Las TIC y la educación actual

Azo dyes represent 60% of all known dye structures (44). The ®rst azo compounds were synthesized by Peter Gries in 1858 using building units designated A, D, E, M, and Z (Fig. 2). Mono-azo dyes were formed via electrophilic attack of a diazotized species (A) on a sulfonated amino-

* BIO-DOT apparatus;Bio-Rad Laboratories, Richmond, CA. Membrane-bound protein was stained using Amido Black 10B or Ponceau Red (0.1% w/v) dissolved in 7% acetic acid and destained with 7% (w/v) acetic acid.

naphthol nucleus (E or Z). By altering the reaction pH, temperature, and concentration of reagents, diazo, triazo, or tetrakisazo dyes may be formed. The structure of azo dyes can written in shorthand: A?Z refers to a mono- azo dye produced by reacting a diazotized aromatic compound (A ˆ benzenediazonium chloride) with Z. Examples of A?Z dyes are Acid Orange 12, T-azo-R, and Acid Orange 1. A typical diazo dye is Amido Black 10B (1-amino-2p-nitrophenylazo-7-phenylazo-8-naphthol-3,6-disulfo- nic acid) with the formula A1_?Z/A2_{. The A}1_{and A}2_{units are attached to}

a central (Z) unit, 1-amino-2-naphthol-3,6-disulfonic acid. These structures are shown in Figs 3±5.

All azo dyes possess one or more azo (22N55N22) groups. The nitrogen-nitrogen double bond allows cis-trans isomerism. The naphthalene 2-hydroxyl group hydrogen bonds to the azo-group nitrogen, thereby stabilizing the trans isomer. The characteristics of some azo dyes are listed in Table 2. The absorptivity for Acid Orange 12 (w/v) is 26% higher than for Orange-G. The molar extinction coef®cients for the two dyes differ by only about 6%. The sulfonic acid group of azo dyes remains ionized at most

FIGURE2 Building blocks for synthesis of azo dyes with a brief explanation of A, D, E, M, and Z notation. (Top) Diazotized (group A) compounds (left) or (right) a tetrazotized (group D) compound. (Bottom) Coupling agents with a capacity to react with one equivalent or two equivalents of a group A compound. M is an aromatic amine that can react with A. The product may be diazotized for a second round of coupling.

accessible pH values. However, the exact acidity of benzenesulfonate or naphthylenesulfonate groups is uncertain (pKaˆ * 0.7±1.5).

4.2. Protein Dye Binding

Azo dyes bind with the guanidino, imidazole, and the e-NH2side chain of

arginine, histidine, and lysine, respectively (4,5,7,45±48). Interactions with wool (composed of the protein keratin) occur via ionic bonding. Further

bonding is by van der Waals and hydrophobic interactions. These increase with the area of contact between the dye and protein (44). Nonionic interactions become more important at high dye/protein ratios.

Formerly, the order of sensitivity for dye-binding assays was given as Amido Black 10B > Acid Orange 12 > Orange-G (21,22,26). Protein assays using Orange G were thought to be 100% less sensitive than assays using Acid Orange 12 because the two dyes bound 2 or 1 mole of arginine per mole of dye, respectively. However, more recent data (47) show that both Orange G and Acid Orange 12 form 1:1 mole complexes with protein basic amino acid residues. For Amido Black 10B the ratio of dye bound to basic amino acids is 1:0.5 (Table 3) (49,50). The small distance of separation between the sulfonate groups of Orange G may exclude binding to two sites.

FIGURE5 The structure of Amido Black 10B.

TABLE2 Characteristics of Some Acid Azo Dyes Used for Protein Assay

Dye AB 10B AO 12 OG C.I. No. 20470 15790 16230 Molecular weight 616.50 350 452.38 Net charge 1 1 2 lmax 620 482 480 e (Abs mL/mg)a _{81.5 (43,684) 59.0 (22,066) 46.9 (20,683)}

AB10, Amido Black 10B;AO12, Acid Orange 12;OG, Orange G.

a_{Extinction coef®cient, absorbance per unit concentration of dye bound (mg mL} 1_{). Value in}

parentheses is molar extinction coef®cient. Source: Refs. 21 and 26.

In summary, careful perusal of information in Table 3 shows that the sensitivities of dye-binding assays using Orange-G, Acid Orange 12, and Amido Black 10B are the same.

