Inhibition of flash rusting of HY80 by a mussel adhesive protein: Characterizing the interaction of MeFP-5 with a high strength low alloy steel

Electrochimica Acta 301 (2019) 411e420

Contents lists available at ScienceDirect

Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta

Inhibition of flash rusting of HY80 by a mussel adhesive protein: Characterizing the interaction of MeFP-5 with a high strength low alloy steel Douglas C. Hansen a, c, *, Kathryn R. Zimlich b, Brooke N. Bennett c a b c

University of Dayton Research Institute, Dayton, OH, 45469, United States University of Dayton Biology Department, Dayton, OH, 45469, United States University of Dayton, Chemical Engineering Department, Dayton, OH, 45469, United States

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 February 2018 Received in revised form 24 January 2019 Accepted 25 January 2019 Available online 30 January 2019

A proteinaceous biopolymer, mussel adhesive protein #5 (MeFP-5), isolated from the common blue mussel (Mytilus edulis L) has been investigated as a model for designing an aqueously soluble candidate corrosion inhibitor system that is non-toxic and environmentally friendly and is capable of inhibiting the flash rusting of exposed high strength low alloy (HSLA) steel surfaces during the paint removal process. In a previous study it was found that a significant amount of corrosion inhibition is possible (nearly 100% inhibition after 7 days) on HY80 steel in a 100% relative humidity environment at 40
C. In an attempt to determine the possible mechanisms of corrosion inhibition, surface characterization of HY80 steel treated with various solutions containing the MeFP-5 biopolymer were performed. Fourier transform infrared (FT-IR) spectroscopy using attenuated total reflectance (ATR) of the protein adsorbed onto glass and metal surfaces indicated that complexation of the iron atoms on the oxyhydroxide covered metal surface involved the amino acid L-dopa and the primary amine of lysine. The involvement of these amino acids in the adsorption of the protein onto glass were much less evident. When the adsorbed protein was treated with a catechol oxidase enzyme, vibration bands consistent with metal complex formation involving L-dopa were not observed, indicating that the treatment with enzyme resulted in mess metal chelation at the surface oxide-protein film interface. Energy dispersive x-ray spectroscopy (EDS) findings indicate that iron content is highest where the MeFP-5 biopolymer is adsorbed onto the steel substrate at pH 5.5, 50% higher than the content of the iron on the steel surface alone. When the biopolymer is enzymatically cross-linked on the steel surface at pH 5.5, the iron content is decreased by 15% of that of the adsorbed biopolymer, suggesting that the iron at the steel surface is undergoing complexation and possible metal-mediated cross-linking, whereas the enzyme cross-linked protein on the steel surface complexes less of the iron. Three-dimensional modeling efforts of MeFP-5 suggest that specific domains of MeFP-5 where two amino acids, L-Dopa and lysine, are concentrated may be involved in adsorption to a metal oxyhydroxide film. These results indicate that it is possible to utilize the biochemistry of a naturally occurring biopolymer isolated from the marine mussel, Mytilus edulis (L), to develop a nontoxic and environmentally friendly corrosion inhibitor. © 2019 Elsevier Ltd. All rights reserved.

Keywords: L-dopa Corrosion MeFP-5 SEM-EDS Modeling

1. Introduction Many marine vessel hulls today are made from high strength low alloy (HSLA) HY80 steel, which has a high yield strength of

* Corresponding author. University of Dayton, 300 College Park, þ0152, Dayton, OH, 45469, USA. E-mail address: [email protected] (D.C. Hansen). https://doi.org/10.1016/j.electacta.2019.01.145 0013-4686/© 2019 Elsevier Ltd. All rights reserved.

552 MPa (80 ksi from which it gets its name) [1]. To protect these metal hulls from corrosion due to the marine environment, coatings are applied when the existing coating begins to deteriorate. These failing coatings must be removed before the new coating is applied, which is done by abrasive blasting, or more recently, ultrahigh pressure water jetting [2]. Unfortunately, HY80 steel is susceptible to flash rusting when exposed to the atmosphere after being water jetted due to fine edges on the steel surface caused by the high water pressure impact, so a temporary corrosion inhibitor

412

D.C. Hansen et al. / Electrochimica Acta 301 (2019) 411e420

must be used after the old coating is stripped and before the new one is applied [3]. Since the hull of the vessel will be wet after the water jetting, an inhibitor is needed that will be able to adhere to the HY80 steel in a wet environment. While moisture usually deters adhesion, one only has to look at how sessile marine organisms adhere to substrates in the tidal zone for inspiration; one of these organisms, the common blue mussel Mytilus edulis (L), has excelled at formulating a superior adhesive strategy on wet surfaces (rocks and pilings) while simultaneously being exposed to a high energy environment (crashing waves, tidal flows) [4,5], by virtue of its attachment structure, the byssus [6]. It has been reported that the byssus structure of the mussels in the Mytilidae family contains roughly 25e30 different proteins with 7e8 of these proteins present at the substrate-byssus interface [7]. The Mytilus edulis foot protein-1 (MeFP-1), the largest of the byssus proteins and containing 15 mol % of the catecholic amino acid L-3,4 dihydroxyphenylalanine (L-Dopa) [8] has been investigated in numerous studies as a corrosion inhibitor. In one study, the corrosion inhibition provided by MeFP-1 adsorbed onto 304 L (S30403) stainless steel was found to be greater than the corrosion inhibition provided by bovine serum albumin (BSA), 3,4dihydroxybenzoic acid, and poly-L-lysine using cyclic potentiodynamic curves and pit depth measurements [9]. A second study compared the corrosion inhibition of a commercial preparation of MeFP-1 to BSA on carbon steel using electrochemical impedance spectroscopy (EIS) and atomic force microscopy (AFM) [10]; in that study, it was demonstrated that MeFP-1 and BSA provided comparable corrosion inhibitor for carbon steel in acidic (pH 4.6) solution conditions. More recently, the corrosive inhibition properties of MeFP-1 on HY80 steel were studied and compared to two other Mytilus edulis foot proteins containing the greatest amount of L-Dopa: MeFP-3 (20e25 mol%) and MeFP-5 (27 mol%) [11]. In that study, mass loss, EIS and elevated temperature and humidity exposure testing were performed on HY80 coupons treated with each of these three protein in a phosphate buffer, both with and without enzyme-assisted crosslinking. The exposure testing showed that the substrates treated with the MeFP-5 had the greatest corrosion inhibition after 7 days in the 100% relative humidity 40
C exposure environment, outperforming a commercially available flash rust inhibitor. That study also showed that cross-linking of the MeFP-5 does increase corrosion inhibition, but was unclear whether the higher L-Dopa content was the cause for the increased corrosion inhibition between the three proteins. In addition to L-Dopa, MeFP-5 contains lysine (20 mol%) [7,11], which when adjacent to L-Dopa in the amino acid sequence has been proposed to improve the adhesion of the catechol by removing hydrated cations from a mineral substrate allowing the catecholic L-Dopa better access to the mineral oxide surface [12]; a similar scenario could exist at a hydrated metal oxide surface. The high mole% of lysine and L-dopa and their close proximity to each other in the MeFP-5 sequence suggests that there may be a synergistic role between these amino acids in the adhesion/adsorption of the protein on the metal surface, resulting in the improved corrosion inhibition of this proteinaceous polymer over the other MeFP's tested previously. Therefore, to better understand and possibly explain the mechanism(s) by which the MeFP-5 confers such significant corrosion inhibition on HY80 steel, the objectives of the current study were to characterize the orientation and interaction of the MeFP-5 protein with the metal oxide-hydroxide substrate and what synergistic effect (if any) exists between the lysine and L-Dopa amino acids on the interaction of the protein with the metal oxidehydroxide substrate.

