INHIBITION BY THIOCYANATE OF LACTOPEROXIDASE-CATALYSED OXIDATION AND IODINATION REACTIONS J. TENOVUO Institute of Dentistry, University of Turku, Turku. Finland
Summary-The thiocyanate effects were pH-dependent. Guaiacol oxidation was inhibited only at pH values less than 6.5, whereas iodide oxidation and tyrosine iodination were inhibited at 3.4-8.0 pH. Guaiacol and SCN- had the same binding site on the enzyme surface which did not bind HzO,. SCN- competitively inhibited both iodide oxidation and tyrosine iodination and was even able to de-iodinate iodinated tyrosine derivatives. The results indicate that guaiacol oxidation is inhibited by CN- produced by oxidation of SCN- catalysed by lactoperoxidase, whereas iodide oxidation and tyrosine iodination were inhibited due to the competitive action of SCN-.
INTRODUCTION
Thiocyanate (SCN), the detoxication product of cyanide, was for long regarded as metabolically inert. However, the large amount of thiocyanate ions in body fluids was later connected with metabolic events in the thyroid gland (reviewed by Wood, 1975). Thiocyanate ions combine with the prosthetic groups of haematin enzymes, e.g. lactoperoxidase (LPO) and thyroid peroxidase, both of which catalyse thiocyanate oxidation in the presence of H20, (Wood, 1975). Thyroid peroxidase and lactoperoxidase are separate enzymes (Ljunggren and Akesson, 1968; Morrison and Steele, 1968). Thiocyanate ions inhibit the oxidation of guaiacol catalysed by thyroid peroxidase (Hosoya, 1963) and the oxidation of iodide to iodine and the iodination of tyrosine catalysed by LPO (Tenovuo, 1976). The uptake of iodide by the thyroid and salivary glands is inhibited by SCN (Myant, 1960). Despite its inhibitory properties, SCN is necessary for the antibacterial action of the LPO system in human saliva (Klebanoff and Luebke, 1965). The inhibitory action is assumed to result from the oxidation of SCN by LPO in the presence of H202 possibly forming hypothiocyanite ions (Hoogendoorn rr al.. 1977). My aim was to examine the inhibition caused by SCN- on the following reactions catalysed by LPO: (1) the oxidation of guaiacol to dihydroxydimethoxydiphenyl: (2) the oxidation of iodide to iodine; (3) the iodination of tyrosine. The inhibitory action of cyanide, an oxidation product of thiocyanate and a common peroxidase inhibitor, and SCN were compared. The inhibition by SCN- is important because these ions in the thyroid and salivary glands inhibit the formation of iodinated proteins in man, thus quenching iodine metabolism. The results may give information about the effect of increased SCN concentration in body fluids resulting from smoking (Tenovuo and Makinen, 1976) on the formation of iodinated tyrosine derivatives. The physiological concentrations of
SCN- and II, as well as peroxidase given in Tenovuo (1978). MATERIALS
Peroxidase
AND
activities,
are
METHODS
and iodine ussays
Guaiacol was oxidized by the methods of Chance and Maehly (1964) and the enzyme units were calculated as described by Makinen, Tenovuo and Scheinin (1976). After enzymic oxidation of iodide to iodine, the increase in absorbance at 350nm due to the formation of triiodide (Hosoya, 1963) was measured. The procedure and reaction mixture have been described in detail (Tenovuo, 1978). 0.5 pg of LPO in 2ml of reaction mixture were used. The enzymic iodination of r_-tyrosine was performed as described by Tenovuo (1978). Chemicals
Milk lactoperoxidase (E.C. 1.11.1.7) and 3-iodo+tyrosine were purchased from Sigma Chemical Co. (St. Louis, Miss.). Guaiacol was a product of BDH Chemicals Ltd. (Poole, England) and r_-tyrosine of Fluka AG (Switzerland). All other chemicals were products of E. Merck AG (Darmstadt, Germany). RESULTS
7’he inhibitory action of cyanide and thiocyanate ions on the oxidation of yuuiacol catalysed by lactoperoxiduse 10 mM pfl-dimethylglutarate buffer, pH 3.G7.0 was used. A marked difference in the influence of pH on the reaction velocity in the presence of inhibitors was found (Fig. 1). Cyanide ions (Fig. 1B) inhibited the oxidation of guaiacol(5 mM) at all pH values studied, whereas SCN- (Fig. 1A) inhibited only at pH values below 6.5, even 0.1 mM being effective. The inhibition by CN- at pH 7 suggests that CNcompeted with guaiacol for the same binding site (Fig. 2A), whereas CN- at 0.5 and l.OmM concentrations did not affect the affinity between LPO and
