Los organismos internacionales:evaluación y calidad educativa…21

The following account is based on Refs. 11±14. The Lowry assay involves two reactions. First, a protein-Cu2‡ _{complex forms. Six peptide bonds}

surround a central Cu2‡_{atom. The high-pH solvent (‰OH Š&0:1M;pH 13)}

induces protein denaturation. Loss of native structure precedes binding with Cu2‡ _{to form a type B protein-Cu}2‡ _{complex (Chapter 2). Protein}

denaturation at high pH also exposes tyrosine and tryptophan residues, which then ionize.

The second stage of the Lowry assay is a redox reaction with Folin- Ciocalteu reagent via two pathways. First, Mo6‡_/W6‡ _{reacts directly with}

amino acid side chains (histidine, cysteine, asparagine, tyrosine, trypto- phan). A high pH is not required for these reactions. Second, Cu2‡_mediates

the dehydrogenation of the polypeptide via metal ion±catalyzed oxidation. The electrons are transferred to Mo6‡_/W6‡_{, leading to a color change.}

Features of the Mo6‡_/W6‡_{reaction with Cu}2‡_{and protein are summarized}

in Table 1.

3.2. Metal Ion±Catalyzed Oxidation of Proteins

The Cu2‡ _{catalyzes the oxidative degradation of polypeptides. The process}

Cu3‡_{by chemical or electrochemical oxidation.}

Cu2‡_?Cu3‡_{‡ e; E}_{ˆ 0:63V vs: NHE* …1†}

Metal ion±catalyzed oxidation (MCO) of tetraglycine-Cu2‡ _{has been}

investigated. One proposal is that in the presence of a strong oxidant (Qx), e.g., sodium chloroiridate …NaIrCl2₆ †, tetraglycine-Cu2‡ _(designated

as RH22Cu2‡_{) is degraded in three stages: (a) RH22Cu}2‡ _{is oxidized to}

RH22Cu3‡ _{[Eq. (2)], (b) RH22Cu}3‡_{rearranges to (IIa) or (IIb), which is a}

dehydropeptide-Cu1‡ _{species [Eq. (3)], (c) compound IIb is hydrolyzed to}

diglycinamide, glycoxyglycine, and Cu1‡_{[Eq. (4)]. It has been suggested that}

the structure for IIb is probably an iminopeptide (Fig. 1).

Qxn‡‡ RH22Cu2‡…I† k_{? Qx}1 …n 1†‡_{‡ RH22Cu}3‡_{II† …2†}

RH22Cu3‡_{II† /}k2_{? ? R22Cu2‡…IIa† ‡ H‡}

/ ?R22Cu1‡…IIb† ‡ H‡ …3†

TABLE1 Some Important Reactions for the Lowry Assay

1. Mo6‡_/W6‡_{reacts with reducing agents via a one-electron [e.g., Fe…CN†}4

6 , Fe2‡,

Sn2‡_{] or two-electron transfer (ascorbic acid and peptides). Reactions with}

tryptophan and tyrosine may involve four-electrons per residue.

2. Reduction of Mo6‡_/W6‡_{proceeds rapidly under acidic conditions. Adjusting the}

pH from 1 to 10 leads to deprotonation followed by a slow structural rearrangement and 1.7-fold increase in color.

3. Cu2‡_{is not required for the reaction of Mo}6‡_/W6‡_{with nonpeptide reductants.}

4. The color yield is 3200 (+100) M 1_cm 1_{per electron transferred to Mo}6‡_/W6‡_.

5. Peptides without oxidizable side chains react with Mo6‡_/W6‡_{only if they can form}

a tetradentate peptide-Cu2‡_complex.

6. Each tetradentate Cu2‡_{complex transfers approximately two reducing equivalents}

to Mo6‡_/W6‡_{. With well-de®ned peptides there is a correlation between the color}

yield from the biuret and Lowry methods.

7. Color yield increases for polypeptides with oxidizable side chains.

8. Color yield decreases with the number of side chains with Cu2‡_{complexing ability}

(glutamate, aspartate).

9. Cu1‡_{is not involved in color formation.}

Source: Summarized from Ref. 14.

