Molecular Size distribution of Fluorophores in Aquatic Natural Organic

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Molecular Size distribution of Fluorophores in Aquatic Natural Organic Matter: Application of HPSEC with Multi-Wavelength Absorption and Fluorescence Detection Following LPSEC-PAGE Fractionation Oleg A. Trubetskoj, Claire Richard, Guillaume Voyard, Victor V. Marchenkov, and Olga E. Trubetskaya Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03924 • Publication Date (Web): 06 Apr 2018 Downloaded from http://pubs.acs.org on April 7, 2018

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Environmental Science & Technology

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Molecular Size Distribution of Fluorophores in Aquatic Natural Organic

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Matter: Application of HPSEC with Multi-Wavelength Absorption and

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Fluorescence Detection Following LPSEC-PAGE Fractionation

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Oleg A. Trubetskoj a, Claire Richardbc, Guillaume Voyardbc, Victor V. Marchenkovd,

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Olga E. Trubetskayae* a

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Institute of Basic Biological Problems, Russian Academy of Sciences, 142290, Pushchino, Moscow region, Russia;

7 b

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Equipe Photochimie, BP 10448, F-63000 Clermont-Ferrand;

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c

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d

13

e

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Clermont Université, Université Blaise Pascal, Institut de Chimie de Clermont-Ferrand,

CNRS, UMR 6296, ICCF, F-63171 Aubiere.

Institute of Protein Research, Russian Academy of Sciences, 142290 Pushchino, Moscow Region, Russia; Branch of Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 142290, Pushchino, Moscow region, Russia.

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*Tel. No.+7-4967-730859; FAX . No.+7-4967-330527; E-mail: [email protected]

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ABSTRACT: Analytical high performance size exclusion liquid chromatography (HPSEC)

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with multi-wavelength absorbance and fluorescence detections was used for the analysis of

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molecular size distribution and optical properties of dissolved natural organic matter.

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Experiments were conducted on Suwannee River organic matter (SRNOM) and its fractions

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A, B, C+D preliminary obtained by combination of preparative low pressure size exclusion

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chromatography and polyacrylamide gel electrophoresis (LPSEC-PAGE) and purified by

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dialysis on membrane with nominal cutoff 10 kDa, the fractions molecular size varied in order

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A > B > C+D > 10 kDa. The multi-step fractionation of SRNOM enabled the size-separation

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of at least five types of humic-like fluorophores within NOM showing emission maxima at

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465, 450, 435, 420 and 405 nm. The decrease of the humic-like emission maxima paralleled

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the decrease of the nominal size of fluorescent SRNOM. The protein-like ACSmolecular Paragon Plus Environment


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fluorescence was split into tyrosine-like and tryptophan-like fluorophores and only detected in

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fractions A and B. This work provides new data on the optical properties of size-fractionated

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NOM, which consistent with the formation of supramolecular NOM assemblies, likely

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controlled by association of low-molecular size components. It is clearly observed for the high

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molecular size fraction A, containing free amino acids or short peptides. The combination of

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several different fractionation procedures is very useful for obtaining less complex NOM

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compounds and understanding the NOM function in the environment.

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ABSTRACT ART

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II. Analytical HPSEC

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I. Preparative

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LPSEC-PAGE

SRNOM bulk

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Fluorescence emission maxima

Large molecular size fraction A

450, 435, 420, 405, B>A. Besides, essential differences in the

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A250/A365 ratios were observed between A, B and C+D fractions (Table 1). This ratio was

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used by several researchers to monitor changes in NOM size (24, 29), a positive relationship

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between elution volumes of chromatographic NOM fractions and their A250/A365 ratios was

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observed. This trend was found for A, B and C+D fractions, where A250/A365 ratio was

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highest in the small molecular size fraction C+D.

Similar SUVA280 distribution between

Taking into account the well-known correlation between SUVA280 and

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Fluorescence excitation-emission matrix (EEM) of A, B and C+D fractions. As it is

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currently accepted, the fluorescence of aquatic NOM consists of two main groups of

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fluorophores (2). One group named protein-like fluorophores is related to aromatic amino

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acids. It usually has an excitation and emission maxima below 305 and 380 nm, respectively.

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The other group corresponds to humic-like fluorophores. It shows excitation within the

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wavelength range 220-360 nm and emission within the wavelength range 380-470 nm.