2. Experimental procedures 2.1. Materials High strength low alloy steel plate with 0.6 cm thickness (HY80, Clifton Steel, Cleveland, OH) was cut into 3.00 cm2 coupons. The given chemical composition (wt%) of the steel was: 95.20e96.45 Fe; 2.50e3.20 Ni; 0.55e0.75 Mn; 0.30e0.60 Mo; and 0.20 (maximum) C. The coupons were wet ground using silicon carbide up to 1200 grit, followed with polishing at 3, 1 and 0.04 mm colloidal silica with an isopropanol rinse, subjected to acetone, methanol, and isopropanol baths with sonication cleaning, dried under compressed air, and stored at 60
C to achieve a mirror finish. 2.2. Solution preparation Phosphate buffer: A buffer of 0.1 M dibasic potassium phosphate was added to 0.1 M monobasic potassium phosphate and adjusted with 5% (vol/vol) glacial acetic acid to obtain a pH of 5.5. Enzyme: Ten milligrams of mushroom tyrosinase (E.C. #1.14.18.1, Sigma-Aldrich) with an activity of >1000 units/mg was dissolved in equal volumes of DI water and buffer at a pH of 5.5 for a total volume of 1.0 ml. MeFP-5 Protein: Purification of MeFP-5 followed protocols published elsewhere [11,13]. The purified MeFP-5 protein was lyophilized and stored at 86
C until needed for the characterization experiments. The lyophilized protein was dissolved in cold DI water at a concentration of 2 mg/mL based on the dry weight of the protein. Depending on the solution conditions required, deionized water (17 MU cm) or phosphate buffer was added in equal parts to obtain a final MeFP-5 concentration of 1 mg/mL. Amino acids: For comparative analysis to the MeFP-5 protein, solutions of L-Dopa and L-Lysine (Sigma Aldrich), which are two of the amino acids that are adjacent to each other in ten locations along the MeFP-5 biopolymer, were prepared similarly to the MeFP-5 solutions. The exception is that for the EDS data, the final concentrations of the L-Dopa and L-Lysine were 25.5 mol% and 20 mol%, respectively, to reflect the nominal concentration of these amino acids in the purified MeFP-5 as reported in the literature [14]. Fig. 1 illustrates the chemical structures of L-dopa and lysine. Incubation of solutions on HY80 substrate: Adsorption of the amino acids or MeFP-5 onto the steel substrate was accomplished by placing 100 ml of one of the prepared solutions onto the prepared HY80 coupon surface and allowing to incubate for 1 h under a watch glass at ambient conditions. For the cross-linking experiments, after 1 h of incubation, 10 ml of the prepared enzyme solution was added to the solution and the sample was allowed to

Fig. 1. Chemical structures of two amino acids in MeFP-5, L-dopa and L-lysine.

D.C. Hansen et al. / Electrochimica Acta 301 (2019) 411e420

incubate for an additional hour. At the end of the incubation time for all experiments, the incubation solution was carefully pipetted off and discarded. The coupon surface was then allowed to dry under a watch glass. The treated coupon was then immediately subjected to surface analysis. 2.3. Surface analysis 2.3.1. Fourier transform infrared spectroscopy Each solution of MeFP-5 protein or amino acid(s) were applied to cleaned glass microscope slides and the polished and cleaned HY80 coupons in 5 mL volume drops, which were allowed to completely dry at ambient conditions before the spectroscopic measurements were made. A background spectrum was collected for each substrate prior to collecting a spectrum on the treated area of each substrate. A Nicolet iS 50 Spectrophotometer with Attenuated Total Reflection (FT-IR ATR) was used, having a top-mounted DLaTGS detector with a KBr window, using a monolithic diamond ATR taking 256 scans per sample from 4000 to 600 cm1 at a resolution of 6 cm1 on control glass and HY80 substrates The data were visualized, collected and processed with OMNIC Series Software.

413

Table 1 Representative summary in mole% and residues per thousand (RPT) for the amino acids identified in the MeFP-5 isolated from Mytilus edulis. Glutamine and asparagine are totally deaminated during acid hydrolysis and therefore are reported as glutamic acid and aspartic/asparagine. L-Dopa values have been corrected for loss due to hydrolysis. AA

Letter Code

Mole%

Amino acid RPT

Ala Arg Asp/Asn Cys Glutamic Acid Gly His Leu Lys Met Phe Pro Ser o-Phos Ser Thr Tyr Val L-Dopa Total

A R D C E G H L K M F P S S* T Y V Y*

4.20 5.80 6.70 0.30 2.60 14.10 4.00 2.60 19.60 0.10 0.20 1.50 3.00 6.60 1.80 2.00 2.90 22.00 100.00

42.00 58.00 67.00 3.00 26.00 141.00 40.00 26.00 196.00 1.00 2.00 15.00 30.00 66.00 18.00 20.00 29.00 220.00 1000.00

2.3.2. Electron dispersive x-ray spectroscopy (EDS) For each sample, the spectra was collected at a 1000 magnification, 15.0 mm working distance, at 20 kV using a Zeiss EVO50XVP environmental scanning electron microscope (ESEM) integrated with an EDAX Genesis 2000 energy dispersive spectroscopy (EDS) system. Spectra were collected on two samples for each solution treatment and adsorbate and the average weight percent of iron calculated. No calibration standards were used, therefore the quantitative weight percent values determined are relative. 2.3.3. Molecular modeling The working MeFP-5 model was created in Chem3D® using the protein's consensus amino acid sequence [15]. This first iteration of the MeFP-5 model was built and analyzed with the assumptions of a constant pH of 5.5 and the lack of a surrounding solution or buffer. To investigate how MeFP-5 approaches and aligns with a metal oxide-hydroxide surface, models of goethite (FeOOH) and hematite (Fe2O3) were created by building and extending the unit cells of the metal oxide-hydroxides. The intramolecular spacing between iron and oxygen atoms of the metal constructs as well as the distance between L-Dopa residues for the MeFP-5 construct were calculated and measured. 3. Results and discussion 3.1. MeFP-5 purification The amino acid composition for the resulting isolated and purified MeFP-5 protein is summarized in Table 1. Numerous batches of purified MeFP-5 were used in this study, thus the amount of the component amino acids varied between the batches. Table 1 is a representative summary of the mole% of each amino acid identified in the protein. It is clear from the amino acid composition that the three most prevalent amino acids are glycine, L-dopa and lysine (14, 22 and 19 mol%, respectively) and are similar to those published previously [14]. It is not uncommon for mussel adhesive protein composition to vary from organism to organism or from population to population [16]. Fig. 2 is a schematic of the consensus sequence of the MeFP-5 protein [15], showing the positive and negative charges associated with the functional groups of the basic and acidic amino acid residues. There are ten instances where an L-Dopa residue is adjacent to a lysine residue, and based on this

