their salts, as well as the nonprotein amino acid, isovaline. The implications of the results of such studies for the Vester-Ulbricht mechanism which proposes longitudinally polarized beta radiation as the origin of molecular chirality, for the cosmological question of the enantiomeric compositions of amino acids in the Murchison meteorite, and for the use of D/L ratios of amino acids for geochronological and geothermal estimates are reviewed briefly. These past radiolysis-radio- racemization studies have involved only monomeric amino acids. The present research, extending such investigations to two homochiral L-leucine polypeptides, (L-Leu)10 and (L-Leu)78, was undertaken to see if a polymer of an amino acid might be more stable to radiolysis and radioracemization than the corresponding monomer. It was found that these polypeptides were more stable to radiolysis than was the leucine monomer, but that the extents of radioracemization in all samples were comparable.

Effect of propentofylline on [3H]-leucine incorporation into protein of postischemic rat brain.

The effect of propentofylline on regional [3H]-leucine incorporation into brain proteins was investigated during early reperfusion following permanent occlusion of vertebral arteries and reversible occlusion of the common carotid arteries of awake rats. In rats subjected to 5 min ischemia/50 min reperfusion the total brain radioactivity and TCA-precipitable radioactivity were reduced in the cerebellum, medulla oblongata, hypothalamus, cortex, striatum and hippocampus and the fractional protein radioactivity was decreased in the cortex, striatum and hippocampus. Propentofylline pretreatment at a dose of 25 mg/kg p.o. daily for 14 days completely reversed the reduction in total radioactivity and partially reversed the reduction in TCA-precipitable radioactivity in all brain regions studied and decreased the reduction in fractional protein radioactivity in the hippocampus. The results suggest that the protective effect of propentofylline on transient ischemia-induced brain damage may be related to improvement of [3H]-leucine incorporation into brain proteins.

The radiolysis and radioracemization of amino acids on clays.

L-Leucine and its hydrochloride salt have been deposited on the clay minerals kaolin and bentonite, and the amino acid/clay preparations have been irradiated in a 3000 Ci60Co gamma-ray source for radiation dosages that achieved 2-89% radiolysis of the leucine. The undecomposed leucine was thereupon recovered and both percent radiolysis and percent radioracemization were determined. Similar studies were made using solid L-leucine and its hydrochloride, and L-leucine in 0.1 M aqueous solution. It has been found that radiolysis and radio-racemization in these and the previously studied leucine systems follow pseudo-first-order rate laws, and the corresponding specific rate constants are evaluated and compared. Leucine and its hydrochloride salt proved to be the most stable to both radiolysis and radioracemization, followed by leucine and its HCl salt on kaolin, followed by leucine and its HCl salt on bentonite, with leucine (and its HCl and Na salts) in aqueous solution being least stable to both radiolysis and (except for the HCl salt) radioracemization. Implications of these observations as regards the Vester-Ulbricht mechanism for the origin of optical activity are discussed.

Comparison of radiation-induced radical yields in short peptides and constituent amino acids by detection of trapped oxygen isotope 18O.

This report compares the radiosensitivity of small polypeptides and constituent isolated amino acids using a novel technical approach. The radiation-induced oxygen isotope 18O trapping was measured by means of nuclear reactions. Most of the peptides reveal a higher sensitivity than a mixture of their constituent amino acids. This observation can be rationalized with a simple model which implies intramolecular energy transfer.

Effect of long-wave ultraviolet light on the lens. IV. Leucine metabolism in normal human lenses in vitro.

A series of lens pairs (each pair from the same donor) from human eyebank eyes were incubated in a medium containing tracer leucine. One lens of each pair was exposed to long-wave ultraviolet light. The other was shielded. The irradiated lenses accumulated free leucine to a level only 53% of that in the shielded lenses. Irradiation depressed leucine incorporation to 29% of the control level. These results show that UV may adversely affect important parameters of human lens protein metabolism in vitro over a period of 10-30 h.

