Ls55 luminescence spectrophotometer

Fluorescence spectra were obtained on aqueous solutions of HA samples at a concentration of 100 mgl⁻¹ after overnight equilibration at RT and adjustment to pH 8 with 0.05 M NaOH [28 ]. Spectra were recorded using a Perkin Elmer LS 55 luminescence spectrophotometer equipped with the WinLab 4.00.02 software (Perkin–Elmer, Inc., 2001, Norwalk, CT) for data processing. Total luminescence spectra, in the form of excitation–emission matrices (EEMs, contour maps), were recorded over the emission wavelength range from 300 to 600 nm by increasing sequentially by 5 mm step the excitation wavelength from 250 to 500 nm. A scan speed of 1200 nm min^-1 was selected for both monochromators. The EEM plots were generated as contour maps from spectral data by using Surfer 8.0 software (Golden Software, Inc., 2002, Golden, CO).
The humification index (HIX) was calculated according to Ohno [29 (link)]. This index is expressed as the ratio between the area in the upper quarter (435–480 nm) and the sum of the area in the lower quarter (300–345 nm) and in the upper quarter of the emission spectra of HA measured at an excitation wavelength fixed at 254 nm.

Pyrene actin polymerization assays were performed as previously described (Jiwani et al., 2013 (link)). Full-length TmeB was expressed as a GST fusion in pGEX-6p-1, purified to homogeneity over GST affinity column, and eluted by cleavage of the GST-tag with PreScission protease (Sigma). Remaining components were from the commercially available pyrene actin polymerization kit (Cytoskeleton). For polymerization assays, monomeric pyrene-labeled actin was prepared by diluting lyophilized pyrene actin (Cytoskeleton) in 5mM Tris (pH 8.0) 0.2mM CaCl₂ 0.2mM ATP (G buffer) and collection of the supernatant of a 90 minute, 100,000 xg, 4°C spin in a Beckman Optima MAX TL Ultracentrifuge using a TLA 55 rotor (Beckman Coulter). Approximately 30 µg of pyrene-labeled actin was mixed with 1-2µg of indicated proteins (TmeB, VCA, Arp2/3, GST) in a volume of 500 µl for 5 min before the addition of 1/20^th volume of polymerization buffer (500mM KCl, 20mM MgCl₂, 10mM ATP). The reaction (contained in a semi-microcuvette and holder assembly) was monitored for 30 min with a LS 55 Luminescence spectrophotometer equipped with the biokinetic accessory and directed by FL Winlab software version 4.0 (Perkin-Elmer, Beaconsfield, Bucks, United Kingdom) with 2.5 nm bandwidth at 365 nm excitation wavelength and 2.5 nm bandwidth at 407 nm emission wavelength.

Following the respective exposures cells were harvested and processed for microsome preparation following the protocol described earlier by us [13] (link). In brief, cells were scrapped in PBS at 4°C and pelleted by centrifuging at 500×g for 10 min. The cell pellet was resuspended in microsomal dilution buffer containing 0.1% (v/v) glycerol, 0.25 mM protease inhibitors cocktail, 0.01M EDTA and 0.1 mM dithiothreitol. The cells were then sonicated thrice at 15 Hz for 10 seconds each. Following sonication, the cells were again centrifuged at 9000×g for 20 min. The supernatant was then further centrifuged at 105,000×g for 60 min, to isolate the microsomal fraction. The microsomal pellet, thus obtained was then resuspended in microsomal dilution buffer and protein estimation was done by Bradford's Reagent (Fermentas Inc., Maryland, USA). The activity of 7-ethoxyresorufin-O-deethylase (EROD) for CYP1A1, 7-pentoxyresourfin-O-dealkylase (PROD) for CYP2B6, and N-nitrosodimethylamine demethylase (NDMA-d) for CYP2E1 were determined by following the methods described earlier by us [12] (link), [13] (link), [14] (link), [52] (link) using a Perkin Elmer LS 55 Luminescence spectrophotometer.