Fig. 2. Schematic of consensus sequence of MeFP-5 showing 5 separate domains of the protein with the charges associated with the functional groups of the basic and acidic amino acids within each domain.

observation and for computational modeling purposes MeFP-5 was compartmentalized into five domains. This approach allowed for characterization of different regions of MeFP-5 based on parameters such as electrostatic attraction and the overall charge density of the regions. These domains are outlined in the colored boxes in Fig. 2. 3.2. Fourier transform infrared spectroscopy (FTIR) In Fig. 3, representative IR spectra of the MeFP-5 protein adsorbed onto glass and HY80 steel are presented; all spectra were baseline corrected and water subtracted. In general, the diminution of the vibrational bands assigned to the primary amine functional group of lysine (-NH3þ) in the amide I and II bands and the catecholic functional group of L-dopa in water on the HY80 substrate vs. the glass substrate indicate the involvement of these two amino acids in the adsorption and interaction of the MeFP-5 protein with the HY80 iron oxy-hydroxide surface. The IR spectroscopy vibration band assignments were made as follows: the stretching of H2O occurs around 3000e3600 cm1 and can interfere with surface

414

D.C. Hansen et al. / Electrochimica Acta 301 (2019) 411e420

Fig. 3. Representative FT-IR spectra of MeFP-5 adsorbed onto glass and HY80 steel. The spectra are baseline corrected and water subtracted.

hydroxyl groups; the broad bands at 3264 cm1 (on glass) and 3170 cm1 (on HY80) can be attributed to adsorbed water molecules since even with baseline correction any individual vibration bands are difficult to resolve [17]. The bands from 2850 to 2951 cm1 (on glass and HY80) have been assigned to the aliphatic -CH2 stretching along the protein polymeric backbone [17]; however, the band at 2951 cm1 (on glass) is absent on the HY80 and the bands at 2929 and 2863 cm1 on glass have shifted to 2920 and 2850 cm1 and appear to be enhanced and better defined. This may suggest that a portion of the protein is oriented away from the metal. The 2321 cm1 (-OH asymmetric stretching) [18] is absent from the HY80 spectra, suggesting that the eOH for this band assignment was specific to the adsorbed hydroxyl ion at the glass surface. The spectra in the region from 1500 to 700 cm1 are representative of the functional groups and side chains of lysine and L-dopa [19]; the overall trend of the absorbance values for these bands decreasing for the spectra taken on the metal substrate vs those on the glass indicate that these amino acids and functional groups are involved in the adsorption of the MeFP-5 to the metal substrate [20]. The “fingerprint” spectral region from 500 to 1700 cm1 is of most importance and is discussed in the following detail, comparing the vibration bands between lysine, L-dopa, and MeFP-5 on HY80 steel in water, buffer and buffer with enzyme; the buffer and enzyme conditions were representative of the corrosion inhibition solutions that were most effective in the previous study [11]. 3.2.1. L-dopa, lysine, L-dopa and lysine, and MeFP-5 in water on HY80 In Fig. 4, the broad band from 1600 to 1700 cm-1

Fig. 4. Representative FT-IR fingerprint spectral regions of L-Dopa, Lysine, L-Dopa and Lysine, and MeFP-5, respectively, dissolved in deionized water and adsorbed onto HY80 steel. The spectra are baseline corrected and water subtracted.

(1635e1699 cm1) were assigned to the amide I associated with the stretching vibration of the C¼O and C-N groups (1699 cm1 shoulder band) in the lysine, L-dopa-lysine, and MeFP-5 and on the metal substrate [21]. The bands at 1635 and 1669 cm1 were assigned to the symmetric and antisymmetric stretching of the protonated primary amine eNHþ 3 functional group of lysine [19] and are present in the lysine, L-dopa-lysine and MeFP-5 spectra, all suggesting interaction of the protonated amine group with the metal surface. The band at 1646 cm1 was assigned to the eNH2 amide that is also present on the lysine primary amine [19]. The pKa of the amine is 10.0, meaning at a pH of 5.5, the alkyl amine can become protonated. These spectra indicate that perhaps both forms are present. The 1622 cm1 band was also assigned to the antisymmetric deformation of the protonated alkyl amine of lysine [19] on glass, which is greatly diminished on the MeFP-5 spectra, suggesting involvement of the protonated amine with the metal oxyhydroxide surface. The broad band from 1500 to 1600 cm1 (1507e1560 cm1) were assigned as follows: the band at 1558 cm1 is coincident in both the L-dopa and lys spectra; therefore it can be assigned to theamide II combination band (N-H bending and C-N stretching) and is derived mainly from in-plane N-H bending and C-N within the individual amino acids as well as the peptide unit of the adsorbed protein [21]. The band at 1540 and 1521 cm1 were assigned to the eNHþ 3 protonated amine of lysine [22], and is prominent in the lysine, L-dopa-lysine and MeFP-5 spectra; however, the 1540 cm1 band is coincident with the same vibration band for L-dopa for the aromatic ring vibration [19]. In the L-dopa spectra, the 1486 and 1417 cm1 bands were assigned to the aromatic C¼C ring stretches, the 1417 and 1342 cm1 to the aliphatic C-H vibrations, and the 1262 and 1222 cm1 bands were assigned to the O-H bending modes in the catechol functional group [20]. These vibration bands are similar to those exhibited by catechol binding to hematite via a coordinated monodentate complex involving one oxygen atom of the catechol and one surface Fe atom as well as hydrogen bonding by the remaining catechol hydroxyl to an adjacent hydroxylated surface Fe atom [23]. The absence of these bands exhibiting strong vibration modes in either the L-dopa-lysine or MeFP-5 solution spectra suggests that perhaps metal binding and complexation under these conditions is not at an optimum in either case, or perhaps that lysine may have an effect. The band at 1120 cm1 for the L-dopa which is also present in the MeFP-5 spectrum, has been assigned to the in-plane deformation of eOH on the benzene ring of the L-dopa [20,24]. The band at 1020 cm1 was also assigned to the in-plane deformation of the eCH on the benzene ring of L-dopa [25]; this band overlaps with the 1017 cm1 (lysine e aliphatic amine), 1027 cm1 (L-dopa) and 1033 cm1 for the L-dopa-lysine spectra. Finally, the band at 737 cm1 for the MeFP-5 has been assigned to the -NHþ 3 rocking of the lysine alkyl amine [18] and the rocking of the -CH2 alkyl group in the side chain of lysine [17], again indicating the incorporation of the lysine side chain into the oxyhydroxide metal surface. 3.2.2. L-dopa, lysine, L-dopa and lysine, and MeFP-5 in buffer on HY80 In Fig. 5, the vibrational bands for the adsorbates dissolved in the 0.1 M potassium phosphate buffer on HY80 were identified and assigned to functional groups that are nearly identical to the vibration bands for the adsorbates dissolved in deionized water and adsorbed onto HY80, with the band absorbance values being lower than in deionized water (Fig. 4). As the concentration of the amino acid and protein solutions were identical in all of the IR measurements, the overall diminishment of the absorbance values of the Ldopa and lysine spectra indicate these amino acids readily adsorb onto the metal surface, more so than is evident in the deionized

