Optimization and Enhancement of the Peroxidase-like Activity of Hemin in Aqueous Solutions of Sodium Dodecylsulfate

Iron porphyrins play several important roles in present-day living systems and probably already existed in very early life forms. Hemin (= ferric protoporphyrin IX = ferric heme b), for example, is the prosthetic group at the active site of heme peroxidases, catalyzing the oxidation of a number of different types of reducing substrates after hemin is first oxidized by hydrogen peroxide as the oxidizing substrate of the enzyme. The active site of heme peroxidases consists of a hydrophobic pocket in which hemin is embedded noncovalently and kept in place through coordination of the iron atom to a proximal histidine side chain of the protein. It is this partially hydrophobic local environment of the enzyme which determines the efficiency with which the sequential reactions of the oxidizing and reducing substrates proceed at the active site. Free hemin, which has been separated from the protein moiety of heme peroxidases, is known to aggregate in an aqueous solution and exhibits low catalytic activity. Based on previous reports on the use of surfactant micelles to solubilize free hemin in a nonaggregated state, the peroxidase-like activity of hemin in the presence of sodium dodecyl sulfate (SDS) at concentrations below and above the critical concentration for SDS micelle formation (critical micellization concentration (cmc)) was systematically investigated. In most experiments, 3,3′,5,5′-tetramethylbenzidine (TMB) was applied as a reducing substrate at pH = 7.2. The presence of SDS clearly had a positive effect on the reaction in terms of initial reaction rate and reaction yield, even at concentrations below the cmc. The highest activity correlated with the cmc value, as demonstrated for reactions at three different HEPES concentrations. The 4-(2-hydroxyethyl)-1-piperazineethanesulfonate salt (HEPES) served as a pH buffer substance and also had an accelerating effect on the reaction. At the cmc, the addition of l-histidine (l-His) resulted in a further concentration-dependent increase in the peroxidase-like activity of hemin until a maximal effect was reached at an optimal l-His concentration, probably corresponding to an ideal mono-l-His ligation to hemin. Some of the results obtained can be understood on the basis of molecular dynamics simulations, which indicated the existence of intermolecular interactions between hemin and HEPES and between hemin and SDS. Preliminary experiments with SDS/dodecanol vesicles at pH = 7.2 showed that in the presence of the vesicles, hemin exhibited similar peroxidase-like activity as in the case of SDS micelles. This supports the hypothesis that micelle- or vesicle-associated ferric or ferrous iron porphyrins may have played a role as primitive catalysts in membranous prebiotic compartment systems before cellular life emerged.


Determination of the cmc of SDS With Pinacyanol Chloride
The cmc of SDS was determined following published protocols.S1-S4 In short, UV−vis spectral changes of pinacyanol chloride were followed with variation of the concentration of SDS.The cmc value was determined in HEPES buffer solution (25 mM, 50 mM, or 100 mM), pH = 7.2, either (i) in the absence or (ii) in the presence of hemin (250 nM) and TMB (300 µM).This comparison was made to find out whether the presence of hemin and TMB affect the cmc value of SDS.The total concentration of pinacyanol chloride was always 5.0 µM, and the total volume 1.0 mL.All spectra were recorded at T = 25 °C with a JASCO V-670 UV−vis−NIR spectrophotometer (using a quartz cuvette of pathlength 1.0 cm).
The cmc value was taken as the SDS concentration at which with increasing SDS concentration A606 started to increase (indicating initiation of pinacyanol chloride binding to the first micelles formed).
As shown in Figures S4 -S6, the cmc of SDS showed dependence on the HEPES concentration and the presence of hemin and TMB resulted in a lowering of the cmc values.For a comparison, the cmc was also determined for a 100 mM sodium phosphate buffer solution, see Figure S7A.The peroxidase-like activity of hemin against TMB in the presence of the phosphate buffer solution was found to be much lower than in the presence of the HEPES buffer solution of the same pH = 7.2, see   The initial rate of CTC formation was higher at [SDS] = 0.4 or 0.8 mM than at [SDS] = 1.4 or 10 mM.In the presence of 100 mM sodium phosphate solution (pH = 7.2) at 0.4 or 0.8 mM SDS, vin(CTC) was ≈15 times lower than in the presence of 100 mM HEPES buffer solution (pH = 7.2) at the optimal SDS concentration of 2.0 mM (≈20 nM s −1 vs. ≈300 nM s −1 , see Figure 6).