899
900
J. TENOVUO
23 20
H -
CONTROL 0.1Ornt.t KSCN 03Ornf.i KSCN
15
10 ? > 5 '0 Y 7 2 o "0 25
8 2 e
20
H
CONTROL
o-0
o.lOrnM
KCN
o-o
030mM
KCN
::
15
-
10
-
-e.i 5c
0
’
I
30
40
,
I
,
60
50
PH
70
Fig. 1. Rate of guaiacol oxrdation as a function of pH in the presence of inhibnors (A) KSCN and (B) KCN. Mean values + SD (n = 6). Hz02 (Fig. 2B) as the apparent K, of O.lOmM was unchanged. Guaiacol and H202, therefore, may have separate binding sites on the enzyme. The inhibitor constant, K,, for CN-, estimated from the Dixon plot, was approx 0.25mM. The inhibitory action of SCNwas studied at pH 5.0 because at higher pH values the ion does not
&a
loo
Fig. 3. Plot of recrprocal velocrty vs recrprocal guaracol concentratron (A) and vs reciprocal H20, concentration (B) for the lactoperoxidase-catalysed oxidation of guaiacol at pH 5.0 in the presence of various concentrations of KSCN. inhibit the oxidation of guaiacol. However, no significant non-enzymic oxidation occurred. As with CN-, SCN- competed with guaiacol (Fig. 3A). Even concentrations of SCN- as low as 0.050.20mM markedly decreased the affinity between LPO and guaiacol indicating the same binding site for guaiacol and SCN-. However, the double reciprocal plot with HzO, at pH 5.0 (Fig. 3B) differed from that at pH 7.0 as it was non-linear. The inhibitor constant for SCNwas 0.50.7mM according to the Dixon plot. The inhibitory action of thiocymate on the oxidation of iodide to iodine cat&wed by lactoperoxidase Guaiacol oxidation and iodide oxidation differed in the pH dependence of inhibition by thiocyanate. With iodide, SCNinhibited at all pH values (Fig. 4): at pH 7 (Fig. 5) inhibition was competitive 30
0
50
100
I
50
I
150 1 200 El M
/
I
h
I
mt.4
I
loo
Fig. 2. Plot of reciprocal velocity vs reciprocal guaiacol concentration (A) and vs reciprocal H,Oz concentration (B) for the lactoperoxidase-catalysed oxidation of guaiacol at pH 7.0 in the presence of various concentrations of KCN.
Fig. 4. Rate of Iodide oxidation as a function of pH m the presence of KSCN at various concentration.
Inhibition
of lactoperoxidase-catalysed
by thiocyanate
!L "I 20
k I
1
I
I
I
01
02
0.3
OX
OS
[KSCN]
WI
only in the presence of H202 (Fig. 6A). Thiocyanate ions released the iodine bound to tyrosine (Fig. 7) or the enzyme (Fig. 6A). indicating competitton between SCN- and iodine. When the iodination of tyrosine was complete, after 30 min. the addition ol SCN _ resulted in de-iodination (Fig. 7B). SCN dciodinated mono-iodotyrosine at a low rate (Ftg. 68). A plot of rO’r, vs [KSCN] gave a stmtlar result to that in Fig. 5. indtcating compettttve mhibttton between SCN and iodide ions. The double rectprocal plots of l/‘r vs li[S] in the presence of SCNand iodide ions produced a similar curve with iodide oxidation
30
10
reactions
mM
Fig. 5. Plot of rojc, against [I] (KSCN) for the lactoperoxidase-catalysed oxidation of iodide at various KI concentrations and at pH 7.0. (0) 10 mM: (0) 20 mM: (a) 50 mM
KI because, had it been non-competitive, all 3 concentrations of KI would have yielded one straight line only. The double reciprocal plots of l/n vs l/[S] were not linear.
The inhibition by thiocyunute Iysed oxldatlon oj yuaiucol
of‘ luctopt~rc~.~~du.\~-~~~ta-
There are two possible explanations for the mhtbitory action of SCN- on the reactions catalysed by LPO: the inhibition is caused by either (I) cyamde. cyanate (CNO) or some other product of the oxidation of SCN. or (2) by a direct inhibitory effect caused by binding to proteins. Thiocyanate is oui-
The inhibitory action of thiocyanate and cyanide ions on the iodination of tyrosine catalysed by lactoperoxidase
Thiocyanate and CN- inhibited the iodination of tyrosine at pH 3.4-8.0. LPO was also iodinated, but
06 0.5 04 03 < -
02
:
0.1
2
0
,‘
06
% 8
0.5
-
04 03 0.2 0.1 0
0
2
4
6 TIME
8
10
12
14
16
(MINUTES)
buffer.pH 7 0. addition of KI (0.6 mM). H202 (0.1 mM) and KSCN (0.1 M). (B) De-iodination of (0.5 M) in 0.01 M phosphate buffer, pH 70 Arrows mdtcate addition of KSCN (0.1 M), Hz02 (0.1 mM) and LPO (0.4 ng;ml).