D.C. Hansen et al. / Electrochimica Acta 301 (2019) 411e420

Fig. 5. Representative FT-IR fingerprint spectral regions of L-Dopa, Lysine, L-Dopa and Lysine, and MeFP-5, respectively, dissolved in 0.1 M phosphate buffer (pH 5.5) and adsorbed onto HY80 steel. The spectra are baseline corrected and water subtracted.

water solution. In the 1500e1600 cm1 range, the similarity in the vibration bands and absorbance values of the L-dopa-lysine and the MeFP-5 suggests that a synergy exists between the L-dopa and lysine amino acids; the increase in the absorbance values for these two amino acids together may be attributed to possible aggregation or coacervation of the molecules at the metal oxy-hydroxide surface [26]. Indeed, in the buffer solution, it is clear that the spectra for the L-dopa-lysine solution is more like that of the MeFP-5 in the signature range (1300e1700 cm1) than of the two amino acids alone. The bands at 1472, 1269, 1201, 1127, 1073, and 1034 cm1 for the MeFP-5 spectrum closely resemble those exhibited by a catechol forming a monodentate Fe complex involving both oxygen atoms with two adjacent surface Fe atoms on hematite and goethite particles [23]. These two mechanisms are possible due to the formation of mixed oxides of iron on the HY80 substrate when the 0.1 M phosphate buffer is present as determined by Raman spectroscopy (data not shown). Of particular interest is the vibration band at 1269 cm1; vibration bands at 1260 cm1 have been shown to correlate with C-O stretching vibration of the side chain hydroxyl groups of L-dopa forming a complex with a metal ion (in this case Cu2þ ions in solution) [27]. In the case for the L-dopa and MeFP-5 in water, there was a strong well defined band at 1262 cm1 for Ldopa, but no discernible band near that wavenumber for the MeFP5. In the buffer solution, the strong, broad band at 1269 cm1 (Fig. 5) is evidence that metal complexation by the L-dopa at the HY80 surface can be occurring. 3.2.3. L-dopa, lysine, L-dopa and lysine, and MeFP-5 in buffer and enzyme on HY80 In Fig. 6 it is readily apparent that the vibration bands in the 1300e1700 cm1 range are greatly diminished as compared to the buffer or water solutions, whereas in the lower wavenumbers (1175e750 cm1) the vibration bands are much higher in absorbance values and well defined as compared to the water and buffer solutions (Figs. 4 and 5, respectively). Vibration bands for catechol oxidase occur within 1481e1590 cm1 wavenumber range and are therefore difficult to distinguish from the L-dopa, lysine and MeFP-5 vibration bands [28]. For the MeFP-5 spectrum, the 1146 cm1 band has been assigned to the eOH in-plane deformation of tyrosine [24], and the 1076 cm1 (aromatic C-H bending), 1030 cm1 (aromatic C-C or C-H stretching), 985 cm1 (aromatic C-C stretching), and 937 cm1 (aromatic eOH out of plane deformation) bands have all been attributed to the catecholic functional groups [17,20,24,25]. In comparison to the FTIR spectrum for the adsorbates in buffer and water (discussed above), the 1262 cm1 vibration band is absent for L-dopa and MeFP-5 in buffer when the enzyme mushroom

415

Fig. 6. Representative FT-IR fingerprint spectral regions of L-Dopa, MeFP-5, and L-Dopa and Lysine, respectively, dissolved in 0.1 M phosphate buffer (pH 5.5) and enzyme added, adsorbed onto HY80 steel. The spectra are baseline corrected and water subtracted.

tyrosinase is added to the adsorption solution. When sodium periodate (NaIO4) was added to the solution in the study by Fant [27] to initiate cross-linking between adjacent polymer strands of the mussel adhesive protein after the metal ions had been complexed, this vibration band disappeared. This indicated that the metal complex formation was stopped and reversed in the presence of a catalyst for the cross-linking mechanism. As the enzyme mushroom tyrosinase used in the present study catalyzes the same reaction on the L-dopa catecholic functional groups of MeFP-5, it is clear from the IR spectra that the absence of the 1262 cm1 band is evidence that the addition of the enzyme stops or reverses the complexation of the iron atoms present in the MeFP-5 protein/oxyhydroxide film. 3.3. Electron dispersive x-ray spectroscopy (EDS) EDS data for the average weight percent of iron obtained from treatments of HY80 steel with the various solutions and adsorbates as compared to a control is shown in Fig. 7. The spectral line intensities and resulting weight percentage values for the iron collected from the HY80 surface are relative, in that no calibration standards for the composition elements of HY80 were analyzed. At an accelerating voltage of 20 kV, the calculated electron beam penetration into the steel substrate is 1.6 mm, using the Kanaya e

Fig. 7. 3-axis plot of average wt% of iron obtained from treatments of HY80 steel with various solutions and adsorbates as comparted to a control. Spectral values collected at a working distance of 15.0 mm with an accelerating voltage of 20 kV. Spectra collected on two samples for each solution treatment; average values are shown in boxes.

416

D.C. Hansen et al. / Electrochimica Acta 301 (2019) 411e420

Okayama formula [29]. Previous measurements of adsorbed, enzymatically cross-linked mussel protein thickness for similar protein/enzyme concentrations yielded films of 7.3 ƞm [30]; therefore the signal being measured by the electron penetration is representative of the surface of the steel substrate treated with the adsorbed protein as well as the underlying substrate. The values presented in the 3-axis plot in Fig. 7 are the average of two separate samples for each solution/adsorbate treatment. The weight percent of iron is the percentage of the signal line that is representative for that element within the area that is being interrogated by the electron beam. The focus of the EDS analysis was on the amount of iron remaining on the surface as a function of solution treatment/ adsorbate treatment. Between the solution treatments, the surface of the HY80 incubated with adsorbates dissolved in deionized water resulted in the highest average weight percent (wt%) of iron with the control being the highest (DI water only) followed by: lysine, MeFP-5, L-dopa and lysine, and L-dopa; this is in contrast to the buffer solution, where the L-dopa and lysine, lysine and MeFP-5 exhibited very similar wt% values of iron, followed by the control and L-dopa having the lowest value. Finally, for the buffer/enzyme solution, the MeFP-5 and control had similar wt% values for iron, followed by Lysine, L-dopa with the L-dopa and lysine combination having the lowest wt% of any solution-adsorbate combination. It is

