Oxidative Cleavage of Tryptophanyl Peptide Bonds during Chemical-and Peroxidase-catalvzed Iodinations*

Abstract Tryptophanyl peptide bonds are oxidatively cleaved by "active iodine" that is generated with H2O2, iodide, and a peroxidase (donor: H2O2 oxidoreductase, EC 1.11.1.7). Complete oxidation of tryptophan derivatives and peptides to the oxindole occurs within 2 min at pH 3 to 5 with either lactoperoxidase or horseradish peroxidase, and a 2.5-fold molar excess of iodide plus H2O2. Under identical conditions, simple tryptophan peptides are cleaved 30 to 40% in 10 min at pH 5.0, and the rate of cleavage is parallel to, but less than, the rate of oxidation. All three constituents (iodide, H2O2, and peroxidase) are required for oxidation and fission to proceed. Iodinating reagents yield similar results, except that they oxidize tryptophan derivatives and peptides (Z-Trp-X, X-Trp-X) over the pH range of 2 to 11. Fission of the tryptophan peptide proceeds in acid media with a maximum at pH 5.0, and no cleavage occurs in alkaline media. A 2.5-fold molar excess of positive halogen reagents, ICl, iodide oxidized with chloramine-T, and N-iodosuccinimide completely oxidize tryptophan peptides at pH 5.0 within 1 min, whereas oxidation by I2 and I3- is 2 and 8 times slower, respectively. Chloramine-T per se oxidizes tryptophan peptides at about the same rate as I2 below pH 6.5, but is unreactive above this pH. All of the iodinating reagents induce complete oxidation within 1 min at pH 9.5. Thirty to forty percent of a tryptophan peptide (e.g. Z-Trp-Gly or Z-Trp-Leu) is cleaved in 10 min at pH 5 by a 2.5- to 10-fold molar excess of all the reagents, except for I3- which promotes half as much cleavage. With all of the iodinating reagents, the rate of cleavage is parallel to, but less than, the rate of oxidation. Iodimetric titrations and pH profiles suggest that the oxidation and oxidative cleavage of tryptophan peptides during iodination proceeds via the mechanism proposed for brominating agents (Patchornik, A., Lawson, W. B., Gross, E., and Witkop, B. (1960) J. Amer. Chem. Soc. 82, 5923). Two equivalents of I+ or I2 convert the indole nucleus to the oxindole, which undergoes spontaneous hydrolysis after cyclization to the iminolactone. Unlike brominating reagents, a third iodine equivalent does not form a stable iodooxindole derivative with several different tryptophan peptide models or oxindole itself. Two exceptions are Z-Trp-Gly and Z-Trp, which are probably iodinated by ICI on the benzyloxycarbonyl moiety. Free tryptophan is oxidized by 2.6 to 2.9 eq of iodine (ICl or I2), but it does not become iodinated in labeling experiments with 131I2 or 131ICl.


SUMMARY
Tryptophanyl peptide bonds are oxidatively cleaved by "active iodine" that is generated with HzOz, iodide, and a peroxidase (donor: HzOz oxidoreductase, EC 1.11.1.7). Complete oxidation of tryptophan derivatives and peptides to the oxindole occurs within 2 min at pH 3 to 5 with either lactoperoxidase or horseradish peroxidase, and a Z&fold molar excess of iodide plus HzOz. Under identical conditions, simple tryptophan peptides are cleaved 30 to 40% in 10 min at pH 5.0, and the rate of cleavage is parallel to, but less than, the rate of oxidation. All three constituents (iodide, Ht02, and peroxidase) are required for oxidation and fission to proceed.
Iodinating reagents yield similar results, except that they oxidize tryptophan derivatives and peptides (Z-Trp-X, X-Trp-X) over the pH range of 2 to 11. Fission of the tryptophan peptide proceeds in acid media with a maximum at pH 5.0, and no cleavage occurs in alkaline media. A Z&fold molar excess of positive halogen reagents, ICl, iodide oxidized with chloramine-T, and N-iodosuccinimide completely oxidize tryptophan peptides at pH 5.0 within 1 min, whereas oxidation by Iz and Is-is 2 and 8 times slower, respectively. Chloramine-T per se oxidizes tryptophan peptides at about the same rate as Iz below pH 6.5, but is unreactive above this pH. All of the iodinating reagents induce complete oxidation within 1 min at pH 9.5. Thirty to forty percent of a tryptophan peptide (e.g. Z-Trp-Gly or Z-Trp-Leu) is cleaved in 10 min at pH 5 by a 2.5-to IO-fold molar excess of all the reagents, except for IB-which promotes half as much cleavage. With all of the iodinating reagents, the rate of cleavage is parallel to, but less than, the rate of oxidation. Iodimetric titrations and pH profiles suggest that the oxidation and oxidative cleavage of tryptophan peptides dur%ng iodination proceeds via the mechanism proposed for brominating agents ( oxindole derivative with several different tryptophan peptide models or oxindole itself. Two exceptions are Z-Trp-Gly and Z-Trp, which are probably iodinated by ICl on the benzyloxycarbonyl moiety. Free tryptophan is oxidized by 2.6 to 2.9 eq of iodine (ICI or I*), but it does not become iodinated in labeling experiments with I3112 or 1311C1.
