Dad g1315b

Polyamine composition was analyzed according to Weiss et al.47 (link). Separations were performed on a C18 column (250 × 4.6 mm, ProntoSIL Hypersorb ODS, F180PY050, Bischoff) by using an Agilent 1100 Series HPLC system (Degasser G1379A, Quat Pump G1311A, Man. Inj. G1328B, COLCOM G1316A, DAD G1315B, FLD G1321A, RID G1362A, Agilent Technologies) controlled by ChemStation software version B.01.03 (Agilent Technologies).
The mobile phase consisted of a gradient (details see Weiss et al.47 (link)) of solvent A (25 mM triethylamine, titrated to pH 4.8 with acetic acid), solvent B (acetonitrile:water 80:20 (v/v)), and solvent C (methanol). Samples of 10 μl were injected into the column and analyzed at a flow rate of 1.3 ml/min at 33 °C with online UV detection at 210 nm and online fluorescence detection at λ_Ex = 248 nm, λ_Em = 398 nm.
Retention times were verified by analysis of calibration mixtures containing approximately 0.8, 8, or 80 pmol DAP, PUT, SPD, and HSP at appropriate time points during the analysis. The general applicability of sample preparation and polyamine content analysis of samples containing BvHSS was verified by performing qualitative HSS activity assays followed by HPLC-based analyses.

The SEC-multi angle light scattering (MALS) measurements were performed using an online MALS detector (miniDAWN Treos, Wyatt Technology Corp.) coupled to an Agilent Technologies 1100 series HPLC system equipped with a refractive index detector (differential refractometer, RID, G1362A, Agilent Technologies) and a multiple wavelength detector (DAD, G1315B, Agilent Technologies). Protein samples (100–500 µg/analysis) were separated on a Superdex 200 10/300 GL SEC column (GE Healthcare Life Sciences) at a flow rate of 0.5 ml/minute in PBS pH 7.4. The MALS detector was normalized with bovine serum albumin (BSA) (Sigma-Aldrich) prior PDI analysis. The molecular weight was calculated based on the MALS and refractive index data by using 0.185 as refractive index increment (dn/dc) and a second virial coefficient of zero by using the Zimm plot. Data acquisition and analysis was performed with the software ASTRA version 5.3.4.10 (Wyatt Technology Corp.).

Absorption spectra were registered in the range of 200–450 nm in a 10 mm quartz cell by means of a PerkinElmer Lambda 40P Spectrophotometer (PerkinElmer, Waltham, MA, USA) under the following conditions: scan rate: 1 nm s⁻¹, time response: 1 s, and spectral band: 1 nm. Light exposure was simulated in a light cabinet, Suntest CPS+ (Heraeus, Milan, Italy), equipped with a Xenon lamp (Atlas Material Testing Technology, Mt Prospect, IL, USA), compliant with the ICH rules. The apparatus was fitted with an electronic device for irradiation, temperature measurement, and control inside the box. The system was able to closely simulate sunlight and appropriately select spectral regions between 300 and 800 nm through the interposition of filters. In particular, the application of the ID65 standard filter limits radiation to about 320 nm.
Chromatographic equipment consists of a HP 1100 pump fitted with a DAD G1315B (Agilent Technologies, Santa Clara, CA, USA) and a Rheodyne 7725 manual injector. The LC column was a C18 Gemini (Phenomenex, Bologna, Italy), 250 × 4.6 mm × 5 μm.