interesting to note that all 3 solutions containing L-dopa alone resulted in three of the four lowest amounts of iron; since the incubating solution for all of the treatments was removed from the surface by pipetting, these results indicate that the L-dopa were able to complex the iron (III) oxy-hydroxides from the HY80 surface into solution resulting in the lower wt% values. Nishimoto et al. has reported the dissolution of iron from surface oxides of steel by catechols present in humic substances [28], and Hansen et al. has reported the dissolution of iron being increased by the adsorption of catechol to the surface of stainless steel [31]. The wt% of iron present after the incubation with two of the three lysine solutions are similar to that of the control in DI water, indicating minimal loss of iron. However, the wt% of iron remaining after incubation of the HY80 surface with the buffer/enzyme solution containing L-dopa, lysine or the L-dopa-lysine combination yielded the lowest average values. Clearly there is a synergistic effect between the L-dopa and lysine amino acids on the relative amount of iron detected on the surface in the buffer solution conditions. To account for the decrease in the wt% of iron on the surface in this case, it is tempting to suspect an increased relative amount of L-dopa and lysine residues becoming resident on the metal surface from solution, due to the enzyme-catalyzed oxidation of L-dopa to quinones, and the subsequent formation of di-Dopa adducts with the involvement of

Fig. 8. (L) 3-dimensional model of possible MeFP-5 conformation in space; (R) 3-dimensional model showing domains where L-dopa and lysine amino acids are adjacent to each other along the MeFP-5 polymer backbone.

D.C. Hansen et al. / Electrochimica Acta 301 (2019) 411e420

the nucleophilic amine of lysine [32]. While this scenario would skew the wt% of iron lower, it has not been definitively shown that free amino acids of L-dopa and lysine do indeed for such adducts in solution or at a solution-substrate interface. An alternative scenario is the involvement of the primary amine of lysine in a nucleophilic substitution with a metal oxy-hydroxide surface [33]. This formation of a metal-amine adduct at the solution pH of 5.5 is certainly viable; however it is unknown at the present time how such an adduct formation in the presence of free L-dopa can result in a greatly reduced wt% of iron on the steel surface. Finally, the wt% of iron on the HY80 surface after the incubation with the MeFP-5 in the three solutions and indicates that similar amounts of iron are present on the surface for both the protein dissolved in DI water as in the buffer/enzyme solution, with the protein in buffer resulting in a higher relative wt% of iron present. Adsorption of this mussel protein onto HY80 metal (either with or without enzyme added) conferred a significant amount of corrosion inhibition and it was determined that once adsorbed onto the steel substrate, the protein was not easily removed even during exposures for up to 7 days at 40
C and 100% RH [11]. Therefore, it should come of no surprise that after incubation and subsequent removal of the bulk solution that there would be a high level of iron still present on the surface. Adsorption and complexation of iron from the surface into the adsorbed protein film would yield a higher wt% iron signal than when the L-dopa or lysine amino acids were allowed to adsorb and dissolve the iron from the surface metal oxy-hydroxide, as previously discussed. Consequently, when the enzyme was added to the incubating solution, the protein not already adsorbed onto the metal surface could undergo enzymatic cross-linking. The resulting multiple adlayers of MeFP-5 stacked on top of the adsorbed protein film would increase the volume of the protein film and thus a lower wt% of iron signal could occur (Fig. 7). 3.4. Molecular modeling The first iterative 3-dimensional theoretical model of the MeFP5 protein generated is presented in Fig. 8a. As stated previously, this model only considers the possible conformation of the protein

417

based upon the consensus amino acid sequence, with no effect of pH or solution. This is not unlike what has previously been done by Qin and Buehler [34]. Their considerations also involved 100% conversion of the tyrosine residues to L-dopa, although no ophosphoserines were considered. However, their work suggested that the secondary conformation of the protein is folded and contains disordered structures, which may explain the inherent difficulty of determining this proteins conformation using conventional structural modeling and analytical techniques [34]. Danner et al. [15] investigated the effect of pH and salt concentration on the adhesion of MeFP-5 onto freshly cleaved mica; they reported that while adhesion was 3-fold higher at pH 2.6 vs. 5.5, they proposed that the decrease in adhesion was due to pH-dependent oxidation of L-dopa, suggesting a critical role of L-dopa in surface interactions and binding of the protein to a surface. In addition, they found that ionic strength of the potassium nitrate buffer solution had little to no effect on the interaction of the protein with the surfaces under study. These findings, while interesting in terms of adhesion and surface interactions of MeFP-5, shed little light on the conformation and structure of the protein in solution. Nonetheless, the lack of definitive confirmatory analysis of proposed secondary and tertiary structures of the protein allows for a continued examination of the possible surface interactions an open, extended conformation of the protein [14] might encounter. The aggregate charges for each domain designated on the MeFP5 protein (Figs. 2 and 8b, Table 2) was constructed using the pKa values of amino acid residues [35,36] present in MeFP-5 [15]. At a pH of 5.5 (the observed pH at the byssal-substrate interface and the pH of the incubation solution), MeFP-5 contains both positively charged (histidine, lysine and arginine) and negatively charged (phosphoserine and glutamic acid) residues, and the aggregate charge of each domain was computed by adding the charges of the individual residues together. From Table 2 it is clear that the MeFP5 protein can have domains where the aggregate charge would allow for electrostatic attraction and coulombic interactions over a surface composed of mixed charges. The surface of the HY80 steel, being composed primarily of Fe (95.20e96.45 wt%) can have a mixture of iron oxides present;

Table 2 Aggregate charges for each domain on MeFP-5 using pKa values of peptidyl amino acids. Domain 1

Domain 2

Domain 3

Domain 4

Domain 5

Amino Acid

pH ¼ 5.5

Amino Acid

pH ¼ 5.5

Amino Acid

pH ¼ 5.5

Amino Acid

pH ¼ 5.5

Amino Acid

pH ¼ 5.5

P-Ser-1 P-Ser-2 Glu-3 Glu-4 Dopa-5 Lys-6 Gly-7 Gly-8 Dopa-9 Dopa-10 Pro-11 Gly-12 Asn-13 Ala-14 Dopa-15 His-16 Dopa-17 P-Ser-18 Gly-19 Gly-20 P-Ser-21 Dopa-22 His-23 Total Charge

e e e e 0 þ 0 0 0 0 0 0 0 0 0 þ 0 e 0 0 e 0 þ 3

Gly-24 P-Ser-25 Gly-26 Dopa-27 His-28 Gly-29 Gly-30 Dopa-31 Lys-22 Gly-33 Lys-34 Dopa-35 Dopa-36 Gly-37 Lys-38 Total Charge

0 e 0 0 þ 0 0 0 þ 0 þ 0 0 0 þ þ3

Ala-39 Lys-40 Lys-41 Dopa-42 Dopa-43 Dopa-44 Lys-45 Dopa-46 Lys-47 Asn-48 P-Ser-49 Total Charge