When an '(active iodine" species, such as I+ or 12, reacts with proteins, substitution and oxidation reactions occur with amino acid side chains. Tyrosine and histidine undergo substitution to form mono-and diiodinated derivatives, while oxidative transformations occur with tryptophan, methionine, cysteine, cystine, and possibly serine and threonine residues (l-11).
Furthermore, it has been discovered that N-iodosuccinimide iodinates and oxidatively cleaves tyrosyl peptides (12) in a manner identical to the oxidative fission promoted by N-bromosuccinimide (13), which also cleaves tryptophan and histidine peptides (14,15).
In a recent report (16) from this laboratory, we found that several different iodinating agents (ICI, chloramine-T-KI, Iz, 13, peroxidase-catalyzed iodination) oxidize tryptophan to the oxindole and that this oxidation results in significant fission of tryptophan peptide bonds at pH 4 to 5. The oxidation and oxidative cleavage of tryptophan peptides by various chemical and peroxidase-catalyzed iodination procedures have been more thoroughly studied and are the subject of this communication.

EXPERIMENTAL PROCEDURE
Materials-All N-benzyloxycarbonyl peptides were obtained from Cycle Chemical, Los Angeles, Calif. nL-Tryptophan and tryptophan tripeptides were products of Schwarz-Mann and P(-indole-3)-propionic acid was supplied by Calbiochem. N-Iodosuccinimide and 2-oxindole (indolin-2-one) were purchased from K & K Laboratories, Hollywood, Calif. N-Bromosuccinimide and chloramine-T were products of Eastman Organic Chemicals.
Solutions of chloramine-T, N-iodosuccinimide, and N-bromosuccinimide were freshly prepared before use. Iodine monochloride (20 mm in 1 M HCl and 2 M NaCl) was prepared as described by Izzo et al. (17); preparation of ICI requires KI rather than KC1 as inadvertently printed in the reference. A stock solution of 15 mM 12 was prepared in ethanol, and 13 consisted of 15 mM I, dissolved in 45 rnM KI. Chloramine-T is the sodium salt of N-chloro-p-toluenesulfonamide and generates sodium hypochlorite in water (16)

RESULTS
Oxidation and Oxidative Cleavage of Z-Trp-Gly by Zodination with Peroxidases-The ultraviolet absorption spectra of Z-Trp-Gly before and after oxidation with active iodine generated by HzOz and horseradish or lactoperoxidases are shown in Fig. 1. The peroxidases induce a 60 to 70% reduction in absorbance within 10 min at 280 nm with a 2.5 molar excess of HzOz and KI. The spectrum of the oxidized Z-Trp-Gly is nearly identical to that reported for the oxindole derivative of Z-Trp (15) and Z-Trp-Gly (16). If enzyme, iodide, or H202 are omitted from the reaction mixture, no alteration in the spectrum of the peptide occurs. Lactoperoxidase activity was destroyed by heating at 100" for 5 min, but horseradish peroxidase activity was unimpaired.
No absorbance peaks for I, at 287 or 350 nm are seen in the mixtures containing iodide, H202, and peroxidase ( Fig. 1) because excess thiosulfate was added to reduce the yellow color of Iz and 13 which would accumulate in the absence of peptide (19). Lactoperoxidase is 2.5 times more efficient on a weight basis than horseradish peroxidase.
The oxidation of Z-Trp-Gly by the peroxidases is identical to that previously reported with chemical iodinating agents (16). Other derivatives that were tried and found to be susceptible to oxidation included Z-Trp, Trp-Gly, nL-tryptophan, and indolepropionic acid. Z-Trp-Gly oxidation as a function of pH is shown at the top of Fig. 2. The maximum rate with horseradish peroxidase is at pH 3 and at pH 5 with lactoperoxidase.
