Complexation of Phenoxyacetic Acids with Bovine Serum Albumin & their Ternary Complexes in Solution

Indian Journal ofChemistry Vol. 22A. August 1983. pp. 677-682

Complexation of Phenoxyacetic Acids with Bovine Serum Albumin & their Ternary Complexes in Solution

R SAHAI* &ANIL K CHAUDHARY

Department of Chemistry, V S S0 College (Kanpur University). Kanpur 208002 Received 22October 1982; revised 8 March 1983; accepted 16April 1983

The interaction of bovine serum albumin (BSA) with active and inactive herbicides like phenoxyacetic acid (PAA). 2- chlorophenoxyacetic acid (CPA), 2,4-dichlorophenoxyacetic acid (2.4-0) or2.4.5-trichlorophenoxyacetic acid(2.4.5- T) and Cu(II) has been studied using ultracentrifuge and spectrophotometric techniques. The ternary complex formation ofCo(JI).

Ni(IJ), Cu(II), Zr(IV) ·and Th(IV) with these herbicides as secondary ligands and adenine, ethylenediaminetetraacetic acid (EDTA), hydroxyethylethylenediaminetriacetic acid (HEDTA) and nitrilotriacetic acid (NTA) as primary ligands has also been studied by pH-titration technique. The affinity constant data. spectral changes and stability constants for protein- herbicide complexed species of both active (CPA. 2. 4-0 and 2.4. 5-T) and inactive (PAA) phenoxyacetic acids are of the same magnitude, thereby ruling out the possibility of chelation as a possible mode of action of these herbicides.

We have earlier shown 1-3 that both active and inactive phenoxyacetic acid herbicides form quite stable binary complexes 1.3with Altl ll),Fe(lII), Co(Il).

Ni(II), Cu(II), Zr(IV), La(III) or Th(IV) as well as ternary complexes+' with Co(Il), Ni(II) or Cu(II) using pyridine bases as the primary Iigands. Muir and Hausch" have suggested a two-point attachment scheme for biological activity of phenoxyacetic acid herbicides. According to them attachment occurs between regulator and cysteinyl unit of the protein resulting in the formation of a complex between 2, 4- dichlorophenoxyacetic acid and sulphur of cysteinyl residue; however, nosubstantial evidence in support of this mechanism was provided. In order to explore the role of protein binding with these herbicides, the interaction of bovine serum albumin (BSA) with phenoxyacetic acid (PAA), 2-chlorophenoxyacetic acid (CPA), 2, 4-dichlorophenoxyacetic acid (2, 4-D) and 2, 4, 5-trichlorophenoxyacetic acid (2, 4, 5-

n

^has

been studied using ultrafiltration and spectrophoto- metric techniques.

Further Rowlands" has supposed some connection between growth inhibition in primary meristems and disturbance of the DNA-cycle induced by herbicides, but there is lack of experimental evidence in support of this assumption. We have, therefore, also investigated the formation of ternary complexes of Co(Il), Ni(II) and Cu(Il) with agenetic base (adenine) as a primary ligand and these herbicides as the secondary Iigands.

The ternary complexes of Zr(IV) and Th(IV) with the phenoxyacetic acid herbicides as secondary ligands and ethylenediaminetetraacetic acid (EDT A),

hydroxyethylethylenediaminetriacetic acid (HEDT A) or nitrilotriacetic acid (NT A) as aprimary ligand have also been investigated.

Materials and Methods

Cohn Fraction Vbovine serum albumin (BSA) and adenine were purchased from Sigma Chemical Co.

EDTA, HEDTA and NTA were obtained from Hopkin and Williams Ltd, England. The other chemicals were essentially the same as used in earlier investigations+:'.