E.S.R. of spin-trapped radicals in gamma-irradiated polycrystalline amino acids. Chromatographic separation of radicals.

The free radicals produced by gamma-radiolysis of polycrystalline amino acids (L-valine, L-leucine, L-isoleucine and L-proline) at room temperature in the absence of air were investigated by spin trapping with 2-methyl-2-nitrosopropane (MNP). The spin adducts produced by dissolving the irradiated solids in aqueous MNP solutions were separated by high-performance liquid chromatography and then identified by e.s.r. Deamination (ring-opening reaction for L-proline) was observed for all amino acid. For L-valine and L-leucine, H-abstraction from the tertiary carbon in the side chains occurred. For isoleucine, H-abstractions from the alpha-carbon of the amino acid and from a non-terminal carbon in the side chain were found.

The radiolysis and racemization of leucine on proton irradiation.

D- and L-Leucine have been subjected to 39-55 percent radiolysis using 0-11 MeV protons, both with the proton beam passing through the sample or being absorbed by it, and with quenching the sample immediately on completion of irradiation or after a 21-day interval. Racemization was small (1.1-1.7 percent) and comparable in all cases, suggesting that radioracemization and secondary degradative effects were not important factors in our recent unsuccessful attempts to induce optical activity in DL-leucine by partial radiolysis using 0-11 MeV longitudinally polarized protons.

The radiolysis and radioracemization of amino acids on silica surfaces.

L-Leucine, deposited on both 1-quartz powder and on a commercial amorphous silica preparation (Syloid 63), has been subjected to irradiation in a 60Co gamma-ray source, and the ensuing radiolysis and radioracemization have been determined gas chromatographically. The radiolysis and radioracemization observed for leucine on 1-quartz were rather similar to those noted for a crystalline L-leucine control. L-Leucine on Syloid 63, however, was vastly more susceptible to radiolysis as compared to the L-leucine control, and radioracemization was also markedly enhanced - each increasing with larger radiation dosage. L-Isovaline showed a similar, but diminished, enhancement of radiolysis sensitivity when adsorbed on the Syloid surface, but underwent no radioracemization whatsoever. The divergent results of the control and quartz-leucine irradiations versus the Syloid-leucine and Syloid-isovaline irradiations are interpreted qualitatively in terms of the surface area parameters of the two silica adsorbents and the amino acid adsorbates.

Search for effect of longitudinally polarized protons on optically active amino acid.

As a test of the Vester-Ulbricht hypothesis that the specific optical activity of contemporary biology arose from a differential action of polarized nuclear particles, DL-leucine has been irradiated with protons of both positive and negative longitudinal polarization. Neither kind of protons caused by measurable decomposition difference in the two optical isomers of the amino acid.

The radiolysis of tryptophan and leucine with 32P beta-radiation.

We have extended earlier experiments on the radiolysis of DL-tryptophan using 32 p beta-radiation to longer reaction times, observing complete destruction of the tryptophan by secondary, non-radiolytic processes. We have also undertaken the irradiation of DL-leucine with 32 P beta's at -196 degrees, achieving radiolyses to the extents of ca. 20-30%, but observing no concomittant asymmetric bias. The implications of these observations are discussed with regard to the Vester-Ulbricht mechanism for the origin of optical activity.

Radiolysis, racemization and the origin of molecular asymmetry in the biosphere.

An investigation has been undertaken to determine whether ionizing radiation might engender racemization of optically active amino acids, along with their usual radiolysis. As prototypes, crystalline D- and L-leucine, as well as aqueous solutions of their sodium salts were exposed to the radiation from a 3000 Ci 60Co gamma-ray source. Gamma-ray doses which caused about 68% radiolysis of solid leucine left a residue which was about 5% racemized, while racemization proved even greater at lower doses for the dissolved sodium salts. In aqueous solution both percent degradation and percent racemization of the sodium salts were proportional to gamma-ray dosage within the range employed (1--27 x 10(6) rads). Implications of these observations for the origin of molecular asymmetry by the beta-decay parity violation mechanism are discussed.