These pH profiles are similar to the pH activity curves for iodide oxidation by the peroxidases.
Oxidation and Oxidative Cleavage of Tryptophan Peptides by ICI, Chloramine-T-KZ, Chloramine T, N-Zodosuccinimide, ZZ, and Is--The effect of pH on the oxidation of Z-Trp-Gly by various iodinating agents and chloramine-T is shown in Fig. 5. N-Iodosuccinimide, ICI, and chloramine-T-K1 had nearly identical pH profiles with 90 to 100% oxidation (equivalent to 77% decrease in absorbance) occurring between pH 2 to 6 and 9.5 to 10.5, whereas two-thirds as much oxidation is seen over the pH range 6.5 to 8.5. Chloramine-T probably oxidizes iodide to ICl (16), The pH profiles for the peroxidase-catalyzed oxidative fission and between pH 2 and 7.5 the order of addition of chloramine-T idase, oxidative fission peaks at pH 4 to 5, out of phase with the tryptophan oxidation pH curve. This difference is probably explained by the fact that the rate of fission is 3 to 4 times faster at pH 5 than at pH 3 (16). Moreover, oxidative fission was measured after 10 min, whereas the oxidation was measured after 1 min.
The kinetics of Z-Trp-Gly oxidation at pH 5 by a 2.5.fold molar excess of H202 and KI with lactoperoxidase and horseradish peroxidases is shown in Fig. 3. Lactoperoxidase oxidizes 90 to 95% of the peptide to the oxindole in 2 min, whereas horseradish peroxidase promotes nearly complete oxidation after about 10 min.
The rates of oxidative fission of Z-Trp-Gly by the peroxidases (Fig. 4) are parallel to, but less than, the rates of oxidation. Lactoperoxidase cleaves 37% of the peptide after 10 min. Glytine formation was verified by thin layer chromatography.  and iodide to the buffered solution of Z-Trp-Gly was not critical. However, if chloramine-T is added to a buffered solution of Z-Trp-Gly and iodide above pH 7.5, diminished oxidation of the peptide is observed at pH 8.5 (dashed line), and no oxidation occurs at pH 9.5 and 10.5. When chloramine-T and iodide are premixed in distilled water and then added to a buffered solution of the peptide at pH 7.5 or greater, the solid line (Fig. 5) was obtained.
Chloramine-T optimally oxidizes Z-Trp-Gly at pH 5.0, but is unreactive at pH 6.5 or above (Fig. 5). However, chloramine-T still effectively oxidizes iodide at pH 7.5 and less so at pH 8.5, as indicated by the curves with chloramine-T and KI. Iz and 13 have similar pH curves, except that 13 is less reactive than Iz below pH 7.5. Very little oxidation with 13is seen here in acid media because the reaction time was only one minute.
1~ and 13 may be converted to a common intermediate in alkali.
This intermediate may be IO-, but cannot be either 103 or 104 since these substances do not oxidize Z-Trp-Gly even after 10 min.' 13 dissociates to Iz and I-, although the equilibrium greatly favors 1, (19). The rate of oxidation of Z-Trp-Gly by a 2.5 molar excess of each iodinating agent was measured at pH 5.0 and pH 7.5 (Fig.  6). ICI, chloramine-T-KI, and N-iodosuccinimide completely oxidize the peptide within 1 min at pH 5.0, whereas chloramine-T and 1~ require 5 and 10 min, respectively. Two-thirds of the peptide is oxidized by I;-at pH 5.0 after 10 min. A slower rate of oxidation is seen at pH 7.5 with all of the agents, except for 13, which is more reactive at pH 7.5 than at pH 5.0, and chloramine-T by itself is inactive toward Z-Trp-Gly at pH 7.5. Similar results were observed with other tryptophan compounds, including Z-Trp-Leu, Z-Trp, Trp-Gly, benzoyl-Trp, indolepropionic acid, Ac-Trp-NH2,2 Gly-Trp-Gly, Glu-Trp-Glu, Lys-Trp-Lys, and Leu-Trp-Leu. As shown in Fig. 7, the rate of oxidative cleavage of Z-Trp-Gly at pH 5.0 by the various iodinating agents is parallel to, but less than, the rate of oxindole formation (Fig. 6), resembling the results obtained with the peroxidases (Figs. 3 and 4). Glycine formation in each reaction mixture was verified by thin layer chromatography.