Fig. 6(A). Iodination and de-iodination of lactoperoxidase (0.4 pg/ml) m 0.01 M phosphate Arrows Indicate mono-iodotyrosme
TIME
Fig. 7(A). Iodination of tyrosine
(MINUTES)
(0.2 M) catalysed by lactoperoxtdase (0.4 pg/ml) in the presence of H202 (0.1 mM). The arrows indicate the addttion of KSCN (0.1 M). (B) De-iodmation of iodinated tyrosine derivatives. Initial reaction mixture contained tyrosine. KI, LPO and HZOz in concentrations presented above. The arrow indicates the addition of KSCN in the following concentrations: (0) 0.01 M: (0) 0.1 M: (@I 0.5 M.
902
J. TENOVUO
The inhibition by thiocyanate of lactoperoxidase-catadized to SOS and either CN or CNO depending upon /ysed oxidation of iodide and the iodination of tyrosine pH, CNO only being produced in very alkaline solutions (Hughes, 1975). Chung and Wood (1970) Iodide oxidation and the iodination of some amino observed with LPO that CN was the initial product acids and proteins are well-known reactions catalysed of the oxidation of SCN by H202, but later an interby peroxidases. Iodine antagonism of SCN occurs in mediate oxidation product of SCN converted CN to the thyroid gland. The nature of the inhibitory mechCNO which is spontaneously hydrolysed NH, and anism is, however, still uncertain. It is only known coz. that SCN may compete with I for oxidizing equivalSCN- ions strongly bind to proteins (Wood, 197% ents and in a secondary way the product of the peroxsometimes even leading to their de-iodination (Carr. idase-catalysed SCN reaction may change the struc1952). The primary site of binding is the arginine resiture of the thyroglobulin, making it less effective for due, the secondary sites being lysine and histidine the biosynthesis of thyroxine (Morrison and Schon(Pande and McMenamy, 1970). Thiocyanate is also baum. 1976). a ligand for metallo-proteins but the bond is generally My results clearly indicate that SCN- inhibits both weak and readily reversible (Wood, 1975). I oxidation and tyrosme iodination independently Therefore. with LPO, both inhibitory mechanisms of pH; furthermore, SCN- de-lodinated tyrosine deare possible, depending on the reaction mvolved. My rivatives, indicating a greater affinity of SCN- than results suggest that guaiacol oxidation is inhibited by I- to tyrosine. The inhibition of I- oxidation by CN- or CNOproduced by oxidation of SCN, SCN- differed from that of guaiacol in two respects: whereas iodide oxidation and tyrosine iodination are (a) the inhibition was not affected by pH, (b) SCNinhibited due to competition of SCN- and iodide was much less effective in inhibiting I- oxidation than ions for the same binding site. With peroxidase-cataguaiacol oxidation. Thus, with I- oxidation, the inhilysed reactions involvmg two substrates, a Michaelis bition was probably not caused by CN- formed by constant was obtained for H202, the first reactant SCN oxidation. With thyroid peroxidase, it is possible with the enzyme, but the velocity was proportional to separate the ability to catalyse the iodination reacto the concentration of hydrogen donor. CN- up to tion whereas guaiacol oxidation activity is unaffected 1.0 M has no effect on the affinity between LPO and (Morrison, Bayse and Danner, 1970). The great affinity of SCN- to proteins appears to produce comHzO, (K, - O.lOmM), but competes with the hydrogen donor guaiacol for the same binding site on the petition with I- and, thus. to inhibit the iodination enzyme. of tyrosme and proteins already at low concenUnlike cyanide or cyanate inhibition, SCNmtrations. With I- oxidation, the SCN- ligand for hiblts the oxidation of guaiacol only in acid solutions metallo-proteins, such as lactoperoxidase. 