0 þ þ 0 0 0 þ 0 þ 0 e þ3

Gly-50 Lys-51 Dopa-52 Lys-53 Dopa-54 Leu-55 Lys-56 Lys-57 Ala-58 Arg-59 Lys-60 Dopa-61 His-62 Arg-63 Total Charge

0 þ 0 þ 0 0 þ þ 0 þ þ 0 þ þ þ8

Lys-64 Gly-65 Dopa-66 Lys-67 Dopa-68 Dopa-69 Gly-70 Gly-71 P-Ser-72 P-Ser-73 Total Charge

þ 0 0 þ 0 0 0 0 e e 0

418

D.C. Hansen et al. / Electrochimica Acta 301 (2019) 411e420

Fig. 9. (Top) 3-dimensional model of goethite (a-FeOOH) along (100) plane and orthorhombic unit cell (inset); (Bottom) 3-dimensional model of hematite (ɣ-Fe2O3) along (001) plane and rhombohedral unit cell (inset).

models of the passive oxide layers on iron have been described previously [37]. An outer layer of the passive film can contain aFeOOH (goethite) [37,38] which has an isoelectric point (IEP) of 5.6e10.2 or g-Fe2O3 (hematite) which has an IEP of 3.0e9.5; and an inner layer of Fe3O4 (magnetite) which has an IEP of 5.1e9.9 [37e39]. Therefore at the pH of 5.5 all three oxides/oxy-hydroxides can be present and can either be negatively or positively charged [39]. Büchler et al. [40] determined that a hydroxide layer can form on the protective iron oxide film, although these results were based upon potential scan and potential step measurements on pure iron. In contrast, work by Davenport et al. [41,42] indicate that the passive film is either composed of ɣ-Fe2O3/Fe3O4 or a spinel related to ɣ-Fe2O3 and Fe3O4. Therefore, since it was not determined on the HY80 substrates tested in the present study what oxide/hydroxide species were present, a theoretical composition was proposed for the modeling of the MeFP-5 interaction at the metal-protein interface. For the purposes of the ensuing discussion, the theoretical interaction of the MeFP-5 protein with either goethite or hematite will be considered, as they could compose the outermost layers of the passive oxide film. In order to determine a possible orientation and adsorption scenario of the MeFP-5 protein onto the HY80 surface, the spacing of Fe and O atoms in a-FeOOH and g-Fe2O3 oxide matrices along the planes (100, 001 and 111) were simulated and measured. As the

Table 3 Iron atom and oxygen atom spacing on simulated goethite (100) and hematite (001,111) planes. Fe Atom Spacing Goethite (g-FeOOH)

Average Length (Å)

Range (Å)

(100) Hematite (g-Fe2O3) (001)

5.23

4.83e5.02

5.1

5.04e5.12

Oxygen Atom Spacing Goethite (g-FeOOH) (100) Hematite (g-Fe2O3) (111)

Average Length (Å)

Range (Å)

2.41

1.70e2.95

2.93

2.73e3.07

crystalline structure of goethite is in an orthorhombic lattice structure [43], it was necessary to construct a repeating unit cell in the (100) plane (Fig. 9a and inset) which would be representative of the oxyhydroxide for spatial measuring purposes; the same was done for hematite, since hematite is classified as a rhombohedral lattice system, (Fig. 9b and inset). These constructs then enabled the determination of Fe atom and oxygen atom spacing, respectively. The distance between the Fe atoms in the goethite (100) plane ranged from 4.83 to 5.02 Å (Table 3). The spacing of the Ldopa amino acid residues along the biopolymer was also determined (measured at the oxygen atom in the para position on the benzene ring, Table 4). Hematite can be derived from the conversion of goethite via the removal of water and cation rearrangement, with three unit cells of goethite giving rise to one unit cell of hematite yielding a highly ordered mosaic of hematite crystallites [44]. The calculated distances between the Fe atoms in the hematite oxide along the (001) plane was 5.04e5.12 Å (Table 3). For the spacing of the oxygen atoms, in both goethite (100) and hematite (111) the range was 1.70e2.95 and 2.73e3.07 Å, respectively. From the data it can be surmised that the spacing of the L-dopa residues in domain 3 (Table 4) can accommodate the Fe atoms for complex formation within the goethite (100) and hematite (001) planes. The atomic spacing distances for iron and oxygen for the goethite and hematite used in the current modeling are similar to the values previously reported [43e47]. It is tempting to speculate on which domain might dominate in the interaction sequence of the MeFP-5 with the HY80 surface

Table 4 L-dopa spacing along MeFP-5 protein sequence. Distance measured between oxygen atom at the para-position on the benzene ring. L-Dopa Residue Spacing on MeFP-5 Domain

Average L-Dopa Spacing (Å)

Range (Å)

Overall 1 2 3 4 5

11.42 10.41 9.78 8.77 12.63 12.77

5.78e19.56 5.78e15.25 6.27e14.62 4.97e11.38 8.79e16.47 9.09e16.05

D.C. Hansen et al. / Electrochimica Acta 301 (2019) 411e420

419

Fig. 10. (L) Close up image of L-dopa and lysine residues in domain 3 where the ε-amine extends out from protein. (R) 3-dimensional model of MeFP-5 approaching a goethite surface; L-dopa functional groups in red, lysine functional groups in green. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

during the incubation of the protein solution on the metal substrate. In an attempt to determine how the MeFP-5 molecule would approach and bind to a goethite or hematite layer, the 3dimensional model of the protein was oriented in space adjacent to either layer and visualized in the 3-dimensional software program. In Fig. 10a, a ball and stick model of MeFP-5 (L-dopa functional groups are in red and lysine functional groups are in green) approaching the surface of a goethite surface is presented. In Fig. 10b, a close up of the MeFP-5 protein and the L-dopa and lysine residues in domain 3 indicates that for the lysine residue at position #47 (Table 2), the ε-amine group extends out from the protein in such a fashion that it could function as an initial site for coulombic interaction with the hydrated iron oxide. In terms of aggregate charge, domain 4 has the highest positive total charge of þ8 (Table 2) which would suggest that this portion of the protein would experience the strongest electrostatic attraction to the oxide surface; domain 3 and 2, while having the same aggregate charge (þ3) is still less than domain 4. However, domain 3 also contains the highest concentration of L-dopa and lysine residues (4 of each residue over a span of 11 residues total). Perhaps it is this domain that could achieve the highest binding to the oxide through the formation of monodentate complexes between the Ldopa and goethite oxyhydroxide [23] and the involvement of the lysine primary amine to form a metal-ammine through a nucleophilic substitution [33]; both reactions would result in the dehydration of the goethite surface, resulting in a more stable passive Fe2O3 layer at the substrate surface [48]. Adsorption of organic ligands like MeFP-5 to iron oxide surfaces can be described by a ligand exchange reaction; structural iron ions at the oxide water interface are partly coordinated by oxygen or hydroxyl ions of the crystal lattice and partly by adsorbed water or hydroxyl anions [49]. Adsorption of ligands and formation of inner sphere surface complexes involves replacement of surface hydroxyl groups or water by a ligand exchange reaction [50]. Since the specific type of iron oxide/hydroxide present on the HY80 surface in this present study has not been definitively established, how the composition of the surface oxide/hydroxide would affect the ability of the MeFP-5 to coordinate to the passive