4.3. Soluble Protein Dye Complexes

Protein-dye complexes can be studied by spectrophotometry or equilibrium dialysis (51). To avoid precipitate formation, the concentration of dye used (1±10 mM) is 350±1000 times below those used for the Udy assay. BSA binding with azosulfathiozole, Orange I, Orange II, methyl orange, and tetrazine yellow was investigated by Klotz et al. (52), Klotz (53), and Sheppard et al. (54). Pesavento and Profumo (55) examined T-azo-R binding to BSA. Other interesting reports describe protein binding to phenol red (3), bromophenol blue (56,57), thymol blue (58), and the reactive dye cibracron blue (59±61).

Protein dye binding shifts the equilibrium between nonionized and ionized dye forms. The extinction coef®cient for the bound dye (eb) increases

while the wavelength for maximum absorption (lmax) shifts to lower values.

The hyperchromic effect is explained by reference to the conjugation theory. The lmaxfor dye molecules is determined by the energy required for p?p*

electron transition. Protein binding alters the degree of conjugation

TABLE3 Dye-Binding Capacity for a Range of Samples for Orange G (OG), Acid

Orange 12 (AO 12), and Amido Black 10B (AB 10B)

DBC (mmoles g 1_cP)a Sample OG AO12 AB 10B BSA 1365.0 1775.7 743.5 HSA 1466.8 1780.0 834.4 HGG 1028.8 1354.3 582.8 k-Casein 692.5 857.1 274.4 Meat meal 630.5 905.7 339.3 Fish meal 708.0 920.0 336.0 Milk protein 800.9 1094.3 490.3 Soybean 984.5 1345.7 555.2 Average 959.6 1254.1 519.5 Ratiob _{0.8 1.0 0.54}

a_{Dye-binding capacity (mmoles of dye bound per gram protein) from Ref. 50.}

b_{Average dye: BAA ratio. BSA, bovine serum albumin;HSA, human serum albimin;HGG,}

involving the p orbital and lowers the energy of the p* state. Lysine, arginine, or histidyl (auxochromic) groups donate electrons to the dye molecule, thereby increasing its conjugation extent. A further explanation centers on the transfer of free dye molecules from a polar low-viscosity solvent phase to a relatively nonpolar or restricted protein phase. Dye transfer to more nonpolar solvents and micelles leads to spectral changes resembling those observed during protein binding (62±64).

Usually, a ®xed concentration of dye is exposed to increasing amounts of protein. Absorbance readings are recorded with a reference cuvette containing a dye solution of the same concentration as the sample cuvette (Table 4). Measuring the ``difference absorption'' (DA) is useful where a dye solution has a high background. The absorbance change for dye reagent depends on the total dye concentration (D), extinction coef®cient (ef), and

the cuvette path length (1 cm) as described in Equation (6).

A1ˆ efD …6†

Df, protein, and the protein-dye complex are in equilibrium. Dbhas its own

extinction coef®cient (eb). The net absorption change is described by

TABLE4 A Summary of Symbols Used in Describing Protein-Dye Binding

Symbol De®nition

A1and A2 Absorbance for dye and dye ‡ protein

DA ( ˆ A2 A1) Difference absorbance

a Fraction of dye bound

D Total concentration of dye

Db Concentration of bound dye

Df Concentration of free dye

ef Extinction coef®cient for free dye

eb Extinction coef®cient for bound dye

De ( ˆ ef eb) Extinction coef®cient difference for the free and

bound dye

eapp Apparent molar extinction change when a fraction of

dye is bound

P, Pf Added, free concentration of protein

Kd Conditional dissociation constant

limax Wavelength for maximum absorbance

liso Isobestic wavelength where De ˆ 0

n, ns Number of dye molecules bound per molecule

Equation (7).

A2ˆ ef…D Db† ‡ ebDb …7†

From Eqs (6) and (7) it can be seen that A2±A1ˆ DA ˆ Db(eb ef) and also

A2 A1

D ˆ

Db…eb ef†

D or eappˆ a…eb ef† ‡ ef …8†

and therefore a ˆ…e_eapp ef†

b ef† …9†

where a is the fraction of dye bound and eapp ( ˆ A2/D) is the apparent

extinction coef®cient change when dye is bound.

The isobestic point (liso) is the wavelength at which bound and free

dye molecules have equal absorptivity (efˆ eb). By running absorbance

spectra with increasing dye or protein concentration, lisocan be identi®ed as

the wavelength at which there is no absorbance change (DA ˆ 0). The existence of an isobestic point is indication that the dye exists as two interconvertible forms (e.g., bound and free). No isobestic point will appear if ef= ebover the wavelength range examined. The corollary is that protein-

dye binding will not generate an absorbance change if De ˆ 0.