1s weak where it is competitive with guaiacol. The rate of and reversible and thus a larger concentration of reaction of SCN- with LPO may be affected by pH SCN- is needed for the inhibltion although I- and because the different ionic forms of the enzyme may SCN- have the same binding site on the enzyme. react at different rates with the inhibitor, or because It appears that SCN- in human saliva competithe inhibitor itself ionizes. The main oxidation tively inhibits the iodination of tyrosine residues in product of SCN at acid pH is CN, and a role for salivary proteins catalysed by peroxidases secreted it in the inhibition caused by SCN cannot be from the salivary glands. This may be relevant to the excluded. A low concentration of SCN is Inhibitory. small amount of organically-bound iodine in human even 0.17~1moles per ml of KSCN produces nearly saliva, although many salivary proteins, especially the maximum amount of CN in the presence of H202 amylase and albumin, are very susceptible to iodina(Chung and Wood. 1970). The assumption that CNtion in vitro (Sarimo and Tenovuo, 1977). is responsible for the inhibitory action of SCN presupposes that CN is not formed to any great extent Ackno~letlyemrnts- This mvestlgation was supported by a in neutral or alkaline solutions. Chung and Wood grant from the Finmsh Dental Society. The skilful techm(1970) did their experiments on CN production at pH cal assistance of Mrs A. LZhteenmBki is gratefully acknowledged. 5.7. Further evidence for the inhibition by CN- or CNO- of the oxidation of guaiacol arises from the fact that, although OSCNis the main oxidation REFERENCES product of SCN- catalysed by LPO, at acid pH, Carr C. W. 1952. Studies on the binding of small Ions OSCN- is rapidly converted to CNO- which is hydin protem solutions with the use of membrane elecrolyzed to CO, and NH: (Hoogendoorn et ul.. 1977). trodes. I. The bmdmg of the chloride ion and other inorThis IS why guaiacol oxidation was inhibited by ganic anions m solutions of serum albumin Archs BioSCN- only in the pH range below 6.5. them. Biophy.5. 40, 28&294. The double reciprocal plot of l/r vs 1/[H202] of Chance B. and Maehly A. C. 1964. In. Methods in Enzwno1oy.r. Vol. II, p. 764. Academic Press, New York. guaiacol oxidation at pH 5.0 is non-linear because of Chung J. and Wood J. L. 1970. Oxidation of thiocvanate the mhibltory actlon of high HzO, concentrations. to cyanide and sulfate by the lactoperoxldase-hydrogen This was not conventional substrate Inhibition, but peroxide system. Archs B~ochem. Biophys. 141, 73-78. inactivation of the enzyme by excess H20,. This was Hoogendoorn H., Piessens J. P.. Scholtes W and Stoddard confirmed by incubating the enzyme with 0.05 M L. A. 1977. Hypothiocyanite ion; the Inhibitor formed H,O, for 1 min before the rate of the oxidation of by the system lactoperoxidase-thlocyanate-hydrogen gualacol was measured. When compared with the peroxlde. Curies Res. 11, 77-84. effect of pre-incubation for 1 min without Hz02. the Hosoya T. 1963. Effect of various reagents including gualacol-oxidizing activity of LPO was 40 per cent antIthyroId compounds upon the activity of thyroid perless in the presence of H,O,. oxidase. J. Biol Chew Tokyo 53, 381-388.
Inhibition
by thlocyanate
of lactoperoxldase-catalysed
Hughes
M. N. 1975. In: Chemistry and Biochemistry o/ Acid and its Deriratws (Edited by Newman A. A.). p. 30. Academic Press, London. Klebanoff S. J. and Luebke R. G. 1965. The antilactobacillus system of saliva. Role of salivary peroxidase Proc. Sot. exp. B~ol. Med. 118, 483-486. LJunggren J.-G. and Akesson A. 1968. Solubihzation. lsolation. and Identification of a peroxidase from the microsomal fraction of beef thyroid. Archs Biochem. Bi0phy.t. Thm-panic
127, 346-353
Mrikinen K. K. Tenovuo J and Scheinm A 1976. Xyhtolinduced increase of lactoperoxidase activity. J. dent. Res. 55, 652-660.
Morrison M. and Steele W F. 1968 Lactoperoxidase. the peroxidase in the sahvary gland. In: Biology of the Mouth (EdIted by P. Person). pp 89--l IO. Amer. Assoc. for Adv Ser.. Washmgton Morrlson M.. Bayse G. and Danner D. J. 1970. The role of mammalian peroxidase m lodmation reactlons. In: Biochemistry of rhe Phayocyric Process (Edited by Schultz J.), pp 51-66. North-Holland. Amsterdam.