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The bulk SRNOM sample exhibited two humic-like peaks with excitation-emission

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wavelength pairs (Ex/Em) 230/450 and 330/450 nm (Table 1, Fig. 2). The highest emission

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intensity was found in the peak with λex = 230 nm. These data are comparable with the ones

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obtained by Alberts and Takács (4) for SRNOM bulk sample. It should be noted that humiс-

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like fluorescence was detected in all aquatic NOM samples investigated, while protein-like

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fluorescence was not always observed in bulk NOM both by EEM (2, 3) and reversed-phase

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HPLC (22, 42). The EEM fluorescence spectra of fractions drastically differed from that of

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the SRNOM both quantitatively and qualitatively. The fluorescence intensity was generally ACS Paragon Plus Environment

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smaller in the fractions than in the bulk SRNOM, meaning that the essential part of

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fluorophore(s) was lost during the dialysis step. Moreover, fractions A and B gave additional

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protein-like fluorescence with Ex/Em - 230/330-335 and 270/340-350 nm, while such

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emissions were not found in the bulk SRNOM and fraction C+D (Table 1, Fig. 2). The first

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explanation could be that fractionation changed the fluorescent properties of fractions, but this

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hypothesis appears unlikely. Alternatively, protein-like fluorophores could be much more

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concentrated in the fractions A and B than in the bulk SRNOM. Due to a detection limit issue

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these protein-like fluorophores could become detectable only when the fractions A and B

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were analyzed separately.

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For a detailed investigation of this phenomenon, we carried out analysis of fractions A,

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B, C+D and bulk SRNOM by HPSEC with information-rich multi-wavelength absorbance

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and fluorescence detection. The excitation wavelength 270 nm and emission wavelengths

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region 300-600 nm were chosen for HPSEC, because they enabled the detection of both

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protein-like and humic-like fluorescent species in a single chromatographic run.

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HPSEC analysis of fractions A, B, C+D and bulk SRNOM with multi-wavelength

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UV detection. The HPSEC chromatogram of bulk SRNOM sample extracted at 270 nm

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showed two small peaks, labeled 2 and 3, and an intense hump (Ve at hump top = 9.6 mL)

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with rather well-resolved peaks labeled 5, 6, 7, 8 on the tailing edge (Table 2, Fig. 3, black

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solid lines). In the large molecular size fraction A the sharp peak 2 and a broad hump (Ve at

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hump top = 7.8 mL) were present. The medium molecular size fraction B had small and

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closely located peaks 2 and 3 and symmetrical hump (Ve at hump top = 8.4 mL). The small

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molecular size fraction C+D exhibited hardly distinguishable peaks 2 and 3 but the hump (Ve

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at hump top = 9.4 mL) was broad with several shoulders at longer elution volumes. To

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summarize, peak 2 was abundant in fraction A, much smaller in fraction B, and detected as

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traces in fraction C+D. The Ve of the humps, accounting for the largest area on the UV

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chromatograms, varied in the order Ve = 7.8 mL for A < Ve = 8.4 mL for B< Ve = 9.4 mL for

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C+D. Therefore, the HPSEC analysis confirmed that the fractions molecular size obtained by ACS Paragon Plus Environment


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preparative LPSEC on Sephadex G-75 varied in the sequence A>B>C+D. During LPSEC

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and HPSEC the separation is commonly described by size exclusion effect (the higher the

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retention time - the lower the molecular size). It is well known that separation of NOM by

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SEC is also influenced by non-size exclusion effects (electrostatic repulsion or interaction and

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sorption processes) which can induce an important variability in detected elution volumes

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(13). However, in our study the HPSEC analysis of fractions obtained by LPSEC has shown

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that irrespective of the SEC conditions (the type of solid and liquid phases and different

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instruments used) the NOM fractionation was based mainly on the size exclusion effect.

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We note that the Ve of fractions humps (7.8 , 8.4 and 9.4 mL) were shifted to shorter

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retention time in comparison with Ve of bulk SRNOM hump (9.6 mL). These results clearly

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indicate that the molecular sizes of A, B and C+D fractions were higher than that of bulk

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SRNOM. This effect could be explained by the fact that before HPSEC during dialysis the

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fractions had lost organic matter with nominal molecular size < 10 kDa (39). A similar result

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was obtained by Remucal et al. (43) who used a membrane with cut-off 100-500 Da for SRFA

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dialysis. The total areas of HPSEC UV-chromatograms of bulk SRNOM and fractions were

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similar, because the samples were used at the same volume and identical optical density at

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270 nm, and did not irreversibly adsorb on the column.

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chromatograms of SRNOM and fractions had rather similar shapes with the same absorbance

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peaks and shoulders at any wavelength detection in the range 210-400 nm.

