Trace metal grade nitric acid

Ferrlecit (NDC 0024-2792-10) (Sanofi U.S. LLC., Bridgewater, NJ, USA) and generic sodium ferric gluconate (SFG) (NDC 0591-0149-87) (Watson Pharma, Inc., Corona, CA, USA), containing 62.5 mg elemental iron per 5 mL (223.8 mM) sterile single-use vial were purchased. All drug stocks used throughout the investigation were from freshly opened vials. Trace metal grade nitric acid and hydrochloric acid were purchased from Fisher Scientific.

10 karat gold wire was purchased from Hoover and Strong (Chesterfield, VA). Trace metal grade nitric acid was purchased from Fisher Scientific (Pittsburgh, PA, USA). Heptakis-(6-deoxy-6-mercapto)-β-cyclodextrin (molecular weight 1247.44 g mol⁻¹) was purchased from Cyclolab Cyclodextrin Research and Development Laboratory Ltd. (Budapest, Hungary). Doxorubicin hydrochloride (molecular weight 579.98 g mol⁻¹) was purchased from Sigma Aldrich (St. Louis USA). A Hummer VI sputter coater (Anatech Ltd) was used to sputter gold. The AFM imaging was performed in tapping mode with a multimode AFM from Veeco (Santa Barbara, California, USA) operated with a Nanoscope IIIa™ controller. TAP300G silicon cantilevers with resonance frequency near 300 kHz were used. Thermogravimetric analysis was performed using a Q500 TGA from TA Instruments (New Castle, DE). UV-Visible absorbance spectroscopy was performed using a Cary 50 UV-Vis spectrophotometer from Agilent (Santa Clara, CA). Scanning electron microscopy (SEM) was carried out using a JSM-6320F field emission SEM from JEOL (Peabody, MA). The surface area of NPG wire was determined by the gold oxide stripping method using cyclic voltammetry between 0.0 –1.6 V at a scan rate of 5 mV s⁻¹.

HPLC-purified metal-Ybt complexes were dried down using a lyophilizer and resuspended in ultrapure water and trace metal grade nitric acid (Fisher). Final concentration of nitric acid was 2% v/v. Samples were diluted 1:10 using 2% nitric acid solution, and metal concentrations were analyzed by high resolution ICP-MS (Agilent 7500 ICP-MS). Machine was calibrated using Environmental calibration standard (Agilent) and PerkinElmer Pure Plus ICP-MS standard (PerkinElmer).

We adapted the protocol described by Peng et al.^{21 (link)} to determine the iron content in 100 μg of the purified VcFeoB protein before and after treatment with 2,2’-bipyridyl. Namely, the protein solutions were digested with 1 mL of 35 % v/v trace-metal-grade nitric acid (Fisher Scientific) in 5 mL Savillex vials at 80 °C for 3 h. Vials were then cooled and allowed to evaporate to dryness at 60 °C overnight. The residue was resuspended in ~ 3 mL of 2% v/v nitric acid to reach a total dissolved solids concentration of approximately 200 ppm, and then incubated at 60 °C overnight. Metal content was measured using an Agilent 7500ce inductively coupled plasma mass spectrometer and analyzed against defined standards at the ICP-MS laboratory in the Jackson School of Geosciences at the University of Texas at Austin. The sample of treated protein was prepared by incubating 100 μg VcFeoB with 100 μM 2,2’-bipyridyl during 1 h at RT, then the sample was filtered through a 50 kDa Amicon^® Ultra-15 Centrifugal Filter Unit (Millipore-Sigma) to remove the chelator agent, and resuspended in the original volume of milliQ water. A blank sample containing no protein was used as control.