reactlons
903
Morrison M. and Schonbaum G. R. 1976. Peroxidase-catalyzed halogenation. Ann. Rer. Biochem 45, 861-888 Myant N B 1960. Iodme metabohsm of sallvary glands. Ann. N.Y. Acad. SCI 85, 208-214 Pande C. S and McMenamy R. H. 1970. Thlocyanate bindmg with modified bovme plasma albumlns 4rch\ Biochem. Bioph,s. 136, 260-267 Sarlmo S and Tenovuo J 1977. Enzymlcall!, lodlnated human sahvaq protems. Fractlonatlon and charactrrlzatlon by column chromatograph! and electrofocualng Blochem
J
167. 23-~29
Tenovuo J. 1976 The peroxldatlc actnIt> m the human oral cavtty. .4cru rdont. wmd 34. suppl. 71. p, I6 Tenovuo J. and Makmen K K 1976. Concentratmn of thiocyanate and lontzable lodIne m saliva of smokers and nonsmokers. J. drnr. Re\ 55, 661-663 Tenovuo J 1978. Lactoperoxrdase-catalysed lodme mctabohsm In human saliva Irchc orul Bwl. 23. 253 75X Wood J. L 1975 In: Chrml.\rr\, end Bwchem~,~rr~ o/ Thro~vanrc 4citl clnd It\ Drrnurrtm (E&ted by Neuman A 4 ) pp. 156221 .Academlc Press. London
Summary-The thiocyanate effects were pH-dependent. Guaiacol oxidation was inhibited only at pH values less than 6.5, whereas iodide oxidation and tyrosine iodination were inhibited at 3.4-8.0 pH. Guaiacol and SCN- had the same binding site on the enzyme surface which did not bind HzO,. SCN- competitively inhibited both iodide oxidation and tyrosine iodination and was even able to de-iodinate iodinated tyrosine derivatives. The results indicate that guaiacol oxidation is inhibited by CN- produced by oxidation of SCN- catalysed by lactoperoxidase, whereas iodide oxidation and tyrosine iodination were inhibited due to the competitive action of SCN-.
INTRODUCTION
Thiocyanate (SCN), the detoxication product of cyanide, was for long regarded as metabolically inert. However, the large amount of thiocyanate ions in body fluids was later connected with metabolic events in the thyroid gland (reviewed by Wood, 1975). Thiocyanate ions combine with the prosthetic groups of haematin enzymes, e.g. lactoperoxidase (LPO) and thyroid peroxidase, both of which catalyse thiocyanate oxidation in the presence of H20, (Wood, 1975). Thyroid peroxidase and lactoperoxidase are separate enzymes (Ljunggren and Akesson, 1968; Morrison and Steele, 1968). Thiocyanate ions inhibit the oxidation of guaiacol catalysed by thyroid peroxidase (Hosoya, 1963) and the oxidation of iodide to iodine and the iodination of tyrosine catalysed by LPO (Tenovuo, 1976). The uptake of iodide by the thyroid and salivary glands is inhibited by SCN (Myant, 1960). Despite its inhibitory properties, SCN is necessary for the antibacterial action of the LPO system in human saliva (Klebanoff and Luebke, 1965). The inhibitory action is assumed to result from the oxidation of SCN by LPO in the presence of H202 possibly forming hypothiocyanite ions (Hoogendoorn rr al.. 1977). My aim was to examine the inhibition caused by SCN- on the following reactions catalysed by LPO: (1) the oxidation of guaiacol to dihydroxydimethoxydiphenyl: (2) the oxidation of iodide to iodine; (3) the iodination of tyrosine. The inhibitory action of cyanide, an oxidation product of thiocyanate and a common peroxidase inhibitor, and SCN were compared. The inhibition by SCN- is important because these ions in the thyroid and salivary glands inhibit the formation of iodinated proteins in man, thus quenching iodine metabolism. The results may give information about the effect of increased SCN concentration in body fluids resulting from smoking (Tenovuo and Makinen, 1976) on the formation of iodinated tyrosine derivatives. The physiological concentrations of
SCN- and II, as well as peroxidase given in Tenovuo (1978). MATERIALS
Peroxidase
AND
activities,
are
METHODS
and iodine ussays
Guaiacol was oxidized by the methods of Chance and Maehly (1964) and the enzyme units were calculated as described by Makinen, Tenovuo and Scheinin (1976). After enzymic oxidation of iodide to iodine, the increase in absorbance at 350nm due to the formation of triiodide (Hosoya, 1963) was measured. The procedure and reaction mixture have been described in detail (Tenovuo, 1978). 0.5 pg of LPO in 2ml of reaction mixture were used. The enzymic iodination of r_-tyrosine was performed as described by Tenovuo (1978). Chemicals