R22Cu1‡_{IIb† ‡ H}

2O ?Cu1‡

‡ diglycinamide ‡ glycoxyglycine …4†

With molecular oxygen as oxidant, reaction (2) would lead to the superoxide radical [Eq. (5)], which probably remains protein bound as a ternary complex (III). RH22Cu2‡_{I† ‡ O} 2 k? RH22Cu1 3‡…II† ‡O2 / ?O 222R22Cu1‡…III† ‡ H‡ …5† Intramolecular oxidation of (III) then generates Cu2‡_{-hydroperoxide [Eq.}

(6)]. O

222R22Cu1‡…III† ?RO222Cu2‡…IV† …6†

FIGURE 1 Suggested mechanism of Cu2‡-mediated dehydrogenation of peptides

leading to an iminopeptide or a dehydropeptide. (Adapted from Ref. 14.)

Formation of (IV) occurs via two-electron reduction of oxygen to produce a protein-bound hydroperoxide anion. Breakdown of the hydroperoxide (Fig. 1) accounts for the carbonyl compounds detectable using 2,4-dinitrophe- nylhydrazine (DNPH).

A superoxide radical may be involved in the initial oxidation of RH22Cu2‡ _{to RH22Cu}3‡ _{[Eq. (2)] provided that O}

2 is released from the

protein in Eq. (5). In summary, RH22Cu2‡ _{can be oxidized by strong}

oxidants (NaIrCl2

6, hydrogen peroxide, and presumably Mo6‡/W6‡). The

formation of RH22Cu3‡ _{can also be initiated by atmospheric oxygen and}

superoxide species …O

2 and RO222Cu2‡†. The RH22Cu3‡may also form via

the disproportionation of 2 moles of RH22Cu2‡_{to produce RH22Cu}3‡_and

RH22Cu1‡_.

Fragmentation of tetraalanine-Cu2‡_{by IrCl}2

6 produces alanylalanine

amide (HAla2NH2) and pyruvyl alanine (PyrAlaOH). These were identi®ed

by reacting with ninhydrin or DNHP and by analysis with high-voltage paper electrophoresis. Peptide fragments are also formed during the analysis of polyalanine by the Lowry method. In one study, the blue molybdate- tungstate complex was removed by adsorption with cross-linked poly- acrylamide (Bio-gel P). The eluted (colorless) product reacted with DNPH. Hydrazone derivatives formed were analyzed by proton nuclear magnetic resonance (NMR) and by gel ®ltration on Sephadex G-25. In this manner, the products of polyalanine MCO were identi®ed as Ala6or Ala8peptide

fragments. Tests showed that DNHP reacts with a-iminoisobutyric acid but not with dehydropeptide analogues such as N-acyldehydroalanine methyl ester. Therefore, MCO leads to an imino peptide and the dehydropeptide (Fig. 1). The initial oxidation involves electron abstraction from the lone- pair electrons on the peptide nitrogen rather than from the methylene group. According to Livitski et al. (12), the compound II is further oxidized as shown in Eq. (7). Hydrolysis of the dehydrogenated Cu2‡_{22R leads to Cu}2‡

[Eq. (8)] and not to Cu1‡_{as shown in Eq. (4).}

_R22Cu2‡_{II† ‡ Qx}…n†‡ k_{? Qx}1 …n 1†‡_‡_R22Cu3‡_?R22Cu2‡_{† …7†}

R22Cu2‡_{‡ H}

2O ?Cu2‡‡ diglycinamide ‡ glycoxyglycine …8†

Evidence for Eq. (8) comes from the deployment of 2.20_-biquinoline

(2,20_{DQ), which should form a purple complex with Cu}1‡_{. Addition of}

2,20_{DQ failed to detect the presence of Cu}1‡_{. Therefore, the copper ion}

probably cycles between oxidation states 2 ‡, 3 ‡, and 4 ‡ under strongly oxidizing conditions. Failure to detect free Cu1‡_{shows that the sequence of}

Eq. (8). The net reaction is shown in Eq. (9). Polypeptide Cu2‡_{‡ Qx}n‡_{‡ H}

2O

?Cu2‡_{‡ Qx}…n 2†‡_{‡ hexapeptide fragments …9†}

MCO reactions involving proteins are discussed further in Chapter 5. 3.3. Kinetics of the Lowry Protein Assay

The duration of the Lowry assay is 40 minutes. Proteins react with Cu2‡

(reagent C) in 10 minutes. A further 30 minutes is needed for the reaction with reagent E before A750readings are recorded. The speed of the Lowry

assay depends on one or more of the reactions described in Sec. 3.2. 1. The time course for the alkali denaturation of proteins can be

prolonged for stable proteins (e.g., lysozyme).