N-Iodosuccinimide, I*, chloramine-T, ICI, and chloramine-T- K1 have similar rate curves and promote 27 to 36% oxidative fission of the peptide in 10 min. The extent of cleavage in those reaction mixtures containing chloramine-T is probably underestimated because this agent oxidizes glycine to a form that no longer produces a color with ninhydrin.
A 5fold excess of chloramine-T over glycine destroys about 15% of the amino acid in 10 min. 1, displays a slower rate than the other iodinating agents but cleaves one-fourth of the peptide at 10 min. The rate of cleavage by Is-doubles if the reaction is performed at 37" rather than room temperature.
A previous statement (16) that Ia does not oxidatively cleave Z-Trp-Gly was in error because glycine was not detected by thin layer chromatography at that time.

1949
The oxidative cleavage of Z-Trp-Gly by varying concentrations of the iodinating agents and chloramine-T with a fixed amount of Z-Trp-Gly is shown in Fig. 8. Cleavage increases with greater oxidant concentrations and reaches a maximum when the ratio is 2 to 2.5 with N-iodosuccinimide, chloramine-T-KI, and chloramine-T.
There is a moderate decrease in cleavage at high concentrations of these agents, probably because they destroy glycine (as indicated above). Oxidative cleavage with Iz, ICI, and 1, increases quickly up to 2 to 2.5 moles of oxidant and continues to increase to a lesser extent up to a ratio of 10, except for Ia-, which declines slightly.
Part of the greater cleavage with I2 appears to be a solvent effect, since the halide is dissolved in ethanol and increasing concentrations of ethanol were in those reaction mixtures containing increasing amounts of iodine. This interpretation is supported by the finding that cleavage with a 5-fold molar excess of ICI increased from 37 to 53% when the ethanol content of the reaction mixture was changed from 10 to 30% by volume.
Other experiments in which the ratio of the oxidant to Z-Trp- Oxidative cleavage of Z-Trp-Gly by varying concentrations of iodinating agents. Each reaction mixture, in a volume of 3.0 ml, contained 300 nmoles of pH 5.0 acetate buffer, 300 rl of 2 mM Z-Trp-Gly, and the necessary volume of 15 mM iodinating agent or chloramine T (Chlor. 2') to give the ratio of oxidant to Z-Trp-Gly listed on the abscissa. ICI was neutralized to pH 5.0 with NaOH just before use. The reactions lasted for 10 min at 23" and were terminated with 50 ~1 of 0.1 M Na&Oa.
One milliliter was analyzed with ninhydrin and corrected for any color elicited with ninhydrin by the complete reaction mixture in which Na2S203 was added prior to the oxidants.
Each point is the mean of duplicate analyses which agreed within 5% of each other. NIS, N-iodosuccinimide.
Gly was increased, as in Fig. 8, also showed that the oxidation of ICI 2-Oxindole 0 0 0 Z-TrpGly linearly reached a maximum at a ratio of 2 to 2.5, IZ 2-Oxindole 0" 0 0 where nearly complete oxidation occurred. NBS 2-Oxindole 0.3 0.7 1.2 Stoichiometry and Kinetics of Reaction of Iodinating Agents with Typtophan, Tyrosine, Histidine, and Methionine Peptides-Using an iodimet.ric titration procedure, data on the rate and extent of reaction of the iodinating agents, chloramine-T, and N-bromosuccinimide with oxidation-sensitive amino acids is presented in Table I. With the exception of Iz-, all of the iodinating agents and chloramine-T reacted very quickly at pH 5.0 with N-acetyltryptophan amide at a ratio of 2 moles of oxidant per mole of Ac-Trp-NH,.
Other experiments not shown in Table I, with an iodide-sensitive electrode (Orion Research Inc., Cambridge, Mass.) indicated that 1 mole of iodide appeared for each mole of ICI that reacted with AC-Trp-NH*.
The rate of disappearance of the iodinating agents by iodimetry correlates well with the spectrophotometric rate of oxidation of the indole nucleus (Fig.  6). Up to 3.2 moles of N-bromosuccinimide react with Ac-Trp-NH2, as reported by Patchornik et al. (14,15), who indicated that 2 Br+ atoms are reduced during the oxidation of the indole, and 1 Br+ atom is incorporated into the indole. Indolepropionic acid and N-benzoyl tryptophan each reacted with 2 moles of ICI or Iz, but no reaction is detected with oxindole.