Milk lactoperoxidase (E.C. 1.11.1.7) and 3-iodo+tyrosine were purchased from Sigma Chemical Co. (St. Louis, Miss.). Guaiacol was a product of BDH Chemicals Ltd. (Poole, England) and r_-tyrosine of Fluka AG (Switzerland). All other chemicals were products of E. Merck AG (Darmstadt, Germany). RESULTS
7’he inhibitory action of cyanide and thiocyanate ions on the oxidation of yuuiacol catalysed by lactoperoxiduse 10 mM pfl-dimethylglutarate buffer, pH 3.G7.0 was used. A marked difference in the influence of pH on the reaction velocity in the presence of inhibitors was found (Fig. 1). Cyanide ions (Fig. 1B) inhibited the oxidation of guaiacol(5 mM) at all pH values studied, whereas SCN- (Fig. 1A) inhibited only at pH values below 6.5, even 0.1 mM being effective. The inhibition by CN- at pH 7 suggests that CNcompeted with guaiacol for the same binding site (Fig. 2A), whereas CN- at 0.5 and l.OmM concentrations did not affect the affinity between LPO and
899
900
J. TENOVUO
23 20
H -
CONTROL 0.1Ornt.t KSCN 03Ornf.i KSCN
15
10 ? > 5 '0 Y 7 2 o "0 25
8 2 e
20
H
CONTROL
o-0
o.lOrnM
KCN
o-o
030mM
KCN
::
15
-
10
-
-e.i 5c
0
’
I
30
40
,
I
,
60
50
PH
70
Fig. 1. Rate of guaiacol oxrdation as a function of pH in the presence of inhibnors (A) KSCN and (B) KCN. Mean values + SD (n = 6). Hz02 (Fig. 2B) as the apparent K, of O.lOmM was unchanged. Guaiacol and H202, therefore, may have separate binding sites on the enzyme. The inhibitor constant, K,, for CN-, estimated from the Dixon plot, was approx 0.25mM. The inhibitory action of SCNwas studied at pH 5.0 because at higher pH values the ion does not
&a
loo
Fig. 3. Plot of recrprocal velocrty vs recrprocal guaracol concentratron (A) and vs reciprocal H20, concentration (B) for the lactoperoxidase-catalysed oxidation of guaiacol at pH 5.0 in the presence of various concentrations of KSCN. inhibit the oxidation of guaiacol. However, no significant non-enzymic oxidation occurred. As with CN-, SCN- competed with guaiacol (Fig. 3A). Even concentrations of SCN- as low as 0.050.20mM markedly decreased the affinity between LPO and guaiacol indicating the same binding site for guaiacol and SCN-. However, the double reciprocal plot with HzO, at pH 5.0 (Fig. 3B) differed from that at pH 7.0 as it was non-linear. The inhibitor constant for SCNwas 0.50.7mM according to the Dixon plot. The inhibitory action of thiocymate on the oxidation of iodide to iodine cat&wed by lactoperoxidase Guaiacol oxidation and iodide oxidation differed in the pH dependence of inhibition by thiocyanate. With iodide, SCNinhibited at all pH values (Fig. 4): at pH 7 (Fig. 5) inhibition was competitive 30
0
50
100
I
50
I
150 1 200 El M
/
I
h
I
mt.4
I
loo
Fig. 2. Plot of reciprocal velocity vs reciprocal guaiacol concentration (A) and vs reciprocal H,Oz concentration (B) for the lactoperoxidase-catalysed oxidation of guaiacol at pH 7.0 in the presence of various concentrations of KCN.
Fig. 4. Rate of Iodide oxidation as a function of pH m the presence of KSCN at various concentration.
Inhibition
of lactoperoxidase-catalysed
by thiocyanate
!L "I 20
k I
1
I
I
I
01
02
0.3
OX
OS
[KSCN]
WI
only in the presence of H202 (Fig. 6A). Thiocyanate ions released the iodine bound to tyrosine (Fig. 7) or the enzyme (Fig. 6A). indicating competitton between SCN- and iodine. When the iodination of tyrosine was complete, after 30 min. the addition ol SCN _ resulted in de-iodination (Fig. 7B). SCN dciodinated mono-iodotyrosine at a low rate (Ftg. 68). A plot of rO’r, vs [KSCN] gave a stmtlar result to that in Fig. 5. indtcating compettttve mhibttton between SCN and iodide ions. The double rectprocal plots of l/‘r vs li[S] in the presence of SCNand iodide ions produced a similar curve with iodide oxidation
30
10
reactions
mM
Fig. 5. Plot of rojc, against [I] (KSCN) for the lactoperoxidase-catalysed oxidation of iodide at various KI concentrations and at pH 7.0. (0) 10 mM: (0) 20 mM: (a) 50 mM
KI because, had it been non-competitive, all 3 concentrations of KI would have yielded one straight line only. The double reciprocal plots of l/n vs l/[S] were not linear.