2. The oxidation of RH22Cu2‡ _{by atmospheric oxygen [Eq. (1)]}

occurs with the rate constant (k1) of 5.5 6 10 4(s 1). The time for

99.9% completion (5/k1) is 151 minutes.

3. Oxidation of RH22Cu2‡ _{by IrCl}

6 (and presumably by Mo6‡/

W6‡_{) occurs with a rate close to the diffusion-controlled limit.}

Reduction of Mo6‡_/W6‡ _{by simple reductants is nearly instanta-}

neous, being delayed only by the reagent mixing time.

4. Reduced tungstate-molybdate undergoes slow deprotonation and structural changes at high pH. At 258C this process takes about 30 minutes.

5. Rearrangement of RH22Cu3‡_{to form a radical species [Eq. (2)] is}

a slow process. With IrCl2

6 as oxidant, k2is 1.66 6 10 3(s 1) with

a 99.9% completion time of 36 minutes. Clearly, it is not possible to identify a single rate-limiting reaction for the Lowry assay of proteins.

A high reaction rate is essential for automated protein analysis. Anderson and Marshall (15) and also Huang et al. (16) adapted the Lowry method for continuous ¯ow analysis. The sample throughput was 30 per hour. 4. CALIBRATION FEATURES

Calibration graphs for the Lowry assay are usually curved. A linear response is obtained for simple compounds (Sn2‡_{, tyrosine, etc.) (14). One}

W6‡ _{is degraded at high pH with a half-life of about 8 seconds (1).}

Assuming this explanation is correct, the color yield during protein analysis re¯ects a balance between Mo6‡_/W6‡ _{decomposition by alkali and its}

reduction to form a blue complex. Inorganic reductants react rapidly with Mo6‡_/W6‡ _{before its degradation. Nonlinearity was also ascribed to the}

declining copper/protein ratio as the concentration of protein is increased. Attempts to improve the linearity by increasing the concentration of copper led to higher readings for the sample blank.

Readings of A750may be converted to protein concentration [P] using

a nonlinear graph. This practice emphasizes data points adjacent to the unknown value. By contrast, a linear graph gives equal weighting to all the experimental points. In chemical analysis, ``linearity is next to cleanliness.'' Most analysts feel a sense of relief where calibration data conform to a linear function;

DA750ˆ F‰PŠ …10†

where F is the slope of the calibration graph. The line described by Eq. (10) passes through the origin and hence

‰PŠ ˆ DA750=F …11†

Stauffer (17) ®tted Eq. (12) or its logarithmic form [Eq. (13)] to data from the Lowry assay.

A700ˆ o‰PŠF …12†

log A700ˆ F log‰PŠ ‡ w …13†

where F and o are constants and w ˆ log o. For a protein concentration range of 4±400 mg mL 1 _{a plot of log A}

700 versus log‰PŠ was linear. The

unknown protein concentration can be determined from Eq. (14).

‰PŠ ˆ 10…Y w†=F _14†

Notice that Y…ˆ log A750† is the value for the unknown sample. Coakley

and Jones (18) used reagent volumes three times higher than in the standard method.* Their calibration results were described by a hyperbolic curve.

* A 0.6-mL portion of protein standard solutions was reacted with 3.0 mL of Lowry reagent C.

After standing for 10 minutes, 0.3 mL of Folin-Ciocalteu regent was added. The A750readings

A750ˆ_{w P‰ Š ‡ F}‰ ŠP …15†

Equation (15) applies for within-cuvette BSA concentrations of 0.01± 0.77 mg mL 1_{and for A}

750values of 0.18±2.2.* The constants F and w were

determined by calibration. The protein concentration can be found using Eq. (16).

P

‰ Š ˆ_{1 wA}FA750

750 …16†

For routine use, Eq. (16) can be linearized using a double-reciprocal transformation. 1 A750ˆ F P ‰ Š‡ w …17†

Equation (17) allows a two-point calibration: (a) analyze the unknown sample with two protein standards and (b) determine the constants F and w from Eqs. (18) and (19).