The binding ofPAA, CPA, 2,4-0 and 2,4, 5-Twith BSA (4.0 x 10-4M) was studied by ultrafiltration technique" at 25"C using these herbicides in the concentration range of 3.5x !0--4to 2.0 x 10'-2M.The complexation of Cu(I1) with BSA (4.0 x \0 -4

Mj

was also studied by this technique using BSA in the concentration range of 4.0 x 10-4M to 4.0 x 10-2M.

The number of binding sites (n) and affinity constant (K) were calculated using Scatchard ' relationship (Eq.I),

[~] =nK - 1'K ... (1)

where Ii

=

number of mol of bound acid per mol BSA;

[A]

=

molar concentration of free acid.

The ternary complexes of metal ions with phenoxyacetic acid herbicides (secondary ligands) and adenine, EDTA, HEDT Aand NTA (primary ligands) were studied potentiometrically in 50% and 70% aq.

dioxan (v/v) for adenine and aminocarboxylic acids, respectively. The potentiometric titrations in pure aqueous medium could not be carried out because the complexes precipitated out immediately on adding secondary ligand to I:I metal-primary ligand solution.

Dissociation constants of adenine and the stability constant'S of its metal complexes were taken from the literature'':", except for complex of Co(I1) which was calculated in this investigation. The value of 10gKco

677

INDIAN J.CHEM..VOL. 22A.AUGUST 1983

was found to be 5.87. The pKa's of EOTA, HEOTA and NTA were also taken from literature10.11• The protonation constants of PAA, CPA, 2, 4-0 and 2, 4, 5-T and the stability constants of binary complexes were taken from our earlier investigations.':".

It is assumed for convenience that complexation of secondary ligand (L) starts after the complete formation of metal-primary ligand I:1 complex. For the calculation of stability constants of ternary complexes, the method of Martell et a/IO•12 was used.

For 1:1:1, 1:1:2 and 1:2:2 ternary complexes (M- adenine-phenoxyacetic acid), the equilibria repre- sented by Eqs. (2-4), respectively, were considered:

M2+ +L-+HA~MLAH+;

[MLAH+]

~LAH= [MH] [L ][HA] ^{... (2)} M2+ +HA+2L -~ML2AH;

M [MLzAH]

KML~AH=rrvrn] [HA] [L

r

MZ++2HA+2L -~ML2A2Hl;

M [ML²A²H^2]

KML~A~H,=[MU] [HA]2

rr-r

...(3)

...(4)

where HA =protonated adenine and L =completely dissociated phenoxyacetic acid herbicide.

Results and Discussion

Binding ofphenoxyacetic acid herbicides with BSA The plots of \i/[A] against I' (see Eq. I) were not linear (Fig. I). A close examination of these plots reveals that BSA contains at least two types of binding sites for these auxin-like substances. The sharply ascending limb (Fig. I,curve AB) at lower I' values indicates a high-affinity type binding site. while the

,c"

c,c~"-

,{

o.oLI"~-=-,-=I----'---~I~J~;;:;:o=~~:::;

0.0 2.0 4.0 6.0 8.0 10.0 12.0

i7

Fig.I+Plots for the bindingof CPA. 2. 4-D and 2. 4.5-T with BSA.

Table I-Binding Parameters ofPAA. CPA. 2. 4-D and 2.4.

5-T with BSA

Ligand ^{11 Kx 10'}

(dm3mol-l)

PAA 1.06±0.03 1.55± 0.07

CPA 1.17±0.02 0.81±0.O8

1.4-D 1.25±0.ll 0.61 ±0.03 2.4.5-T 1.31±0.08 0.54±0.04

near horizontal limb (Fig. I.curve BC) at higher \i values indicates a low-affinity type binding site. The affinity constants for these two binding processes have been estimated from the slopes of Scatchard plots (Fig. I. curve AB + BC) and the values are listed in Table I. It is evident from the data in Table I that the high-affinity group contains a single binding site and the low-affinity group contains a large number of binding sites for these phenoxyacetic acid herbicides.