The inhibition by thiocyunute Iysed oxldatlon oj yuaiucol
of‘ luctopt~rc~.~~du.\~-~~~ta-
There are two possible explanations for the mhtbitory action of SCN- on the reactions catalysed by LPO: the inhibition is caused by either (I) cyamde. cyanate (CNO) or some other product of the oxidation of SCN. or (2) by a direct inhibitory effect caused by binding to proteins. Thiocyanate is oui-
The inhibitory action of thiocyanate and cyanide ions on the iodination of tyrosine catalysed by lactoperoxidase
Thiocyanate and CN- inhibited the iodination of tyrosine at pH 3.4-8.0. LPO was also iodinated, but
06 0.5 04 03 < -
02
:
0.1
2
0
,‘
06
% 8
0.5
-
04 03 0.2 0.1 0
0
2
4
6 TIME
8
10
12
14
16
(MINUTES)
buffer.pH 7 0. addition of KI (0.6 mM). H202 (0.1 mM) and KSCN (0.1 M). (B) De-iodination of (0.5 M) in 0.01 M phosphate buffer, pH 70 Arrows mdtcate addition of KSCN (0.1 M), Hz02 (0.1 mM) and LPO (0.4 ng;ml).
Fig. 6(A). Iodination and de-iodination of lactoperoxidase (0.4 pg/ml) m 0.01 M phosphate Arrows Indicate mono-iodotyrosme
TIME
Fig. 7(A). Iodination of tyrosine
(MINUTES)
(0.2 M) catalysed by lactoperoxtdase (0.4 pg/ml) in the presence of H202 (0.1 mM). The arrows indicate the addttion of KSCN (0.1 M). (B) De-iodmation of iodinated tyrosine derivatives. Initial reaction mixture contained tyrosine. KI, LPO and HZOz in concentrations presented above. The arrow indicates the addition of KSCN in the following concentrations: (0) 0.01 M: (0) 0.1 M: (@I 0.5 M.
902
J. TENOVUO
The inhibition by thiocyanate of lactoperoxidase-catadized to SOS and either CN or CNO depending upon /ysed oxidation of iodide and the iodination of tyrosine pH, CNO only being produced in very alkaline solutions (Hughes, 1975). Chung and Wood (1970) Iodide oxidation and the iodination of some amino observed with LPO that CN was the initial product acids and proteins are well-known reactions catalysed of the oxidation of SCN by H202, but later an interby peroxidases. Iodine antagonism of SCN occurs in mediate oxidation product of SCN converted CN to the thyroid gland. The nature of the inhibitory mechCNO which is spontaneously hydrolysed NH, and anism is, however, still uncertain. It is only known coz. that SCN may compete with I for oxidizing equivalSCN- ions strongly bind to proteins (Wood, 197% ents and in a secondary way the product of the peroxsometimes even leading to their de-iodination (Carr. idase-catalysed SCN reaction may change the struc1952). The primary site of binding is the arginine resiture of the thyroglobulin, making it less effective for due, the secondary sites being lysine and histidine the biosynthesis of thyroxine (Morrison and Schon(Pande and McMenamy, 1970). Thiocyanate is also baum. 1976). a ligand for metallo-proteins but the bond is generally My results clearly indicate that SCN- inhibits both weak and readily reversible (Wood, 1975). I oxidation and tyrosme iodination independently Therefore. with LPO, both inhibitory mechanisms of pH; furthermore, SCN- de-lodinated tyrosine deare possible, depending on the reaction mvolved. My rivatives, indicating a greater affinity of SCN- than results suggest that guaiacol oxidation is inhibited by I- to tyrosine. The inhibition of I- oxidation by CN- or CNOproduced by oxidation of SCN, SCN- differed from that of guaiacol in two respects: whereas iodide oxidation and tyrosine iodination are (a) the inhibition was not affected by pH, (b) SCNinhibited due to competition of SCN- and iodide was much less effective in inhibiting I- oxidation than ions for the same binding site. With peroxidase-cataguaiacol oxidation. Thus, with I- oxidation, the inhilysed reactions involvmg two substrates, a Michaelis bition was probably not caused by CN- formed by constant was obtained for H202, the first reactant SCN oxidation. With thyroid peroxidase, it is possible with the enzyme, but the velocity was proportional to separate the ability to catalyse the iodination reacto the concentration of hydrogen donor. CN- up to tion whereas guaiacol oxidation activity is unaffected 1.0 M has no effect on the affinity between LPO and (Morrison, Bayse and Danner, 1970). The great affinity of SCN- to proteins appears to produce comHzO, (K, - O.lOmM), but competes with the hydrogen donor guaiacol for the same binding site on the petition with I- and, thus. to inhibit the iodination enzyme. of tyrosme and proteins already at low concenUnlike cyanide or cyanate inhibition, SCNmtrations. With I- oxidation, the SCN- ligand for hiblts the oxidation of guaiacol only in acid solutions metallo-proteins, such as lactoperoxidase. 