Purification and Structural Characterization of "Simple Catechol", the NGAL-Siderocalin Siderophore in Human Urine

Abstract

The identification of ligands that bind the protein Neutrophil Gelatinase-Associated Lipocalin (NGAL, Siderocalin, Lipocalin-2) have helped to elucidate its function. NGAL-Siderocalin binds and sequesters the iron loaded bacterial siderophore enterochelin (Ent), defining the protein as an innate immune effector. Simple metabolic catechols can also form tight complexes with NGAL-Siderocalin and ferric iron, suggesting that the protein may act as an iron scavenger even in the absence of Ent. While different catechols have been detected in human urine, they have not been directly purified from a biofluid and demonstrated to ligate iron with NGAL-Siderocalin. This paper describes a "natural products" approach to identify small molecules that mediate iron binding to NGAL-Siderocalin. A 10K filtrate of human urine was subjected to multiple steps of column chromatography and reverse-phase HPLC, guided by NGAL-Siderocalin-iron binding assays and LC-MS detection. The co-factor forming a ternary structure with iron and NGAL-Siderocalin was identified as authentic simple catechol (dihydroxybenze) by ESI-HR-Mass, UV, and NMR spectrometric analysis. Comparison of the binding strengths of different catechols demonstrated that the vicinal-dihydroxyl groups were the key functional groups and that steric compatibilities of the catechol ring have the strongest effect on binding. Although catechol was a known NGAL-Siderocalin co-factor, our purification directly confirmed its presence in urine as well as its capacity to serve as an iron trap with NGAL-Siderocalin.

Keywords: Binding; Catechol; Enterochelin; Ferric Iron; Lipocalin-2; NGAL; Purification; Siderocalin.

Figure 1

The general purification procedures of NGAL binding siderophores: from raw urine to pure catechol by different column chromatography.

Figure 2

Formation of NGAL-Siderocalin:Siderophore:⁵⁵Fe³⁺ complexes by (A) five fractions of the first MCI gel Liquid Chromatography (LC) column compared with the negative control, apoNGAL-Siderocalin and with the positive control, NGAL-Siderocalin:Ent; (B) the Sephadex LH-20 LC fractions; (C) the first HPLC fractions; (D) the Silical Gel LC fractions.

Figure 3

LC-HR-ESI-MS⁻ (negative mode) of the SSF1 fraction and the standard catechol positive control: (A) Total Ion Chromatography of SSF1, (B) Total Ion Chromatography of standard catechol, (C): ESI⁻ (negative mode)-HR-MS of the peak at RT=12.211 min. (D) Automatic calculation of the molecular formula by G3335AA MassHunter Qualitative Analysis Software B.01.03 based on high resolution (HR)-MS.

Figure 4

Separated chromatograms listing LC-DAD (280 nm) of SSF1 (S, Green), o- (Catechol, dark blue), m- (Resorcin, light blue), and p- (Hydroquinone, orange) dihydroxybenzenes.

Figure 5

Purification of catechol from SSF1 by HPLC: (A) HPLC spectrum; (B) HPLC analysis of purified catechol eluting at RT = 20.7min, overlaying the first peak found in A.; (C) UV spectrum of purified catechol demonstrating its characteristic peak at 274 nm; (D) ¹H NMR of purified catechol (DMSO-d6 as solvent: solvent δ ppm: 2.477).

Figure 6

The binding strength of catechol and its derivatives (% retention of ⁵⁵Fe³⁺), was tested with NGAL-Siderocalin, ⁵⁵Fe³⁺ and 10 μM compounds (see Materials and Methods). Compounds 1: catechol, 2: 2,3-DHBA, 3: pyrogallol, 4: guaiacol, 5: 1,2-dimethoxybenzene, 6: catechol cyclic sulfate, 7: catechol sulfate sodium, 8: pyrogallol-2-Sulfate, 9: 3-methoxycatechol, 10: pyrogallol-4-Sulfate, 11: gallic acid, 12: 3-methylcatechol, 13: 4-methylcatechol, 14: 3,4-dihydroxybenzoic Acid (A) Basic siderophores (B) Protected hydroxyl group(s) dramatically decrease binding strength (C) Bulky steric groups decrease the binding strength (D) The para-position substitution has a greater effect than the ortho-position substitute (E) The steric compatibilities may have greater importance than electrostatic interactions. [14]