ICI
Z-Trp-Gly 1.   Table I). The results with chloramine-T may whereas this molar ratio increases to 2.4 with ICI and to 2.9 with reflect either chlorination or oxidation of Z-Trp-Gly beyond the oxindole.
To determine whether the reaction of more than 2 moles of ICI with Z-Trp-Gly represented covalent attachment of iodine to the peptide, an experiment with labeled ICl was performed.
Z-Trp-Gly was incubated with a 5-fold excess of lalIC1 for 10 min and the unreacted halide was reduced to iodide with excess thiosulfate.
Radioactivity is incorporated into a substance that is adsorbed by a column (15 x 25 mm) of Amberlite XAD-2, after correct,ion for nonspecific adsorption of radioactivity to the column with a reaction mixture that contained excess thiosulfate prior to the addition of 1311C1. This adsorbed substance presumably represents peptide containing covalently linked iodine, since XAD-2 adsorbs Z-Trp-Gly, but not iodide. Similar results were obtained with Z-Trp and Z-Trp-Leu and with peroxidase-catalyzed iodinations. Further experimentation is required to determine whether iodine is attached to the indole or benzyloxycarbonyl moieties of Z-Trp-Gly. Two moles of Iz or N-iodosuccinimide react with Z-Trp-Gly and only 0.9 mole of 13 reacts after 30 min. N-Bromosuccinimide reacts to the extent of 4. Free tryptophan reacts rapidly with 1~ or ICl and consumes 2.7 and 3.0 moles of each oxidant, respectively, after 30 min (Table  I). Analysis with Amberlite XAD-2 of reaction mixtures containing 131ICl or 13112 and tryptophan (or Ac-Trp-NHz) indicated that very little iodine was covalently linked to tryptophan. It thus appears, that with iodination, free tryptophan is oxidized beyond the oxindole and explains our earlier finding (16) that 3 eq of Ia-disappeared for every mole of tryptophan.
Additional results in Table I demonstrate that Z-Tyr-Gly, Z-Met-Gly, and cysteine, but not Z-His-Gly, react rapidly with ICI or Iz at pH 5.0. Free glycine was not detected with ninhydrin in those reaction mixtures that contained any of the benzyloxycarbonyl peptides and thus, no peptide bond cleavage occurs. Inasmuch as diiodophloretylglycine and Z-Tyr-Gly are cleaved by N-iodosuccinimide (12), the failure to detect fission of Z-Tyr-Gly by ICl was unexpected; however, cleavage of diiodophloretylglycine is promoted by IC1.l We may have failed to detect cleavage of Z-Tyr-Gly with ICl because a precipitate (Z-IzTyr-Gly and possibly its dienone lactone) settled out of the reaction mixture, thus preventing further reaction with ICI.
Competition experiments at pH 5.0 revealed that no inhibition of fission of Z-Trp-Gly occurred in 10 min when equimolar amounts of Z-Met-Gly, Z-His-Gly, and Z-Tyr-Gly were mixed separately with Z-Trp-Gly and a 5-fold excess of ICI. At pH 7.5, Z-Tyr-Gly partially inhibited the oxidation of Z-Trp-Gly because tyrosine is more effectively iodinated at an alkaline pH. Further experimentation is necessary to assess fully the relative rates of reaction of the sensitive amino acids with the iodinating agents.

DISCUSSION
The oxidation and oxidative cleavage of tryptophanyl peptides during iodination probably occurs by the mechanism suggested for brominating agents (14,15,20). The reaction scheme is shown in Fig. 9 and is illustrated with Z-Trp-Gly (I). Two equivalents of active iodine (Iz or I+) convert tryptophan to the oxindole (IV) via oxidative and hydrolytic reactions over a wide pH range. At pH 5.0 the oxindole cyclizes to an iminolactone (V), which spontaneously hydrolyzes to the lactone (VZ) and glycine. No significant hydrolysis occurs above pH 7, because iminolactone formation is hindered. A third iodine equivalent of ICl, but not 12, becomes covalently linked to Z-tryptophan derivatives either on the indole or benzyloxycarbonyl moieties, a conclusion supported by iodimetric titration data and labeling experiments. It would appear that the iodine is bound to the benzyloxycarbonyl portion because ICl does not iodinate 2-oxindole, whereas N-bromosuccinimide does (Table I). Moreover, derivatives such as Ac-Trp-NHz, indolepropionic acid, and benzoyltryptophan react with only two equivalents of ICl or Iz (free tryptophan is an exception and has been discussed above). These results indicate that the oxidation potential of ICI is less than that of the brominating agents, since 3 eq of N-bromosuccinimide react with either Z-Trp-Gly or Ac- 15,20,21). The lower oxidation potential of iodinating agents is also reflected in the finding that histidyl peptides are oxidatively cleaved at pH 5 during bromination (13, 22), but not during iodination. In fact, little or no iodination of histidine with ICI or Iz occurs at p1-I 5.0 in 10 min, although iodohistidine formation proceeds readily at an alkaline pH (7).
