Materials

Pierce™ Protease Inhibitor Mini Tablets, Coomassie Protein Assay, Imperial™ Protein Stain, TCEP, and DTT were from Thermo-Fisher (Waltham, MA). Mini-Protean® TGX™ precast protein gels, nitrocellulose blotting membranes, Affi-Gel 10, Bio-Gel P-6, Laemmli sample buffer, and Mini Bio-Spin columns were from Bio-Rad (Hercules, CA). WesternSure® Premium chemiluminescent blotting substrate was from Li-Cor Biosciences (Lincoln, NE). All primary antibodies (sc-48345, sc-136178, sc-21738, sc100812, sc-365062, and sc-515428) and the mouse IgG kappa binding protein [m-IgGκ BP; (sc-516102)] conjugated to horseradish peroxidase (HRP), were from Santa Cruz Biotechnology (Santa Cruz, CA). All other reagents were from Millipore-Sigma (Burlington, MA).

Collection and storage of the rat brains under study

Brains used for the comparison of thiol oxidations in the enzymes of interest and for the investigation of interprotein disulfide crosslinks involving Prx-2 were obtained from male Sprague–Dawley (SD) rats that were 4–5 weeks and 6–7 weeks old, respectively. Following euthanasia by decapitation, severed heads were dropped immediately into liquid nitrogen and brains were removed from the frozen heads using a chisel. In addition to these immediately-frozen brains, brains that had been frozen following intentional delays to freezing of 3 min and 15 min were also obtained from the 4–5-week-old rats. All frozen brains were transferred to a -80 °C freezer for storage prior to homogenizations and fractionations.

Brains used for the aging study were from Fischer 344 (F344) male rats, of 4, 18, and 28 months of age, and were obtained from the National Institute on Aging (NIA), shipped to us overnight on dry ice. According to NIA protocols, the animals had been euthanized by CO2 asphyxiation followed by cervical dislocation and brains had been removed within 5 min of anoxia and stored at -80 °C.

Preparation of alkylated protein fractions

For all experiments other than the trapping of interprotein disulfide bonds involving Prx-2, brains were weighed, partially thawed, and homogenized, at 5 mL/g of tissue, in Tris–EDTA buffer (TEB; 200 mM Tris, 10 mM EDTA, 1 mM benzamidine, pH 7.0) to which was added Triton X-100 (1% v/v), N-ethylmaleimide (NEM; 200 mM), and a Pierce™ Protease Inhibitor Mini Tablet. As previously described in detail (Foley et al. 2014, 2016), homogenates were alkylated for 1 h at room temperature (21–23 °C) and centrifuged for 65 min at 100,000 × g at 4 °C to yield supernatants containing a combination of soluble and Triton X-100-solubilized protein.

For study of the interprotein disulfides involving Prx-2, brains were homogenized in 20 mM acetic acid, pH 4.0, containing a pre-dissolved Pierce™ Protease Inhibitor Mini Tablet. Aliquots of the homogenates were diluted 1:1 with TEB containing 1% (v/v) Triton X-100, and 1 mM benzamidine and either none, 0.2 M NEM, or 0.4 M NEM. Following a 30 min incubation period at 21–23 °C, to permit alkylation of protein thiols in NEM-containing samples, homogenates were centrifuged at 100,000 × g for 65 min at 4 °C. The resulting supernatants were diluted with the Tris–EDTA buffer to 1 mg protein/mL, further diluted 1:1 with 2x Laemmli sample buffer containing either no reducing agent or 20 mM DTT, and heated for 5 min at 95 °C.

Fractionation of soluble proteins by redox PAO-affinity chromatography

All procedures were conducted, with minor modifications, as described in detail previously (Foley et al. 2020). Briefly, unreacted NEM was removed from the 100,000 × g supernatants by centrifugal gel filtration. One-mL volumes of NEM-free supernatants, containing 3 mg total protein, were incubated with immobilized PAO for 90 min at 21–23 °C followed by fractionation to yield flow-through (FT), washes, and DTT-eluted proteins formerly containing operationally-defined disulfide bonds. Aliquots of pre-column (Pre) fractions, together with the FT, last wash (LW) and DTT-eluted fractions were diluted 1:1 with 80% (v/v) glycerol and stored at -20 °C until analyzed.

Measurements of protein

Protein concentrations in the 100,000 × g supernatants and in the DTT-eluted fractions generated during immobilized PAO-affinity chromatography were quantified, using the Coomassie Protein Assay (Thermo-Fisher), from standard curves generated using bovine serum albumin.

Analyses of protein from the PAO-affinity fractions

To confirm the presence of the proteins of interest in the DTT-eluted fractions, a representative sample from an immediately frozen brain, contained in an excised but unresolved gel band, was shipped to MS Bioworks LLC (Ann Arbor, MI) for alkylation of reduced thiols with iodoacetamide, in-gel tryptic digestion, and protein identification by LC–MS/MS as described in detail earlier (Foley et al. 2020). Proteins in the gel band were alkylated with iodoacetamide, subjected to in-gel tryptic digestion, and identified by LC–MS/MS using a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive. Data were searched using Mascot and filtered using a 1% protein and peptide FDR and requiring at least two unique peptides per protein. Protein cysteine residues labeled by NEM were considered to contain thiols that were reduced in the brain. Protein cysteine residues labeled with iodoacetamide, giving rise to carbamidomethyl groups, were assumed to contain thiols that had been reversibly oxidized [i.e., Cys(ox)] prior to the on-column reduction of disulfide bonds by TCEP during the PAO-affinity fractionation described above. Analysis of oxidized and reduced thiols was performed using Scaffold software.

Protein gel electrophoresis and western blotting were performed as described previously (Foley et al. 2020). Blots were developed using WesternSure® Premium chemiluminescent blotting substrate and the C-DiGit Blot Scanner (Li-Cor Biosciences) and analyzed using Image Studio™ software (Li-Cor Biosciences).

Prx-2 redox blots

Samples prepared as described above in non-reducing and reducing Laemmli sample buffer, and containing 5 µg protein each, were resolved on 4–20% precast gels and transferred to 0.2 µm nitrocellulose blotting membranes. After blocking with 5% (m/v) nonfat dry milk, blots were incubated overnight at 4 °C with primary antibody to Prx-2 at a 1:200 dilution, washed 3 × for 5 min each with Tris-buffered saline containing 0.1% Tween-20, and incubated for 2 h at room temp with HRP-conjugated secondary antibody at a 1:1000 dilution. Blots were developed as described above.

Statistical analyses

For study of the effects of delays to tissue freezing on protein thiol redox states, brains that had been immediately-frozen, and frozen following delays of 3 and 15 min, were experimentally paired by processing one brain from each of the three groups on days of tissue fractionations. Statistically-significant differences of the means (N = 5) between the control (immediately-frozen) and the delayed groups, and between the 3 and 15 min groups, were assessed by paired student t-test. Statistically-significant differences among the means (N = 3–5) for the three age groups for total protein, creatine kinase B, and alpha-enolase were assessed by one-way ANOVA.