4.4. Analysis of Protein Dye-Binding Reactions

The protein dye-binding reaction is summarized by the following equation

Df‡ nPf„ Db …10†

Replacing Dbwith DA/De, we can de®ne the dissociation constant (Kd) as

Kdˆ…D DA=De†…nP DA=De†_DA=De …11†

The concentration of dye species changes with pH and ionic strength. Therefore, Kdis a conditional constant with a value that depends on the pH

and ionic strength (52). Depending on the protein/dye ratio Eq. (11) takes on the two forms described in Cases 1 and 2.

Case 1, Low dye/protein ratio. With excess protein we have nP (DA/De) & nP in Eq. (11). This approximation is also justi®ed if the number of binding sites is large;hence,

Kdˆ…D DA=De†nP_DA=De

and

DA ˆ nDePD

Kd‡ nP …12†

For high protein concentrations (nP >10Kd) DA reaches a maximum

(DAmax) where

De ˆDAmax

D …13†

Equation (13) is the chief means by which De and also ebmay be determined

(65). First, invert all terms in Eq. (12). The resulting double-reciprocal relation [Eq. (14)]* allows the determination of DAmax by graphical means

(see the following). 1 DAˆ Kd DeDnP‡ 1 DeD …14†

Multiplying the former relation by DADeD gives Eq. (15). Other linearized forms result from multiplying Eq. (15) by 1/(DeKd) or 1/(DeKdD).

DA ˆ DeD DAKd nP …15† DA nPDeˆ D Kd DA DeKd …16† DA PDDeˆ n Kd nDA DeDKd …17†

* The transformation is analogous to linearization of the Michaelis-Menten equation to give the Lineweaver-Burke double reciprocal plot, Eadie plot, Hanes plot, etc.

Finally, Equation (17) may be restated as G ˆnD_K d nGP Kd …18† where G ˆ Db/P.

To evaluate DAmax, De, and Kd/n, proceed as follows:

1. Add varying concentrations of protein to a ®xed concentration of dye (D).

2. For each sample measure DA.

3. Using Equation (14), plot a graph of 1/DA (Y-axis) versus 1/P (X-axis). From the X ˆ 0 intercept ®nd 1/DAmax( ˆ 1/DeD).

4. Use the estimate for DAmax and ®nd De from Eq. (13).

5. From the slope and known values for De and D calculate Kd/n. It

is not possible to determine Kdindependently using Eqs (14)±(17).

An alternative stratagem is to translate DA values to Db (e.g.,

Dbˆ DA/De) and Df ( ˆ D DA/De). Thereafter, use Eq. (18) to

evaluate all binding parameters.

Note that Eqs (12)±(18) are valid only at high protein/dye ratios. Under these circumstances, only high-af®nity protein sites (strong sites) are occupied. Binding parameters therefore relate to strong sites. The number of strong binding sites (ns) is distinct from the total number of sites (n).

Case 2. High dye/protein ratio. With excess dye D (DA/De) & D in Eq. (11);therefore,

DA ˆ_KDenPD

d‡ D …19†

Eq. (19) describes protein ligand binding when a small ®xed concentration of protein is exposed to varying concentrations of dye. As the concentration of dye increases (e.g., D > 10Kd), DA increases to a maximum value (DAmax)

and Eq. (19) becomes*

DAmaxˆ nDeP …20†

Using the De value determined before (see Case 1), ®nd the total number of binding sites (n) as follows:

1. Add varying concentrations of dye to a ®xed concentration of protein.

2. For each mixture measure DA.

3. Plot a graph of 1/DA (Y-axis) versus 1/D (X-axis). The X ˆ 0 intercept yields 1/(nDeP) and the slope is Kd/nDeP.

4. Calculate the number of binding sites from Eq. (21).

n ˆ DAmax=…DeP† …21†

Dividing the graph slope by the intercept gives Kd under

conditions such that both strong and weak binding sites are ®lled. In summary, two different experimental designs and analyses for protein dye binding are possible. Case 1 employs a ®xed (low) concentration of dye and varying amounts of protein. The estimates of De, Kd, and n

obtained are related to high-af®nity sites. Case 2 employs a ®xed (low) concentration of protein and varying (high) concentrations of dye. This study is useful mainly for determining the total number of binding sites. Values of Kdare average parameters for both weak and strong dye-binding

sites (57,58,65).