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It should be noted that UV-

The UV spectra at Ve, corresponding to peak’s tops, were extracted from the multi-

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wavelength absorbance detector data.

A gradual absorbance decrease with increasing

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wavelength without any essential maxima was detected for all peaks. Differences in the ratio

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A250/A365 nm were however observed (Table 2). After preparative LPSEC fractionation of

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bulk SRNOM positive relationship between Ve and A250/A365 was observed for fractions A,

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B and C+D (Table 1). However, after analytical HPSEC this correlation was no longer

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observed (Table 2). We assume therefore that absorbance ratio A250/A365 may be used only

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for a rough estimation of aquatic NOM’s molecular size. ACS Paragon Plus Environment

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HPSEC analysis of humic-like and protein-like fluorescence distribution with multi-wavelength fluorescence detection.

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The HPSEC fluorescence chromatograms of the bulk SRNOM and its fractions at

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excitation/emission wavelength pairs (Ex/Em) - 270/450 nm for detection of humic-like

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fluorophores is shown on Fig.3. In contrast to UV chromatograms (solid black lines) that

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showed similar total peak areas, the total area of humic-like fluorescent chromatograms

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(dashed black lines) of SRNOM and fractions differed greatly and varied in the order:

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SRNOM>C+D>A>B.

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SRNOM and its fractions using EEM fluorescence analysis (Fig. 2, Table 1).

These data coincides with the fluorescence intensity obtained for

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The humic-like fluorescent chromatogram of SRNOM (Fig.3, dashed black line) showed

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several rather well resolved peaks, labeled 5, 6, 7 and 8 with emission maxima at 435, 430,

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420 and 410 nm, respectively (Table 2). Wu et al. (22) and Her et al. (24), who conducted

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chromatographic experiments at excitation wavelength 350-337 nm, observed that the

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emission maxima of several aquatic NOM tended to decrease with decreasing the molecular

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size. In fractions A and B the fluorescence intensity of peaks 5, 6, 7 and 8 were considerably

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smaller than that of the bulk SRNOM. On the other hand, a new peak 4 was found in fraction

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B, and peak 5 was dominant in fraction C+D. In spite of similar elution volumes, peaks 5, 6

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and 8 showed different emission maxima in bulk SRNOM versus to fractions. In all fractions

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the emission maxima of peaks 5 and 6 were red-shifted (from 435 to 450 nm and from 430 to

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435 nm, respectively) in comparison with SRNOM. On the contrary the emission maximum

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of peak 8 in fractions A and B was blue-shifted from 410 to 405 nm (Table 2). These results

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could be explained by dialysis and by the fact that a great part of fluorophores with emission

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maxima 435 and 420 nm (peaks 6 and 7) with nominal molecular size less than 10 kDa were

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lost before HPSEC. On the other hand, fluorophore(s) with emission maxima 465 and 450

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nm (peaks 4 and 5) still remained in fractions B and C+D, respectively and had therefore

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nominal molecular size higher than 10 kDa. The normalized fluorescence emission spectra of

ACS Paragon Plus Environment


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peaks 4, 5, 6, 7, 8 of fraction B are presented in Fig. 4a. The differences in peaks emission

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maxima reached 60 nm (from 465 nm in peak 4 to 405 nm in peak 8).

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The aquatic NOM protein-like fluorescence has been classified into tryptophan-like

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(emission maximum of about 350 nm) and tyrosine-like (about 300 nm) nature (2, 3, 44). The

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analytical HPSEC fluorescence chromatograms of the bulk SRNOM and its fractions were

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extracted at Ex/Em - 270/345 nm for visualization of protein-like fluorescence emission ( Fig.

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3, red line). For the bulk SRNOM and the small molecular size fraction C+D the protein-like

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fluorescence chromatograms (red line) were identical to the humic-like fluorescence

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chromatograms (dashed black lines), but they showed lower intensity. It means that both

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samples did not contain the protein-like fluorophores.