1s weak where it is competitive with guaiacol. The rate of and reversible and thus a larger concentration of reaction of SCN- with LPO may be affected by pH SCN- is needed for the inhibltion although I- and because the different ionic forms of the enzyme may SCN- have the same binding site on the enzyme. react at different rates with the inhibitor, or because It appears that SCN- in human saliva competithe inhibitor itself ionizes. The main oxidation tively inhibits the iodination of tyrosine residues in product of SCN at acid pH is CN, and a role for salivary proteins catalysed by peroxidases secreted it in the inhibition caused by SCN cannot be from the salivary glands. This may be relevant to the excluded. A low concentration of SCN is Inhibitory. small amount of organically-bound iodine in human even 0.17~1moles per ml of KSCN produces nearly saliva, although many salivary proteins, especially the maximum amount of CN in the presence of H202 amylase and albumin, are very susceptible to iodina(Chung and Wood. 1970). The assumption that CNtion in vitro (Sarimo and Tenovuo, 1977). is responsible for the inhibitory action of SCN presupposes that CN is not formed to any great extent Ackno~letlyemrnts- This mvestlgation was supported by a in neutral or alkaline solutions. Chung and Wood grant from the Finmsh Dental Society. The skilful techm(1970) did their experiments on CN production at pH cal assistance of Mrs A. LZhteenmBki is gratefully acknowledged. 5.7. Further evidence for the inhibition by CN- or CNO- of the oxidation of guaiacol arises from the fact that, although OSCNis the main oxidation REFERENCES product of SCN- catalysed by LPO, at acid pH, Carr C. W. 1952. Studies on the binding of small Ions OSCN- is rapidly converted to CNO- which is hydin protem solutions with the use of membrane elecrolyzed to CO, and NH: (Hoogendoorn et ul.. 1977). trodes. I. The bmdmg of the chloride ion and other inorThis IS why guaiacol oxidation was inhibited by ganic anions m solutions of serum albumin Archs BioSCN- only in the pH range below 6.5. them. Biophy.5. 40, 28&294. The double reciprocal plot of l/r vs 1/[H202] of Chance B. and Maehly A. C. 1964. In. Methods in Enzwno1oy.r. Vol. II, p. 764. Academic Press, New York. guaiacol oxidation at pH 5.0 is non-linear because of Chung J. and Wood J. L. 1970. Oxidation of thiocvanate the mhibltory actlon of high HzO, concentrations. to cyanide and sulfate by the lactoperoxldase-hydrogen This was not conventional substrate Inhibition, but peroxide system. Archs B~ochem. Biophys. 141, 73-78. inactivation of the enzyme by excess H20,. This was Hoogendoorn H., Piessens J. P.. Scholtes W and Stoddard confirmed by incubating the enzyme with 0.05 M L. A. 1977. Hypothiocyanite ion; the Inhibitor formed H,O, for 1 min before the rate of the oxidation of by the system lactoperoxidase-thlocyanate-hydrogen gualacol was measured. When compared with the peroxlde. Curies Res. 11, 77-84. effect of pre-incubation for 1 min without Hz02. the Hosoya T. 1963. Effect of various reagents including gualacol-oxidizing activity of LPO was 40 per cent antIthyroId compounds upon the activity of thyroid perless in the presence of H,O,. oxidase. J. Biol Chew Tokyo 53, 381-388.
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Tenovuo J. 1976 The peroxldatlc actnIt> m the human oral cavtty. .4cru rdont. wmd 34. suppl. 71. p, I6 Tenovuo J. and Makmen K K 1976. Concentratmn of thiocyanate and lontzable lodIne m saliva of smokers and nonsmokers. J. drnr. Re\ 55, 661-663 Tenovuo J 1978. Lactoperoxrdase-catalysed lodme mctabohsm In human saliva Irchc orul Bwl. 23. 253 75X Wood J. L 1975 In: Chrml.\rr\, end Bwchem~,~rr~ o/ Thro~vanrc 4citl clnd It\ Drrnurrtm (E&ted by Neuman A 4 ) pp. 156221 .Academlc Press. London