The affinity constant data show that 2. 4. 5-T is bound

to BSA to the highest extent among all the phenoxyacetic acids.

The UV spectra of these acids exhibit peaks in the regions 276-297 nm (IB2" <- IAIg' IL"<-IA) and 269- 289 nm (IB'a <-IAIg' ILa<-IA)' J. However, in the presence of BSA. these peaks become more intense and also undergo bathochromic shifts and appear at 290- 303 and 280-290 nmrespectively. indicating some sort of interactions between phenoxyacetic acids and BSA.

BSA has N-terminal units in the sequence NHz- aspartic acid-threonine and C-terminal units in the sequence HOOC-alanine-Ieucine'4. These interactions may involve phenoxy group and carboxylate group of the herbicides with hydrocarbon portion and - NH2 group of BSA, respectively. These interactions of phenoxyacetic acids with BSA appear to involve hydrophobic and ionic bondings. The hydrophobic

bonding may be for lower-affinity type of binding site and the ionic bonding may be for the higher-affinity type. of binding site. Our observations find further support from the work of Mildvan and Cohn 15 who observed that the interactions between phenoxyacetic acids and centrimide were between phenoxy group of herbicide and hydrocarbon portion of micelle and the carboxylate group of the herbicide and the quarternary ammonium group of the micelle.

Binding of Cu(/l) with BSA

Scatchard plot for binding of CU(II) with BSA is shown in Fig.2. The extent of binding of Cu(II) with BSA is confined to approximately one site (n

=

1.15

±

0.04) with high affinity (K=1.02x 10-3). The visible spectrum of Cu(II) in acetate buffer (PH

=

5.5) exhibits a peak at 820nm which has been assigned to a transition from d".

d/

and d,=,d..to o-antibonding

SAHAI & CHAUDHARY: METAL-ADENINE-HERBICIDE TERNA'RY SYSTEMS

14.0r·

12.0

~ 10.0

::: f.

~ I

'S2 8.oL

~~

_ 6.0

I~

o o

4.0

20L ~

080 0'2~4' 06 08 1.0

V

Fig. 2~Scatchard plot for the binding of Cu(ll) with BSA.

1.2

J

0.9,---,

08

A

~0.5f\

~ ~\

£J \

(;0.4 \

•.

^\

£J \

<: \

0.3[ \

\

\

r I

,I

-,,, ,,,

,

,,

B

I I I

,I I I I

,I I I

0.2f I I

°t

O.~8"0---C4""'6"0....lI-'s.-Jl'"0,-1'-=62"'0;-'--=7 0"'0---7c:ca0"

~ (nm)

Fig.:1- Visible spectra of: (A)Cu(NO,), (4 x 10~·M). (B)Cu(NO)), (4x 10~·M)+BSA (4.0x 10~5M) in acetate buffer.

, I

a6~0

and half-filled d"-l' levellb. In the presence ofBSA the peak at 820 nm is shifted to 700 nm (Fig. 3).This blue shift clearly indicates that Cu(l!) is coordinated to BSA. The possibility of binding of Cu(I!) with carboxylic group is ruled out because at pH 5.5, the protons of carboxylic groups are s-amino groups 17.So we may expect that the nitrogen involved in the binding of Cu(l!) with BSA is via the immidazole side chain nitrogen 18.19, t-hough binding via z-amino group nitrogen can not be excluded.

Cu(I!) binding with BSA inacetate buffer is evident from spectrophotometric data. However, the interpretation of the data on a quantitative basis in terms of intrinsic constants and number of binding sites isbeset with many difficulties, as is evident even in simple cases of binding of Cu(I!) with glycine and glycylaldehyde/".