Effect of SDS on the Stability of Aqueous Hemin Solutions Kept Inside Polystyrene Cuvettes
On the basis of previous findings about the adsorption of hemin from aqueous solution onto plasticware, S6 we investigated whether the presence of SDS has an influence on this adsorption process, and whether the SDS concentration dependence of the peroxidase-like activity of hemin measured with TMB (Figure 6) could be explained by differences in the rate and/or extent of hemin adsorption to the polystyrene cuvettes used for the activity measurements (see section 2.4).For this, aqueous 100 mM HEPES buffer solutions of pH = 7.2 containing hemin (250 nM) and different amounts of SDS (up to 3.0 mM) were added to polystyrene cuvettes, and the UV−vis absorption spectra and the activity of these solutions were measured from time to time during an incubation at RT of up to 2 h, see Figure S10.In all cases, the Soret band intensity of hemin at λ = 396 nm, A396, decreased with storage time at similar rate (Figure S10A), and in all cases, vin(CTC) decreased with storage time with similar rate (Figure S10B).Therefore, we conclude that hemin adsorption on the polystyrene cuvette wall was not the reason for the variation of vin(CTC) with SDS concentration shown in Figure 6.(0.3 mM) were added after the indicated time, and the initial change in A652 was measured and then converted into units of nM CTC formed per second (nM s -1 ) by using ε652 (CTC) = 39,000 M −1 cm −1 .S5 Comment: Based on literature, S6 the decrease in A396 and in vin with storage time can be ascribed to the adsorption of hemin onto the inner polystyrene wall of the cuvettes used.4) after a reaction time of 60 min.The CTC yields were calculated by using ε652 (CTC) = 39,000 M −1 cm −1 .S5 The absorption spectra shown were measured by withdrawing volumes of 350 µL from the reaction mixtures and placing the aliquots inside a quartz cuvette of pathlength 0.1 cm, i.e., neither dilution nor work-up of the reaction mixtures was required.
Results: The yields of the charge transfer complex (CTC) after 60 min were 87 % for (1), 94 % for (2), 1.6 % (for 3), and 0.1 % for (4).).The progress of the reaction was followed for 60 s by recording the entire UV−vis absorption spectrum of the reaction mixture every 2 s and monitoring A652 from which vin(CTC) was calculated using ε652 (CTC) = 39,000 M −1 cm −1 .S5 Result: Inactivation of hemin by hydrogen peroxide occurred in a period of less than one minute, and the degree of hemin inactivation remained approximately the same for all incubation times tested.The UV−vis absorption spectra were measured every 3 s for 60 s.Results: For all three SDBS concentrations used, bonds cantered around 370, 650, and 900 nm developed with time, in agreement with the formation of the CTC, see Figure 2. Depending on the SDBS concentration, however, there were differences in (i) the rate at which the intensity of the three bands increased, (ii) the ratio of A652 to A900, (iii) the situation at λ ≈ 850 nm in the case of 2.0 mM SDBS (reproducibly observed for all three measurements at 2.0 mM SDBS, with a kink in the A652 vs. time line, see Figure S20), and (iv) the appearance of a small peak at λ ≈ 460 nm.

Vesicle Dispersions
The SDS/dodecanol vesicle dispersions prepared by polycarbonate extrusion are only kinetically stable and not thermodynamically.Therefore, checking the reproducibility of any type of experiment with vesicular dispersions is important.We measured the peroxidase-like activity of hemin under the "optimal conditions" towards TMB as reducing substrate by using five different batches of prepared SDS:dodecanol (3:7) vesicles.The determined initial rates of CTC formation for the five reaction mixtures are given in Figure S27.The values of vin(CTC) varied between ≈300 and ≈480 nM s -1 ; see also Figure S26A (entry "all").As a conclusion, it is recommended to use for one comparative set of measurements always the same batch of vesicles.This was done for the data shown in Figure S28 (effect of added L-His on the activity of hemin in the SDS/dodecanol vesicle dispersion).
Figure S7.(A) Determination of the cmc of SDS with pinacyanol chloride (5 µM) in the presence of 100 mM sodium phosphate buffer solution, pH = 7.2 at T = 25 °C, either (i) in the absence of hemin or TMB (■), (ii) in the presence of 250 nM hemin (•), or (iii) in the presence of 250 nM hemin and 0.3 mM TMB (▲).The SDS concentration was varied between 0.2 and 1.4 mM (in steps of 0.2 mM).Result: In the presence of hemin and TMB, the cmc of SDS was about 0.6 − 0.8 mM.(B) Peroxidase-like activity of hemin against TMB as reducing substrate in the presence of SDS in phosphate buffer solution at pH = 7.2.Reaction conditions: [phosphate] = 100 mM; [hemin] = 250 nM; [TMB] = 0.3 mM; [H2O2] = 0.3 mM; RT.The absorption spectrum of the reaction mixture was recorded every 3 s for a total of 90 s.A562 is plotted against reaction time as a measure of the formation of the CTC.Using ε652 (CTC) = 39,000 M −1 cm −1 , S5 the initial rate of CTC formation, vin(CTC), in the presence of 0.4 or 0.8 mM SDS was calculated to vin(CTC) ≈ 20 nM s −1 .Results:

Figure S8 .Figure S9 .
Figure S8.Effect of SDS on the initial rate of CTC formation from TMB as reducing substrate, monitored by recording A652 as a function of reaction time (N = 3).Reaction conditions: [HEPES] = 100 mM; pH = 7.2; [SDS] = 0.5, 1.5, 2.0, 2.5, or 4.0 mM; [hemin] = 250 nM; [TMB] = 0.3 mM;[H2O2] = 0.3 mM; RT.Results: For the chosen SDS concentrations, the fastest initial increase in A652 was for 2.0 mM SDS, correlating with the determined cmc of SDS in the presence of 100 mM HEPES (1.9 − 2.0 mM, see FigureS4).The TMB conversion after 130 s (arrow) was ≈38 µM, i.e., ≈12.5%.The reaction was low at 4.0 mM SDS.At 2.5 mM SDS, the A652 vs. time curve showed a kink after ≈70 s, the CTC formation becoming faster.There was no indication of a rapid leveling-off of the CTC formation as observed in the absence of SDS, please compare with Figure4.

Figure S12 .
Figure S12.Effect of L-His on the initial rate of CTC formation from TMB as reducing substrate with hemin as catalyst in the absence of SDS, monitored by recording A652 as a function of reaction time.Reaction conditions: [HEPES] = 100 mM; pH = 7.2; [hemin] = 250 nM; [L-His] = 4.0, 8.0, 20, or 50 mM; [TMB] = 0.3 mM; [H2O2] = 0.3 mM; RT; N = 3. Results: A652 increased with time with a trend to level off.The highest initial rate of CTC formation within this set of experiments was ≈ 170 nM s −1(in the presence of 20 mM L-His), which is significantly lower than in the case of the optimal system in the presence of 2.0 mM SDS (8.0 mM L-His: vin(CTC) ≈ 850 nM s -1 ), see Figure9aand FigureS11.

Figure S14 .
Figure S14.TMB concentration dependence of the initial rate of CTC formation from TMB as reducing substrate and hemin as a catalyst in the presence of SDS and L-His by recording A652 as a function of reaction time.Due to the non-linear initial increase of A652 with time, vin(CTC) was determined from the changes occurring after 25 s using ε652 (CTC) = 39,000 M −1 cm −1 .S5 .Reaction conditions: [HEPES] = 100 mM; pH = 7.2; [SDS] = 2.0 mM; [hemin] = 5 nM; [L-His] = 8.0 mM; [TMB] = 0, 50, 75, 100, 200, 300, and 400 µM; [H2O2] = 1.0 mM; N = 3; RT.The UV−vis absorption spectra of the reaction mixtures were recorded every 2 s for 60 s.For the sake of clarity, only one measurement is shown for each TMB concentration.A plot of vin(CTC) vs. [TMB] is shown in Figure 12 with a fit of the experimental data to the Michaelis-Menten equation (using OriginPro, Version 2021; OriginLab Corporation, Northampton, MA, USA).The fit yielded vmax,app = 193 ± 16 nM s −1 , i.e., kcat,app = vmax,app / [Hemin] = 39 ± 3 s −1 , and KM,app = 253 ± 37 µM.Comment: The non-linear change of A652 with time indicates a complex kinetic behavior, possibly due to influences of the formed product on the reaction.

Figure S17 .
Figure S17.Peroxidase-like activity of hemin (5 nM) in 100 mM HEPES buffer solution (pH = 7.2) in the presence of 2.0 mM SDS, and 8.0 mM L-His, measured with TMB (0.3 mM) as reducing substrate and H2O2 as oxidizing substrate at RT. (A) For [H2O2] = 1.0, 3.0, and 7.0 the time progress is shown for up to 3 min.The reaction mixtures were prepared by mixing appropriate stock solutions of the different compounds in the following order: 1. HEPES; 2. SDS; 3. Hemin; 4. L-His; 5. TMB; 6. H2O2.Results: For high concentrations of H2O2, the increase of A652 with time was not linear.A652 leveled-off the earlier the higher the H2O2 concentration was.(B) Preincubation experiments with 3.0 mM H2O2.Except for TMB, all components were first incubated for either 1, 2, 3, 4, or 5 min at RT before a stock solution of TMB in DMSO was added to start the reaction (N = 3, except for 2 min incubation: N = 2).The progress of the reaction was followed for 60 s by recording the entire UV−vis

In 50 mM HEPES buffer solution, pH = 7.2 Figure S5.
Determination of the cmc of SDS in 50 mM HEPES buffer solution at T = 25 °C.(A)

cmc = 2.8 -2.9 mM. In 25 mM HEPES buffer solution, pH = 7.2 Figure S6.
Determination of the cmc of SDS in 25 mM HEPES buffer solution at T = 25 °C.(A)