The application of these relations to a study of BSA binding with T-azo-R is described next. A ®xed concentration of dye (D ˆ 10.8 mM in 5 6 10 3_{M HCl solvent, pH 2.3) was titrated with increasing concentrations}

of BSA (see Case 1). Fig. 6 shows the pattern of binding of T-azo-R to BSA (55). At concentrations of dye above 100 mM, an insoluble protein-dye complex formed. Apparently T-azo-R binding to BSA could not be analyzed using the Scatchard plot (55). I have reanalyzed such data and others from Refs. 57, 58, and 65 using Eqs (14), (15), and (18) (Figs 6 and 7). Parameters for BSA binding with T-azo-R, bromophenol blue, and thymol blue are shown in Table 5.

The original studies were not designed to measure the total number of (low- and high-af®nity) dye-binding sites. The proportion of basic amino acids (*110 per mole BSA) functioning as high-af®nity binding sites for T-azo-R did not exceed 50%. With bromophenol blue there was dye binding to a very small proportion of basic amino acids.

4.5. Solubility Relations for Protein Dye Complexes*

The reaction between a charged dye molecule (D ) and protein (P‡₎

produces a soluble, complex [PD]AQ that later forms an insoluble complex

* There is no strict adherence to the use of squared brackets to indicate concentration. Brackets are included only where their presence renders equations more readable.

FIGURE 6 Analysis of protein-dye binding. A ®xed concentration T-azo-R dye

(D ˆ 10.8 mM) was titrated with increasing concentrations of bovine serum albumin (P ˆ 0±6 mM). (Top graph) Difference absorbance

changes monitored at 510 nm (DA510) plotted versus total added protein

concentration (P). (Lower graph) Determination of binding parameters using a double reciprocal plot of 1/P versus 1/DA. The Y-intercept is 1/DAmax. Furthermore, DAmax/D ˆ De [see Eq. (13)]. The slope ˆ

FIGURE7 Analysis of T-azo-R binding with bovine serum albumin. Same data as

shown in Fig. 6. (Top graph) Determination of binding parameters in

accordance with Eq. (15). DA is plotted versus DA/P. The slope is Kdand

intercept is DAmax. (Bottom graph) Determination of binding parameters

according to Eq. (18). As P ? 0, then Db? nP and Kd* D. Under such

[PQ]S.

P‡_{‡ D „ PD}

AQ; Kdˆ ‰P‡Š‰D Š=‰PDŠAQ …22†

PDAQ„ PDS; K2ˆ ‰PDŠAQ=‰PDŠS …23†

The net reaction is P‡_{‡ D „ ‰PSŠ}

S …24†

The overall process is comparable to isoelectric precipitation (4). Precipita- tion occurs when suf®cient numbers of dye molecules bind to neutralize all protein charges. In contrast, excess protein produces a ``colloidal protective effect'' that maintains the solubility of protein-dye complexes. From the de®nition of solubility product (KS) we have

KSˆ KdK2ˆ ‰P‡Š‰D Š …25†

The activity for [PQ]Sis given a value of 1. To describe the effect of pH on

protein-dye interactions, consider the ionization of dye-binding sites (pKa

*12.5);

P ‡ H‡_{„ P}‡_{; K}

a ˆ ‰PfŠ‰H‡Š=‰P‡Š …26†

The total protein concentration (P) ˆ [P‡_{) ‡ [P}

f] and after substituting for

Pf,

Kaˆ …‰PŠ ‰P‡Š†H‡=P‡ …27†

TABLE5 Binding Parameters for Soluble Serum Albumin±Dye Complexes

Dye, equation Kd n…mM 1† De (M 1cm 1) n T-azo-R Eq. (14) 0.162 4709 Ð Eq. (15) 0.177 5024 Ð Eq. (18) 0.181 Ð 62.a Bromophenolblue Eq. (14) 3.11 87787 Eq. (15) 2.03 69250 Eq. (18) 2.23 6.0 Thymolblue Eq. (18) 69.0 1430 26

and

P‡_ˆ P

…Ka=‰H‡Š† ‡ 1 …28†

The concentration of P‡ _{changes with the concentration of H}‡ _in

accordance with Equation (28) and consequently KSis given by Equation (29).

KSˆ_K P‰D Š

a=‰H‡Š† ‡ 1 …29†

To attain very low concentrations of soluble protein (in equilibrium with the protein-dye precipitate) requires a high dye concentrations at a low pH. Strong protein-dye binding (small Kd) will also facilitate quantitative

precipitation of protein from solution. At pH 4.84 the gelatin complex with Amido Black 10B yields KS& 4 6 10 12. Under higher acidity conditions

the value KSwas too low to determine (4). Refer to Skoog and West (66) for

more information on KS-solubility relations.

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