Metal-adenine-phenoxyacetic acid ternary systems The potentiometric titration curves of I: I:I metal- adenine-phenoxyacetic acid ternary systems show inflections atIII

=

I(Ill

=

No. of mol of base added/No. of mol of ligand or meta\) and III

=

3 indicating the formation of ternary complexes in two steps; the first step involves the formation of 1:I metal-adenine complex, folfowed by the coordination of phenoxy- acetic acid to I:I system to form I: 1:I ternary system ina separate step. The calculation of stability constant of these complexes was done between m

=

3 and III

=

4 using Eq.(2). These results are summarized inTable 2.

The stability constants of ternary complexes with respect to secondary ligands follow the trend: PAA

>CPA>2, 4-0>2, 4, 5-T, and with respect to metal ions the order is Cu(l I)>Ni(I I)>Co(II), in accord with Irving-Williams sequence21.

It is of interest to consider ~logK values+' (Table 2) defined by Eq. (5), which is a measure of stability of ternary complexes with respect to binary complexes.

~log K=log K~~L-log K~L ...(5)

Except for Ni(lI). where ~log Kis small and negative, the values of Alog K for Cotl l) and Cu(II) are positive, indicating extra stabilisation of I:I:I as compared to the I:I metal-adenine complex. On statistical consideration ':'. I: 2 or I: I:I ternary complexes should be less stable than a I: I complex. This extra stabilisation of I: I:I metal-adenine-phenoxyacetic acid complexes indicates that ligand containing oxygen as donor atoms form more stable complexes

Table :2 Stability Constant Data of Ternary Metal Complexes of Some Phenoxyacetic Acid Herbicides with

Adenine (Primary Ligand) in 50"" Water-Dioxan (v/v) [Temp. =35 C: II=0.1 M(KNO)]

Metal ion logK~~: logII~AL tllog KM

P AA assccondarv ligand

Co(ll) 3.01 8.88 0.13

Ni(I I) 2.12 8.30 0.03

Cu(l I) 1.64 10.58 0.56

CPA as secondary ligand

Co(ll) 3.01 8.90 0.32

Ni(II) 2.75 8.93 0.00

Cu(II) 1.22 10.16 1.13

2.4-D as secondary ligand

Co(I1) 2.90 8.77 0.40

Ni(II) 2.07 8.25 -0.03

CuOI) 1.09 10.03 1.20

2.4. 5-T as secondary ligand

Co(I1) 2.89 8.76 0.42

Ni(ll) 1.79 7.79 -0.20

CuO I) 1.24 10.18 1.26

• Astandard deviation of ±0.01 to 0.09 has been estimated inthese values.

679

INDIAN J. CHEM., VOL. 22A, AUGUST 1983

with Mtadenine)" + than with Mtaq.):' +. This statement finds support from the work of Sigelz4.z5.

Based on the above observations the ternary complexes may be represented either by structure (I) or (II).

The potentiometric titration curves were also obtained for the solutions containing I: 2: I; I:I: 2 and I:2: 2 rnetal-adenine-phenoxyacetic acids. The titration curves show inflections at m=3 with a slight lowering or increase inpH. In the presence of excess of adenine, the inflection occurs at slightly higher pH; in the presence of excess of phenoxyacetic acid concentration slight lowering in pH isnoted during the titration. This clearly indicates that only MAHzL type of complex is formed irrespective of the concentrations of both the primary and secondary ligands. The formation of I:2: 2 (M :A:L)complex is not possible as both the ligands are bidentate and each bivalent metal ion can have only a coordination number of six.

For MAzHzL and MAHzL2, the structures (III and IV) have been suggested. Structures (III) and (IV) are likely to be unstable because there can not be hydrophobic or stacking interaction of ligands adenine-adenine or adenine-phenoxyacetic acid in these complexes. Stacking is thus appreciable only in those aromatic ligands that have comparatively free rotation about the metal-ligand bond and can take up a configuration for maximum molecular interaction.