F ˆy1 y2

x1 x2 …18†

where y1ˆ 1/A750 (1)and y2ˆ 1/A750 (2). Likewise, x1ˆ 1/ [P]1and x2ˆ [P]2.

The concentrations for the protein standards should be [P]1ˆ 0.1 mg mL 1

and [P]2ˆ 5±10 mg mL 1BSA. The intercept of Eq. (17) (w) is found from

averages for y1, y2and x1, x2.

w ˆ y Fx where

y ˆ…y1‡ y₂ 2† and x ˆ…x1‡ x₂ 2† …19† The previous treatment applies for a protein concentration between 0.1 and 10 mg mL 1_{. With protein concentrations below 0.2 mg mL} 1_{, a straightfor-}

ward calibration graph can be used with little loss of accuracy.

* Allowing for the 6.5 times dilution during the Lowry assay, a protein stock solution of 0.065±

5 mg mL 1_{leads to a ``within-cuvette'' concentration of 0.01±0.77 mg mL} 1_{. Investigators are}

5. INTERFERENCE COMPOUNDS

Interference compounds for the Lowry assay are either chelators or reducing agents. Chelators diminish color formation by sequestering Cu2‡_{. Reducing}

agents react with the Folin reagent to create extra color. Some interfering compounds act as both chelators and reducing agents. Dyes having an absorbance maximum near 600±750 nm are also interfering compounds. Peterson classi®ed interfering compounds into more than 13 classes comprising over 180 chemicals (Table 2).

Many of these compounds are encountered in raw and processed foods.* Ascorbic acid and other reducing compounds are found in foods of plant origin (fruits, juices, pastes, concentrates). Wines and certain beverages have high concentrations of phenols and related compounds. Carbohydrates (simple sugarsÐsucrose, glucose, and fructose) and poly- saccharides (pectin or starch) occur in foods including jams, preserves, and

TABLE2 Some Potential Interfering Compounds for the Lowry Assay

Biochemical classi®cation Food additives

1. Amine derivatives Acids and acidulants

2. Amino acids Amino acids

3. Buffers Colors

4. Chelating agents Dyes

5. Detergents (e.g., Triton X-100,

Tween) Sweeteners

6. Drugs Arti®cial antioxidants (BHA, BHT, etc.)

7. Hexosamines Starch

8. Lipids and fatty acids Polysaccharides

9. Miscellaneous compounds Sulfur dioxide, sul®tes

10. Cryoprotectants Uric acid

11. Polyvinylpyrrolidone Fe2‡

12. Nucleic acids 13. Organic solvents 14. Phenols and polyphenols 15. Polysaccharides

16. Reducing agents 17. Salts

18. Sugars

* However, the analyst may not be aware of the presence of interfering compounds in a given sample.

wheat products. Synthetic reductants, acidulants, and dispersants are just some of the other additives associated with foods. Low-molecular-weight interfering substances can be removed by dialysis. Another approach is to recover the protein from the surrounding solvent medium by TCA precipitation (Method 3). Where possible, sample pretreatment should be avoided for routine and high-throughput analysis. The effects of selected interferences on the Lowry assay are discussed in the following.

5.1. Buffers, Chelating Agents, and Detergents

Peters and Fouts (19) showed that BICINE [N,N-bis- (2-hydroxethyl) glycine] and HEPES [N-(2-hydroxyethylpiperazine-N-2-ethanesulfonic acid)] produce color in proportion to their concentration. Ethylene diamine tetraacetic acid (EDTA), tris-hydroxythethylaminomethane (Tris), TRI- CINE, citrate, and Triton X-100 also produced similar effects (20). These interfering compounds increase the absorbance value for the reagent blank. However, they reduce the color yield from proteins. These effects are attributed to complex formation with copper. Interference by buffer components can easily be corrected using the appropriate buffer solution as the reagent blank. Sodium phosphate buffer had no effect on the Lowry assay.

5.2. Carbohydrates

Reducing sugars (mainly hexoses) react with Cu2‡ _{and the Folin reagent.}

Nonreducing sugars (sucrose, oligosaccharides, polysaccharides) form complexes with Cu2‡_{. Tagatose, sucrose, and inulin interfered with the}

Lowry assay after exposure to hot alkali or acid (21). These solvents are routinely used to dissolve proteins. As well as reducing Cu2‡_{to Cu}1‡_{, sugars}

reduce tungstate-molybdate. The effects of simple sugars are signi®cant at concentrations above 1 mM. The order of effectiveness is fructose > sorbose > xylose > rhamnose > mannose > glucose (22).

