J 710 spectropolarimeter

All CD spectra were recorded on a Jasco J-710 spectropolarimeter equipped with a temperature controller, using a 0.5 mm path-length cell, with a 1 nm bandwidth and 1 s response time. For measurements of standard far-UV CD spectra, the sample solutions containing 0.2 mM of each protein in a reaction buffer (50 mM HEPES, 0.5 mM ZnSO₄, 0.5 mM MgSO₄, and 0.5 mM DTT) at pH 7.4 were incubated at 35 °C for 30 min, followed by dilution into a CD buffer (10 mM sodium phosphate at pH 7.4). Subsequently, the far-UV CD of the diluted sample solutions was measured at 35 °C. Three individual scans taken from 260 to 190 nm with 0.2 nm step resolution and 100 nm/min scan speed were added and averaged, followed by subtraction of the solvent CD signals. Finally, the recorded CD intensity was normalized to molar ellipticity using the following equation: $$\left[\theta \right]=\frac{{10}^6\theta }{cl}$$
where $$\left[\theta \right]$$ and $$\theta$$ are the molar ellipticity in units of deg·cm²·dmol⁻¹ and the observed CD intensity in mdeg, respectively, with the path length $$l$$ in mm at the sample concentration $$c$$ in μM.
Time-course CD changes were monitored at 222 nm at every 0.5 s, with 15 μM of protein samples in 10 mM sodium phosphate buffer (pH 7.4) containing 50 μM ZnCl₂, 1 mM MgSO₄, 1 mM DTT, and 10 mM NaCl.

To examine laser-induced alterations in the secondary structure of fibrinogen protein, circular dichroism measurements were made. Immediately before and after irradiation, an aliquot of the fibrinogen sample was diluted (1∶20 for the 2.5 mg/ml sample and 1∶10 for 1.5 mg/ml and 0.6 mg/ml samples) and transferred to a 1 mm quartz cuvette (Starna Cells, Atascadero, CA, USA) and circular dichroism spectra were measured at room temperature using J-710 spectropolarimeter (Jasco Instruments, Easton, MD). Far UV spectra from 200 nm to 250 nm were obtained by averaging over eight scans, with a 1 nm bandwidth, 0.5 nm pitch and 50 nm/min scan speed, and then buffer subtracted. Fibrinogen concentration was estimated from the absorption spectra of undiluted samples, measured before and after laser irradiation. These values were used to convert CD signals to mean residue ellipticity (MRE). We note that a slight decrease in the signal amplitude was observed each time the samples were transferred from one container to another. This was consistent with the expected loss of protein due to adsorption of fibrinogen to the surface of containers [22] .

The folding state and stability of NvCI was investigated on the natural and recombinant forms by CD. The proteins, either in 0.1% TFA (v/v) or 20mM sodium phosphate (pH 2.0, 6.5 or 8.0) were analyzed at 0.05–0.06 mg/ml, from 25 to 90 °C in a Jasco J-710 spectropolarimeter (Pfugnstadt, Germany) in the 190–320nm range. Additional analyses were made under folding conditions (20mM sodium phosphate, pH 8.0) in the absence and presence of reducing reagent (1 or 20 mM DTT), a redox-pair (1mM Cys + 0.5mM cystine), and either absence or presence of denaturant (8 M urea). Complementary ¹H-NMR experiments were performed in a 500MHz NMR (Bruker Advance) on rNvCI in 20 mM sodium phosphate (pH 2.0 and 6.5), in 90% ¹H₂O + 10% D₂O, at 298 K. DQF-COSY, NOESY, TOCSY and HSQC spectra were obtained in a TCI cryoprobe of 5mm and used for provisional backbone assignment.

CD spectra were measured in 0.1 cm cuvettes with 0.15 mg/mL protein in 50 mM NaP_i, pH 7.4 at 20 °C as a sum of 50 single spectra with a Jasco J-710 spectropolarimeter (Jasco, Gross-Umstadt). A buffer baseline was subtracted from all datasets, units were converted to mean residue ellipticity and the mutant spectra normalized to the wildtype spectrum.