film and provide the observed corrosion inhibition needs to be considered. Surface complexation of Fe (III) on iron oxide surfaces by “hard ligands” is evidenced by high affinities of ligands with oxygen donor atoms for adsorption [51]. Examples of these bidentate hard ligands with high affinity for Fe(III) are catecholate and hydroxamate functional groups of bacterial siderophores, citric acid, ascorbic acid, salicylic acid, oxalic acid [50] and of course, the L-dopa containing polyphenolic proteins [52]. Therefore, due to the metal complexation through the ligand exchange reaction, and the formation of inner sphere complexes with a high affinity for adsorption on iron oxides/hydroxides [50], it is readily apparent that the composition of the passive film on iron (oxide vs. hydroxide) would have little effect on the adsorption of the mussel protein and concomitant corrosion inhibition of the iron surface. Other residues and adjacent domains along the MeFP-5 polymer could provide additional binding sites resulting in a strongly adhered, integrated metal-polymer film. Subsequent treatment of the bulk solution of MeFP-5 above the tightly bound MeFP-5 film with a catechol oxidase or other catalyst would result in multiple cross-linked adlayers. Indeed, adsorption of a MeFP-1 protein onto highly ordered pyrolytic graphite (HOPG) with subsequent treatment with mushroom tyrosinase resulted in minimal displacement of the adsorbed and cross-linked film with an AFM cantilever [30]. 4. Conclusions The FTIR analysis and EDS data presented in this study support the conclusion that the adsorption of the MeFP-5 protein involves the functional groups of the amino acid residues of lysine and Ldopa. A synergy was found to exist between lysine and L-dopa in the interaction of the mussel adhesive protein at the HY80 metaloxide interface and that these amino acids are involved in the complexation and dissolution of iron. This is dependent on solution chemistry and pH, and the pH of the incubation solution plays a role in the electrostatic interaction and adsorption of the MeFP-5 onto an iron oxide film. The results also indicate that treatment of the adsorbed MeFP-5 with a catechol oxidase enzyme results in a decrease in the wt% signal of iron, possibly due to an increase in the

420

D.C. Hansen et al. / Electrochimica Acta 301 (2019) 411e420

volume of the adsorbed and cross-linked amount of MeFP-5 on the metal substrate. Five domains have been identified where cumulative charges present on the protein polymer could play a role in electrostatic attraction and coulombic interactions to the metal oxide surface. Three-dimensional modeling of the goethite and hematite surfaces that are present on the treated HY80 surface along with a model of the MeFP-5 protein indicates that the molecular spacing of the L-Dopa residues align well with the iron atoms in the (001) plane of both oxide layers. A simulation of a possible orientation of the protein to either oxide surface indicates that one possible initial binding site involves the ε-amine of lysine. While further work is required to make quantum modeling calculations of the surface oxide-protein interactions, these results suggest this particular mussel adhesive protein could be used as a model to design a polymer that can meet the requirements for a low volatile organic content, aqueously soluble and environmentally benign corrosion inhibitor. Acknowledgements The authors wish to thank Dr. Airan Perez, Program Manager, of the Office of Naval Research for funding this effort under contract N00014-14-1-0537. References [1] E. A Chernyshov, A.D. Romanov, E.A. Romanova, High strength shipbuilding steels and alloys, Metallurgist 60 (2016) 186. [2] L.M. Frenzel, A comparison of surface preparation for coatings by water jetting and abrasive blasting, in: WJTA Conference, 1999. Houston, Texas. [3] H. Teimourian, Technical and economical comparison of waterjet and abrasive blast methods to be used in de-coating and cleaning processes, in: WJTAIMCA Conference and Expo, 2013. Houston, Texas. [4] J.H. Waite, N. Holten-Andersen, S.A. Jewhurst, C.J. Sun, Mussel adhesion: finding the tricks worth mimicking, J. Adhes. 81 (2005) 297. [5] H.G. Silverman, F.E. Roberto, Understanding mussel adhesion, Mar. Biotechnol. 9 (2007) 661. [6] J.H. Waite, The formation of mussel byssus: anatomy of a natural manufacturing process, Results Probl. Cell Differ. 19 (1992) 27. [7] B.P. Lee, P.B. Messersmith, J.N. Israelachvili, J.H. Waite, Mussel-inspired adhesives and coatings, Annu. Rev. Mater. Res. 41 (2011) 99. [8] S.W. Taylor, J.H. Waite, M.M. Ross, J. Shabanowitz, D.F. Hunt, trans-2,3-cis-3,4dihydroxyproline in the tandemly repeated consensus decapeptides of an adhesive protein from Mytilus edulis, J. Am. Chem. Soc. 116 (1994) 10803. [9] D.C. Hansen, S. Dexter, J. Waite, The inhibition of corrosion of S30403 stainless steel by a naturally occurring catecholic polymer, Corros. Sci. 37 (1995) 1423. [10] F. Zhang, J. Pan, P. Claesson, Electrochemical and AFM studies of mussel adhesive protein (MeFP-1) as corrosion inhibitor for carbon steel, Electrochim. Acta 56 (2011) 1636. [11] W.F. Nelson, D.C. Hansen, Investigations of mussel adhesive proteins as flash rust inhibitors, J. Electrochem. Soc. 163 (2016) C553. [12] G.P. Maier, M.V. Rapp, J.H. Waite, J.N. Israelachvili, A. Butler, Adaptive synergy between catechol and lysine promotes wet adhesion by surface salt displacement, Science 349 (2015) 628. [13] L.M. Rzepecki, S.S. Chin, J.H. Waite, M.F. Lavin, Molecular diversity of marine glues: polyphenolic proteins from five mussel species, Mol. Mar. Biol. Biotechnol. 1 (1991) 78. [14] J.H. Waite, X.X. Qin, Polyphenolic phosphoprotein from the adhesive pads of the common mussel, Biochemistry 40 (2001) 2887e2893. [15] E.W. Danner, Y. Kan, M.U. Hammer, J.N. Israelachvili, J.H. Waite, Adhesion of mussel foot protein mefp-5 to mica: an underwater superglue, Biochemistry 51 (2012) 6511. [16] K. Inoue, J.H. Waite, M. Matsuoka, S. Odo, S. Harayama, Interspecific variations in adhesive protein sequences of Mytilus edulis, M. galloprovincialis and M. trossulus, Biol. Bull. 189 (1995) 370. [17] A. Sumayya, C.Y. Panicker, H.T. Targhese, B. Harikumar, Vibrational spectroscopic studies and ab initio calculations of L-glutamic acid 5-amide, Rasayan J. Chem. 1 (2008) 548. [18] G. Davidson, K.B. Dillon, B.E. Mann, D.W.H. Rankin, H.E. Robertson, Spectroscopic properties of inorganic and organometallic compounds, Royal Soc. Chem. 34 (2001) 330. [19] K. Rahmelow, W. Hubner, T. Ackerman, Infrared absorbances of protein side chains, Anal. Biochem. 257 (1998) 1. [20] D.C. Hansen, E. McCaffety, C.W. Lins, J.J. Fitzpatrick, An FT-IR investigation of