Toldra (23) considered protein analysis in connection with the production of high-fructose syrup. Effective control of this process requires the determination of a-amylase and glucoamylase speci®c activity (hence protein concentration) in samples containing up to 30±40% sugar. A solution of 1% (w/v) glucose or maltose produced an A750 response

equivalent to that of 18.8 or 16.6 mg mL 1 _{BSA. Protein analysis is also}

necessary to determine the activity of fungal pectinases produced in submerged culture using high concentrations of pectin as inducer (24). Concentration of 0.1±1.0% (w/v) pectin interfered with both the classical and modi®ed Lowry assays. Calibration graphs showed a decrease in the

slope and increased intercept. Assay sensitivity decreased while the LLD increased with increasing pectin concentration (Chapter 1). The color with protein-free samples increased linearly with the amount of added pectin. These results imply complex formation between pectin and Cu2‡_{. The}

Bradford method (Chapters 6 and 7) coped better with samples containing < 0.5% (w/v) pectin. In our experience, pectin forms a gelatinous mass during the Lowry assay. To avoid this problem, samples are exposed 0.1 M CaCl2and then centrifuged before protein analysis. Berg (25) warned that

``anyone attempting to determine protein contamination in polysaccharide preparations by the Lowry procedure could be misled by positive results which may be attributed to the carbohydrate.''

5.3. Chlorophyll

Leaves, stems, and green fruit contain chlorophyll. Addition of chlorophyll (100 mg mL 1_{) to BSA (300 mg mL} 1_{) increased A}

750 values by 400% (26).

For accurate analysis, leaf protein must be separated from chlorophyll.* 5.4. Cryoprotectants, Sucrose, Glycerol, and

Polyhydroxyalcohols

Ethylene glycol, polyvinylpyrrolidone (PVP), and dimethyl sulfoxide (DMSO) are used to protect proteins against freeze damage. Glycerol, probably acting in a manner similar to sucrose, inhibits color formation. In the absence of copper, PVP reacts with Mo6‡_/W6‡_{, leading to a blue}

coloration (27). Glycerol (2±5%) interferes with the Lowry procedure (28) but DMSO has no effect.

Many foods and condiments contain very high concentrations of sucrose. Sucrose is also used for gradient ultracentrifugation. Sucrose by itself gives a positive response with the Lowry assay (29). However, protein analysis in the presence of sucrose leads to a reduced color yield. The interference becomes progressively worse with 0±65% sucrose. Effects can be reduced to tolerable levels (< 5% analytical error) by (a) diluting samples to < 10% sucrose or (b) doubling the concentration of copper in Lowry reagent B. Quadrupling the copper concentration led to an unstable reagent with a tendency to form colloidal copper (30).

* Leaf protein can be extracted with 0.1 N alkali. Next, precipitate the protein with 10% (w/v) TCA. A second approach is to treat freeze-dried leaf powder with acetone. This decolorized powder can be stored before analysis (this work is discussed in detail later).

Sucrose (and other polyhydroxy compounds) appear to complex with Cu2‡_{when monitored by ultraviolet absorbance spectrophotometry. In the}

absence of copper, sucrose or glycerol does not interfere with the tyrosine reaction with Folin-Ciocalteu reagent. Hydrolysis of sucrose by heat, alkali, or invertase generates fructose and glucose, which affect the Lowry assay as reducing sugars (31). Ficoll (synthetic polymer of sucrose) yields an intense blue color when analyzed by the Lowry procedure (32) (Fig. 2). Concentrations of 2±36% (w/v) ®coll are used for density gradient centrifugation of cell fractions. Because of its high average molecular mass (*400 kDa), it is not removed from samples by dialysis. Addition of the Folin-Ciocalteu reagent to ®coll leads to formation of color with a time course of over 18 hours at room temperature. Ficoll had no effect on the yield of color when added to protein samples after the Folin-Ciocalteu reagent. In the presence of ®xed concentrations (0.21±1.88%) of ®coll, the calibration graph of standard BSA solutions had progressively lower slopes while the intercept increased. Ficoll forms a UV-detectable complex with copper.