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chromatograms extracted at Ex/Em - 270/345 nm revealed the presence of new peaks labeled

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1, 9 and 10 in the large molecular size fraction A and peak 1 in the medium molecular size

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fraction B (Fig.3). In fraction A the broad emission spectrum of peak 9 had a maximum at

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390 nm and a shoulder near 350 nm, while peak 10 had a maximum at 350 nm with a shoulder

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at about 440 nm (Fig. 4b, Table 2). Thus the shapes of both spectra seemed to be the result of

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the overlapping of tryptophan-like and humic-like fluorescence, but the tryptophan-like

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fluorescence was more pronounced in peak 10. Taking into account that Ve of peaks 9 and 10

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(11.9 and 12.6 ml, respectively) were less than Vp of the column (13.1 ml) we could assume

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that these peaks may correspond to the short tryptophan-contained peptides or free tryptophan

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amino acid bound to humic-like part of aquatic NOM with apparent molecular size more than

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5 kDa (according to protein molecular weight standard and fractionation range of the column).

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The detection of the late-eluted fluorescent peaks 8, 9, 10 only in the highest molecular size

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fraction A is surprising. These fluorophores should have been lost altogether during dialysis,

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or should be present in the small molecular size fraction. However, these results are well

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consistent with the formation of supramolecular assemblies by NOM molecules, likely

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controlled by association of low-molecular size components (45, 46, 47).


However, the fluorescence

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The emission maximum of peak 1 in fractions A and B was located in the wavelength

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range shorter than 330 nm (Fig. 4b, Table 2). Under the experimental conditions used in this

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study, we cannot detect the exact emission maximum at wavelength less than 330 nm,

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nevertheless we could attribute this peak to tyrosine-like fluorophores because the emission

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maximum of tyrosine is around 300 nm (44). The peak 1 eluted at about Ve = 5.6 mL (the

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excluded peak of the HPSEC-column) might correspond to the tyrosine-like chromophores

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bound to larger molecular size part of SRNOM with nominal molecular size higher than

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molecular size of globular proteins with MW = 150 kDa. The exact chemical structure of the

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fluorophores could be determined by further analysis of isolated humic-like and protein-like

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peaks of aquatic NOM using ultrahigh-resolution Fourier transform ion cyclotron resonance

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mass spectrometry.

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ENVIRONMENTAL IMPLICATION

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Analytical HPSEC with multi-wavelength fluorescence and absorbance detectors was

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used for the characterization of SRNOM and fractions A, B, C+D obtained by preparative

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LPSEC-PAGE fractionation and dialysis. The results confirmed that fractions differed greatly

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on molecular size and fluorescence intensity and allowed us to detect at least five types of

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humic-like fluorophores size-separated within NOM with different emission maxima at 465,

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450, 435, 420 and 405 nm. The decrease of the emission maxima paralleled the decrease of

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nominal molecular size of fluorescent NOM. The presence of protein-like fluorescent signals

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in the large molecular size fraction A and medium molecular size fraction B was detected as

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well.

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chromophores bound to SRNOM showing nominal molecular size higher than 150 kDa. The

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second group of protein-like fluorophore(s) may be the result of interactions of short

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tryptophan-contained peptides or free tryptophan with SRNOM showing apparent molecular

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size more than 5 kDa. These results provide new information about the fluorescent and

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absorptive properties of NOM, consistent with the formation of supramolecular assemblies by

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NOM molecules (27, 31, 38, 45, 46). These results could be achieved only by using multi-step

One group of protein-like fluorophore(s) might correspond to tyrosine-like



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fractionation of SRNOM that includes: (i) LPSEC-PAGE setup in 7M urea, (ii) dialysis

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through cellulose tubing with nominal cutoff 10 kDa, (iii) HPSEC with multi-wavelength

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absorbance and fluorescence detectors. The detection of several size-separated humic-like and

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protein-like fluorophores within NOM provides detailed information that may help better

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trace NOM origin and biogeochemical processing in the environment. It is clear that the

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isolation of specific fluorophores by combination of several chromatographic procedures will

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be useful for elucidating the structure and source/reactivity of the various components that

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comprise NOM.

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ACKNOWLEGMENTS

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The work has been supported by (1) Russian Foundation for Basic Research (project 18-016-

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00078-a) in part of methodology developing, (2) funding from CNRS-RAS cooperation in

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part of HPSEC fractionation; (3) funding in framework of state assignment.

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High-performance

liquid

466

chromatography (HPLC)-size exclusion chromatography (SEC) for qualitative detection

467

of humic substances and dissolved organic matter in mineral soils and peats in Lithuania.

468

Intern. J. Environ. Anal. Chem. 2015, 95 (6), 508–519.

469

33. Peuravuori, J.; Pihlaja, K.; Trubetskaya, O.; Reznikova, O.; Afanaseva, G.; Trubetskoj, O.

470

SEC-PAGE characterization of lake aquatic humic matter isolated with XAD-resin and

471

tangential membrane ultrafiltration. Intern. J. Environ. Analyt. Chem. 2001, 80 (2), 141–

472

152.