This can only be possible when the two ligands (adenine and phenoxyacetic acid) have their aromatic rings parallel to each other. The interaction is less in

I: 2 metal-adenine system, since adenine can become rigid in a bidentate coordination to the metal ion. For

M= Co (Ill , Ni (II) Or Cu (II)

the same reason, there isno extra stabilization in I:I: 2 metal-adenine-phenoxyacetic acid systems because these Iigands are also rigidly coordinated inabidentate manner to the metal ion with no possible molecular stacking interaction between adenine and the phenoxyacetates in these complexes.

From the above discussion, it is clear that this explanation is' true for structures (I) and (II), but structure (I) appears to be the only possible structure for the mixed-ligand complexes.

Using Sanderson's:" approach, theoretically calculated values of electronegativities (as stability ratio, SR) ofCu(lI) and Zn(lI) have been found to be 5.67 and 9.85 respectively and the difference in electronegativities of Cu '" and Curadenine)?" species are less than those of Zn2+ and Zruadenine):' ⁺ species, thereby indicating that the stability ratio of Cu' +isnot decreased due to coordination with adenine as is observed in the case of Zn2 ". For example, the stability ratio of Cu2 + decreases from 5.67 to 4.35 (or by 1.12 unit) in forming Cutadenine)? +, while it decreases from 9.85 to 4.52(or by 5.32 unit) in forming Zntadeninel/ + from Zn2 + Thus. better n-donor properties of Cu2+ in Cuiadenine):' + species as compared to those of Zn2 + in Zruadenine)!" species are due to comparable electronegativities of Cutadenine)? + and Cu2 ". This explains why we could not calculate the stability constants of ternary systems of Zn2+ in solution.

Metal-aminocarboxvlic acids-phenoxyacetic acid ternary systems

The potentiometric titration curve (Fig. 4, curve B) of 1:1:1 Zr(IV)-EDTA-PAA complex shows in- flections at m

=

2 and m

=

4, thereby indicating the formation of I:I Zr(IV)-EDTA complex in the first step followed by the coordination of secondary ligand (PAA) in the second step. It has been further verified by the perfect match between the titration curve of I:I Zr(IV)-EDTA complex with the mixed-ligand curve upto m

=

2 (Fig. 4, curves A and B). In the second buffer region (m=2 to 4),the carboxylic group ofPAA is bound to Zr(IV)-EDTA complex to form ternary chelate which isstable upto pH 11.0. A comparison of titration curves of Zr(IV)-EDTA-PAA and Zr(IV)- EDT A systems (Fig. 4, curves A and B) shows that latter undergoes hydrolysis/? in the pH range 5.5-6.4 whereas addition of second ligand effectively prevents hydrolysis in the former. A similar type of titration curve has also been obtained for Th(IV)-EDTA-PAA system (Fig. 4, curve C) and other chloro substituted phenoxyacetic acid systems (not shown).

The mixed-ligand potentiometric titration curve (not shown) for I:I:2 molar ratio ofZr(IV)/NTA/PAA