FIGURE 2 Effect of increasing ®coll concentrations on the Lowry assay. Boxed

5.5. Lipids

Lipids increase sample turbidity and A750 values. The products of lipid

autoxidation also react directly with the Folin reagent. Heating lipids with alkali (a common method of protein solubilization) leads to Lowry-positive products. The problem has been examined with arachidonic acid, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol (33). Samples may be delipidated by shaking with chloroform or petroleum ether and then redissolved with 0.5 M NaOH by storing at 378C overnight or by heating at 1008C for 30 minutes. Solvent extraction will not be wholly successful if the protein and proteolipid complexes dissolve within an organic solvent phase. Emulsion formation can also lead to apparent loss of protein from the sample.

Lees and Paxman (34) modi®ed the Lowry assay for proteolipids and lipoproteins. Their method is fast and does not require heating.* Markwell et al. (35) added 1% SDS directly to Lowry reagent C to dissolve membranes and other lipid. The concentration of copper sulfate was also increased from 0.5 to 4% (w/w) to reduce interferences from sucrose and EDTA. The modi®ed method gave results identical to those obtained with lipoprotein samples delipidated using petroleum ether. However, the new method allowed higher rates of analysis and greater convenience. There was no interference from 40±200 mM sucrose. SDS also features in Method 3, which is therefore able to deal with membrane lipids routinely. According to Kirazov et al. (36), treating membrane-containing fractions with 1 M NaOH or 0.5% SDS reduced the stability of A750readings and did not reduce the

interference from lipids.

5.6. Sulfhydryl Agents and Other Reducing Compounds

The effects of various SH compounds are also related to their standard electrode potential (E8) measured against a normal hydrogen electrode (NHE) or a calomel electrode. At concentrations below 1 M, the redox potential is described by the Nernst equation:

E ˆ E_‡RT

nF ln C …20†

where n is the number of electrons transferred, E the observed redox potential corresponding to an activity of C (mol L 1_{), F the Faraday}

* Treat protein samples (20±70 mg) with 0.5 mL of alkaline SDS solution (5% SDS, 0.5 M NaOH), vortex, and let stand at room temperature for 3 hours. Add 2.5 mL of Lowry reagent C followed

constant, and R the gas constant. For dilute solutions, C becomes equal to concentration. Therefore, the effect of reducing compounds on the Lowry assay is related to the ``intrinsic'' redox properties (E8 and n) and the concentration of reducing agent.

Dithiothreitol (DTT), 2-mercaptoethanol (2ME), reduced glutathione (GSH), and oxidized gluthathione (GSSG) produce color with the Lowry assay (37). Reducing compounds react directly with the Folin-Ciocalteu reagent. Table 3 shows the degree of color formation with some SH compounds. As the underlying reaction is a redox process, calibration graphs for SH compounds will be nonlinear. For each SH compound De (M 1_cm 1_{) was determined from the steepest slope in Fig. 3. Values for De}

determined in this manner (Table 3, top half) agree with results from Ref. 14. DTT is one of the most effective interfering SH compounds. Samples with > 0.2 mM DTT have high A750 high background absorbances.

TABLE3 The Color Yield from the Lowry Assay of Some Reducing Compounds

Reducing agent
" (M 1_cm 1₎a _nb _E 1/2(mV)c Dithiothreitol 7625 (176) 2 2-Mecaptoethanol 3900 1 Glutathione (reduced) 3333 1 Cysteine 3400 1 0.398 Glutathione (oxidized) 1870 (760) 0.5 Cystine 1702 (822) 0.5 Cysteine 3150 1 Fe2‡ _{3150 1} Sn2‡ _{6100 2} Ascorbic acid 6700 2 Phenol 12400 4 Tyrosine 12800 2 (4) 0.398 Indole 13000 2 (4) 0.253 Tryptophan 13200 2 (2) 0.205

a_{The De (M} 1_cm 1_{) values in the top half of Table 3 are calculated from the maximum slope in}

Fig. 3. Data in the bottom half of Table 3 from Ref. 14.

b_{n ˆ electrons transferred to Mo}6‡_/W6‡_{from one molecule of reducing compound. Values for n}

were determined by colorimetry,c_{Half-wave electrode potentials were determined by cyclic}

voltammetry, using standard calomel electrode (i.e., ‡ 0.242 V vs. NHE), from Ref. 78. Note

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