473

34. Trubetskoj, O.A.; Trubetskaya, O.E.; Richard, C.

Photochemical activity and

474

fluorescence of electrophoretic fractions of aquatic humic matter, Water Resources 2009,

475

36 (5), 518–524.

476

35. Artinger, R.; Buckau, G.; Kim, J.I.; Geyer, S. Characterization of groundwater humic and

477

fulvic acids of different origin by GPC with UV/Vis and fluorescence detection. Fresenius

478

J. Anal. Chem. 1999, 364 (8), 737-745.

479

36. Richard, C.; Trubetskaya O.; Trubetskoj O.; Reznikova O.; Afanas’eva G.; Aguer J.-P.;

480

Guyot, G. Key role of the low molecular size fraction of soil humic acids for fluorescence

481

and photoinductive activity. Environ. Sci. Technol. 2004, 38 (7), 2052-2057.

482

37. Zanardi-Lamardo, E.; Clark, C.D.; Moore, C.A.; Zika, R.G. Comparison of the molecular

483

mass and optical properties of colored dissolved organic material in two rivers and coastal

484

waters by flow field-flow fractionation. Environ. Sci. Technol. 2002, 36 (13), 2806-2814. ACS Paragon Plus Environment

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19 485

38. Cuss, C.W.; Gueguen, C. Relationships between molecular weight and fluorescence

486

properties for size-fractionated dissolved organic matter from fresh and aged sources.

487

Water Research 2015, 68, 487–497, doi:10.1016/j.watres.2014.10.013

488

39. Trubetskaya, O.E.; Richard, C.; Voyard, G.; Marchenkov, V.V.; Trubetskoj, O.A. RP-

489

HPLC and spectroscopic characterization of Suwannee river water NOM after

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concentrated urea treatment and dialysis. Desalination and Water Treatment 2016, 57

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(12), 5358-5364.

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40. Traina, S.J.; J. Novak, J.; Smeck, N.E. An ultraviolet absorbance method of estimating

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the percent aromatic carbon content of humic acids. J. Environ. Qual. 1990, 19 (1), 151-

494

153.

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41. Trubetskaya, O.E.; Trubetskoj, O.A.; Richard, C. Hydrophobicity of electrophoretic

496

fractions of different soil humic acids. Journal of Soils and Sediments 2014, 14 (2), 292-

497

297.

498

42. Khundzhua, D.A.; Patsaeva, S.V.; Trubetskoj, O.A.; Trubetskaya, O.E. An analysis of

499

dissolved organic matter from freshwater Karelian lakes using reversed-phase high-

500

performance liquid chromatography with online absorbance and fluorescence analysis.

501

Moscow University Physics Bulletin 2017, 72 (1), 68–75.

502

43. Remucal, C. K.; Cory, R M.; Sander, M.; McNeill, K. Low molecular weight components

503

in an aquatic humic substance as characterized by membrane dialysis and orbitrap mass

504

spectrometry. Environ. Sci. Technol. 2012, 46(17), 9350–9359. DOI:10.1021/es302468q

505 506 507 508 509 510

44. Lakowicz, J.R. Principles of fluorescence spectroscopy; Springer Science: New York., 2006. 45. Piccolo, A. The supramolecular structure of humic substances. Soil Sci. 2001, 166, 810832. 46. Sutton, R.; Sposito, G. Molecular Structure in Soil Humic Substances: The New View. Environ. Sci. Technol. 2005, 39 (23), 9009-9015. DOI: 10.1021/es050778q



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20 511

47. Trubetskaya, O.E.; Richard, C.; Trubetskoj, O.A. High amounts of free aromatic amino

512

acids in the protein-like fluorescence of water-dissolved organic matter. Environmental

513

Chemistry Letters 2016, 14 (4), 495-500.

514 515 516

517

518

519

520

521

522

523

524

525

526

527

528

529

530

531 ACS Paragon Plus Environment

Page 21 of 26


21 532

533 534 535 536

2

537 538

540 541 542 543

Absorbance

539

C+D

1

SRNOM

B

544

A

545 546 547 548

0 200

300

400

500

600

700

Wavelength, nm

549 550 551 552

Figure 1. Absorbance spectra of bulk SRNOM, fractions A, B and C+D in 10 mM phosphate buffer, pH=6.5, at a concentration of dry sample = 50 mgL-1.