parabactin adsorbed onto aluminum, Appl. Surf. Sci. 84 (1995) 85. [21] S. Krimm, J. Bandekar, Vibrational spectroscopy and conformation of peptides, polypeptides and proteins, Adv. Protein Chem. 38 (1986) 181. [22] N. Kitadai, T. Yokoyama, S. Nakashima, In situ ATR-IR investigation of L-lysine adsorption on monmorillonite, J. Colloid Interface Sci. 338 (2009) 395. [23] H.A. Al-Abadeh, Review of the bulk and surface chemistry of iron in atmospherically relevant systems containing humic-like substances, Royal Soc. Chem. Adv. 5 (2015) 45785. [24] A. Barth, Infrared spectroscopy of proteins, Biochim. Biophys. Acta 1767 (2007) 1073. [25] H. Gulley-Stahl, P.A. Hogan, S.J. Wall, A. Buhrlage, H.A. Bullen, Surface complexation of catechol to metal oxides: an ATR-FTIR, adsorption and dissolution study, Environ. Sci. Technol. 44 (2010) 4116. [26] V. Serrano, W. Liu, S. Franzen, An infrared spectroscopic study of the conformational transition of elastin-like polypeptides, Biophys. J. 93 (2007) 2429. [27] C. Fant, J. Hedlund, F. Hook, M. Berglin, E. Fridell, H. Elwing, Investigation of adsorption and cross-linking of a mussel adhesive protein using attenuated total internal reflection Fourier transform infrared spectroscopy (ATR-FTIR), J. Adhes. 86 (2010) 25. [28] R. Nishimoto, S. Fukuchi, G. Qi, M. Fukushima, T. Sato, Effects of surface Fe(III) oxides in a steel slag on the formation of humic-like dark-colored polymers by the polycondensation of humic precursors, Colloids Surf., A 418 (2013) 117. [29] K. Kanaya, S. Okayama, Penetration and energy-loss theory of electrons in solid targets, J. Phys. D Appl. Phys. 5 (1972) 43. [30] D.C. Hansen, S.G. Corcoran, J. H Waite, Enzymatic tempering of a mussel adhesive protein, Langmuir 14 (1998) 1139. [31] D.C. Hansen, G.W. Luther III, J.H. Waite, The adsorption of the adhesive protein of the blue mussel Mytilus edulis L onto type 304L stainless steel, J. Colloid Interface Sci. 168 (1994) 206. [32] L.A. Burzio, J.H. Waite, Cross-linking in adhesive quinoproteins: studies with model decapeptides, Biochemistry 39 (2000) 11147. [33] I. Gallardo, J. Pinson, N. Villa, Spontaneous attachment of amines to carbon and metallic surfaces, J. Phys. Chem. B 110 (2006) 19521. [34] Z. Qin, M.J. Buehler, Molecular mechanics of mussel adhesion proteins, J. Mech. Phys. Solid. 62 (2014) 19. [35] Carey, A. Francis, Robert M. Giuliano, Organic Chemistry, ninth ed., McGrawHill, 2014. [36] L. Stryer, Protein structure and function, in: Biochemistry, fourth ed., W.H. Freeman and Co., New York, 1995, p. 23. Ch. 2. [37] E. McCaffety, Models for the passive oxide film on iron, in: Introduction to Corrosion Science, first ed., Springer NY, 2010, p. 225. Ch.9. € ssbauer spectroscopy study of the potential [38] J. Eldridge, R.W. Hoffman, A Mo dependence of passivated iron films, J. Electrochem. Soc. 136 (1989) 955. € ssbauer study of the passive oxide film on iron, [39] W.E. O'Grady, Mo J. Electrochem. Soc. 127 (1980) 555. €hni, Iron passivity in borate buffer, [40] M. Büchler, P. Schmuki, H. Bo J. Electrochem. Soc. 145 (1998) 609. [41] A.J. Davenport, M. Sansone, High Resolution in Situ XANES investigation of the nature of the passive film on iron in a pH 8.4 borate buffer, J. Electrochem. Soc. 142 (1995) 725. [42] A.J. Davenport, L.J. Oblonsky, M.P. Ryan, M.F. Tone, The structure of the passive film that forms on iron in aqueous environments, J. Electrochem. Soc. 147 (2000) 2162. [43] M. Kosmulski, in: A.T. Hubbard (Ed.), Chemical Properties of Material Surfaces, Surfactant Science Series, vol. 102, Marcel Dekker, New York, 2001. [44] R.M. Cornell, U. Schwertmann, Crystal structure, in: The Iron Oxides: Structure, Properties, Reactions, Occurrences and Uses, second ed., Wiley-VCH, Weinheim, Germany, 2003. [45] S.K. Ghose, G.A. Waychunas, T.P. Trainor, P.J. Eng, Hydrated goethite (aFeOOH) (100) interface structure: ordered water and surface functional groups, Geochem. Cosmochim. Acta 74 (2010) 1943. [46] M. Wang, H. Hu, Q. Chen, G. Ji, FT-IR, XPS, ad DFT study of adsorption mechanism of sodium acetohydroxamate onto goethite or hematite, Chin. J. Chem. Phys. 29 (2016) 308. [47] M.P. Ryan, R.C. Newman, G.E. Thompson, An STM study of the passive film formed on iron in borate buffer solution, J. Electrochem. Soc. 142 (1995) L177. [48] B. MacDougall, M.J. Graham, in: P. Marcus (Ed.), Growth and Stability of Passive Films in: Corrosion Mechanisms in Theory and Practice, second ed., Marcel Dekker, New York, 2002, p. 192. [49] W. Stumm, J.J. Morgan, The surface chemistry of oxides, hydroxides and oxide minerals, in: Aquatic Chemistry: Chemical Equilibria and Rates in Natural Waters, third ed., John Wiley & Sons, New York, 1996, p. 533. [50] S.M. Kraemer, Iron oxide dissolution and solubility in the presence of siderophores, Aquat. Sci. 66 (2004) 3. [51] R.C. Hider, Z.D. Liu, H.H. Khodr, Metal chelation of polyphenols, Methods Enzymol. 335 (2001) 190. [52] S.W. Taylor, D.B. Chase, M.H. Emptage, M.J. Nelson, J.H. Waite, Ferric ion complexes of a DOPA-containing adhesive protein from Mytilus edulis, Inorg. Chem. 35 (1996) 7572.