Iodinating and brominating agents react similarly toward tyrosine, methionine, and cysteine. Some iodinating agents, such as N-iodosuccinimide (12) and ICI, or chloramine-T-KI' oxidatively cleave diiodotyrosyl and diiodophloretyl peptide bonds, whereas, 1~ and 13 substitute onto the phenolic ring, but do not cleave these peptides. Differences between brominating agents in effecting the oxidative cleavage of tyrosyl peptides have also been reported (13,20). Thus, ICI, chloramine-T-KI, and N-iodosuccinimide appear to be similar to N-bromosuccinimide in this respect, while 1~ and Ia-behave more like 2,4,6tribromo-4-methylcyclohexadienone (20). Whether the iodinating agents could be employed as selective agents for peptide cleavage in the same manner as the brominating agents remains to be established. Methionine is oxidized during iodination to the sulfoxide, and cysteine oxidation probably ultimately proceeds to cysteic acid.
The oxidation and oxidative cleavage of simple t)ryptophan peptides during iodination cannot necessarily be extrapolated to larger peptides and proteins that possess secondary structures (16, 23). But numerous examples of tryptophan oxidation in various proteins have been described. Lysozyme is a noteworthy example in which essential tryptophan residues (Nos. 62 and 108) are readily oxidized at pH 4.7 to 5.5 to the oxindole by a small molar excess of 13 per mole of enzyme (24,25). This finding indicates that exposed tryptophan residues may be particularly susceptible to oxidation during iodination. Modification of tryptophan residues during iodination in alkaline media has also been reported for casein (2), thyroglobulin (2), serum albumin (3), and y-globulin (4). Thus, the destruction of biologically active peptides and proteins undergoing chemical or peroxidase catalyzed iodination for metabolic studies and radioimmunoassays (26-32) may be due to the modification of essential tryptophan residues. As previously discussed (1,16,23), the extent and quality of iodination of proteins is dependent on several factors: (a) pH, (b) temperature, (c) iodinating agent, (d) concentration of iodinating agent, (e) nature of the protein.
The peroxidase-catalyzed oxidation and oxidative cleavage of tryptophan peptides with iodide and H202 establishes a new property for this class of enzymes, but the relevance of this phenomenon to biological systems remains to be determined. A possible, pertinent example is the bactericidal (33, 34) and virucidal (35) action of myeloperoxidase or lactoperoxidase with iodide and H,Oz, which could be explained by the oxidation of tryptophan residues in the bacterial or viral proteins by peroxidase-generated active iodine. This possibility seems attractive since optimum killing activity (33, 34) and tryptophan peptide oxidation and cleavage with peroxidase-iodide-Hz02 both proceed at pH 5.0. However, modification of other amino acid residues (e.g. tyrosine (33), methionine, cysteine, and cystine) on the bacterial or viral surfaces could also account for the killing process. Parenthetically, oxidation of tryptophan would be expected by the "active chlorine" generated with a myeloperoxidasechloride-Hz02 mixture that is employed in bactericidal studies (33,36).
A peroxidase in the thyroid gland oxidizes iodide with H202 for iodotyrosine and iodothyronine synthesis (37). Results with a partially purified beef thyroid peroxidase preparation indicate that it oxidizes Z-Trp-Gly to the oxindole in the same manner as lactoperoxidase. 1 A priori, no relationship between tryptophan oxidation and iodotyrosine or thyroid hormone synthesis is obvious at this time. However, it is conceivable that the peroxidase-catalyzed cleavage of tryptophanyl (and possibly diiodotyrosyl) peptide bonds might assist in the intracellular hydrolysis of thyroglobulin, an essential step for the release of free thyroid hormones from the thyroid into the blood plasma for transport to the peripheral tissues. This hydrolysis is primarily accomplished by intracellular proteases and peptidases, but peroxidase may assist this process by forming smaller peptides from thyroglobulin or thyroglobulin fragments, thus, enhancing digestion by proteolytic enzymes.