553 554 555 556 557 558 559 560



Page 22 of 26

22 561 562 563

564

565 566 567 568 569

Figure 2. Fluorescence EEM of bulk SRNOM, fractions A, B and C+D at A270=0.05 in 0.1 mM phosphate buffer, pH 6.5.

570 571 572 573 574 575 576


Page 23 of 26


579 580 581

Absorbance at 270 nm

578

12

587


586


593 594

601 602 603 604 605 606 607

7

8

9

10

11

2

0 .0 8

12

13

6 5

F r a c tio n A

4 0 .0 6

6 7

0 .0 4 0 .0 2

3

5

1

8

9 10

2 1 0

5

6

7

8

9

10

11

12

13

F r a c tio n B

6 5

0 .0 8

4 0 .0 6 3 0 .0 4

5

4

1 2 3

0 .0 2

6

2

7 8

1 0

5

6

7

8

9

10

11

5

0 .1 0


600

6

12

13

F r a c tio n C + D

12

6

0 .0 8

8

0 .0 6

7

0 .0 4

4 0 .0 2

2

3

0 .0 0

Fluorescence intensity

599

5

0 .0 0

596

598

0

0 .0 0

595

597

3

2


592

4

0 .0 2

0 .1 0

589

591

8

0 .0 0

588

590

8

0 .0 4


585

7

5

0 .0 6

0 .1 0

584

16

0 .0 8

582 583

23

S R N O M


577

6

A bs270 E x /E m 2 7 0 /4 5 0 n m E x /E m 2 7 0 /3 4 5 n m

0 .1 0

0 5

6

7

8

9

10

11

12

13

Elution volume, ml Figure 3. HPSEC UV chromatograms with detection at 270 nm (solid black lines), and fluorescent chromatograms, with detection at Ex/Em wavelengths 270/450 nm (dashed black lines) and Ex/Em wavelengths 270/345 nm (red lines) for bulk SRNOM and its fractions A, B and C + D. ACS Paragon Plus Environment


Page 24 of 26

24 608 609

60 nm

a).

611 612 613 614 615 616

Normalized fluorescence intensity

610

8 7 6 5 4B 1

0.5

0 350

617

400

450

500

Wavelength, nm

619 620 621 622 623 624 625

Normalized fluorescence intensity

618

b).

1

9A 10A 1B

0.5

1A

0 350

626

400

450

500

Wavelength, nm

627 628 629

Figure 4. Emission spectra of HPSEC peaks 4, 5, 6, 7 and 8 of fraction B showing different

630

humic-like fluorescence maxima (a), and of peaks 1, 9 and 10 with different protein-like

631

fluorescence maxima (b). Spectra were extracted from the data of multi-wavelength

632

fluorescent detector and normalized at maximum of emission.

633 634 635 636


Page 25 of 26


25 637 638

Table 1. Mass balance (%) and specific UV absorbance coefficients at 280 nm (SUVA280)

639

based on dry weight of NOM samples; absorbance ratio at 250 and 365 nm (A250/A365);

640

protein-like and humic-like fluorescence maxima (λex/λem) at

641

wavelengths for bulk SRNOM and fractions A, B, C+D obtained by LPSEC-PAGE setup.

different excitation

642

Absorbance Sample name

SRNOM

Mass balance based on dry weight

SUVA280

(%)

(based on dry weight at a concentration 50 mgL-1)

100

0.61

A250/A365

Fluorescence maximum (nm)

Protein-like

Humic-like

(λex/λem)

(λex/λem)

-

230/450

-

270/455

4.58

330/450 Large molecular size fraction A

4

0.26

3.16

230/335

230/440

270/350

270/440 330/440

Medium molecular size fraction B

10

0.75

3.20

230/330

230/460

270/340

270/465 330/460

Small molecular size fraction C+D

35

0.89

3.76

-

230/455

-

270/465 330/460

643


44


26

Table 2. Elution volumes (Ve), absorbance ratios (A250/A365) and fluorescence emission maximum (Em max) of peaks 1-10, obtained during HPSEC analysis with multi-wavelength absorbance and fluorescence detectors of bulk SRNOM and fractions A, B and C+D.

45

Ve Peak number

at peak’s top

SRNOM A250/A365

(ml)

Large molecular size fraction A

Em max

A250/A365

(nm)

Em max

Medium molecular size fraction B A250/A365

(nm)

Em max

Small molecular size fraction C+D A250/A365

(nm)

Em max (nm)

1

5.6

-

-

3.83

Molecular Size distribution of Fluorophores in Aquatic Natural Organic

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