has a buffer region at low pH values followed by a

SAHAI & CHAUDHARY: METAL-ADENINE-HERBICIDE TERNARY SYSTEMS

10

C 8

~~

'" 6 .!?,

B 4

~~~~~--~2-J--~3-J--~4----75~~

me mol of bas. add.d mol of ligand orrnetot

Fig. 4-Potentiometric titration curves of mixed ligand chelate systems

[(A) Zr:EDTA (1:1); (B) Zr:EDTA:PAA (1:1:1) and (C) Th: EDTA: PAA (I: I: I)]

sharp well-defined inflection at m ~5 which can be explained as follows. It has been observed earlier that Zr(IV) forms a stable Zr(PAA)4 complex 1with a sharp inflection at m=4and with NT A it forms Zr(NTAh. a bis-complex

i".

^{with inflection at m=6. These}

observations taken together provide an explanation for the observed inflection at m ~5 (deprotonation of three protons of NT A and one proton each from two PAA). For I: I:I Zr(IV)-NTA-PAA system there is a higher buffer region which terminates in another inflection at m=^4.

The potentiometric titration curve (not shown) containing equimolar ratio of Zr(IV)-HEOTA-PAA shows inflection at m ~5 and 6. thereby indicating the formation of a binary complex followed by ternary complex formation.

The stability constant data of mixed-ligand complexes. listed in Table 3. show that the order of stability constants of these ternary complexes with respect to metal ions follows the sequence: Zr(IV)

>Th(lV) which is in accordance with their charge!

radius ratio. For a given combination of M and A. the stability constants of MAL complexes are nearly equal (Table 3). From this result. one can say that phenoxy oxygen is not involved in bonding.

The representative species distributions for M(lV)- EOTA-PAA and M(IV)-NTA-PAA (M =Zr or Th) systems have been carried out using the programme of Perrin and Sayce28.1t has been observed that atpH ~4 more than 90°0of the species are present in the form of MAL. The inflection in the titration curve at this pH is therefore. understandable.

As the overall stability constants (fJMAJ of biologically active (CPA. 2. 4-0 and 2. 4. 5-T) and inactive (PAA) herbicides are almost of the same magnitude. it indicates that herbicidal activity may not be due to the formation of ternary complex of these herbicides with polyvalent ligands (primary ligand) present in biological fluid.

Table 3-Stability Constants of Ternary Complexes of Zr(IV) and Th(lV) with Phenoxyacetic Acid Herbicides and

Aminopolycarboxylic Acids [Temp. =25 C:Ji=0.1 M(KNO)lJ

Metal log K~:~L

Secondary Ligand

log/l~:,•••

PAA

ion

EDT Aas primary ligand

ZriIV) 10.62 40.12

Th(lV) 10.42 33.63

ZriIV) 10.54 40.04

Th(lV) 10.21 33.41

ZriIV) 10.48 _} 39.98

Th(lV) 10.51 33.77

ZrOV) 10.42 39.92

Th(IV) 10.40 33.60

NT A as printarv ligand

ZriIV) 9.71 28.64

Th(lV) 9.93 23.23

ZriIV) 9.57 2l!.50

Th(IV) 9.83 23.17

ZrOV) 9.46 2l!.39

Th(lV) 9.75 23.05

ZrOV) 9.39 28.32

Th(IV) 9.63 21.93

H EDTrI as pril/WIT ligand

ZrOV) 9.87 28.37

Th(lV) 9.52 28.76

ZrOV) 9.90 28.40

Th(lV) 9.42 28.66

ZrOV) 9.58 28.08

Th(lV) 9.42 28.66

ZrOV) 9.51 28.01

Th(lV) 9.31 28.55

CPA

2.4-D

2.4.5-T

PAA

CPA

2.4-D

2.4.5-T

PAA

CPA

2.4-D

2.4. S-T

From above discussions, it is evident that phenoxyacetic acid herbicides form stable complexes with BSA and ternary metal complexes with adenine and aminopolycarboxylic acids. The affinity constant data and the spectral changes for binding of active (CPA. 2. 4-0 and 2. 4, 5-T) and inactive (PAA) phenoxyacetic acid herbicides are almost of the same magnitude. The stability constants of ternary metal complexes of these active and inactive phenoxyacetic acids with adenine and aminocarboxylic acids are also of the same magnitude. These observations suggest that metal-protein-herbicide or metal-genitic base- herbicide model for herbicidal activity of these phenoxyacetic acid herbicides may not be appropriate one. These observations. thus, lend further support to our view that complexation may not be the possible mode of action of phenoxyacetic acid herbicides I 3.

Acknowledgement

The authors are thankful to ICAR. New Delhi. for financial assistance. The authors are grateful to Profs P. R. Singh and P.C. Nigam. lIT. Kanpur for providing some experimental facilities and constant encouragement.

681

INDIAN J.CHEM ..VOL. 22A. AUGUST 1983

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