BIOCHEMI(‘Al. MEDICINE 18, 330-343 (1977)

Entrapment of Phenylalanine Hydroxylase in a Polyacrylamide Matrix

BENJAMIN WEISS,’ MARIA Hur, AND ABEL LAJTHA

Institute of Neurochemistry, Rockland Research Institute Ward’s Island, New York 10035, and

Department of Biochemistry, Columbia University, College of Physicians and Surgeons,

New York, New York IO032

Received April 23. 1977

The basic components of the rat liver hydroxylating system, which catalyzes the conversion of phenylalanine to tyrosine, consists of two enzymes, phenylalanine hydroxylase (Phe H) and dihydropteridine reduc- tase, and two coenzymes, a tetrahydropteridine, and a reduced pyridine nucleotide (Eqs. [l] and [2]) (l-3).

Phenylalanine + tetrahydropteridine + 0, Phe F: tyrosine + dihydropteridine + H,O. 111

Dihydropteridine + TPNH + H+ dihydropteridine tetrahydropteridine + reductase

TPN+ 121

The reaction is irreversible, and, under certain conditions, oxidation of the tetrahydropteridine can be uncoupled from tyrosine formation (4). During hydroxylation, hydrogen, or other substituents such as bromine or chlorine, migrates from the para to the metu position (5). Whether the oxygen molecule undergoes homolytic or heterolytic cleavage during incorporation is not known as are, also, other intimate details of the reaction mechanism.

In our studies on the immobilization of Phe H, it was decided to examine first the method of physical entrapment within a polymer lattice. The advantages of lattice entrapment are experimental simplicity, re- quirement for small amounts of material to produce a water-insoluble enzyme conjugate, and maintenance of the intrinsic properties of the enzyme since no chemical modifications are involved. Polyacrylamide

’ To whom correspondence should be addressed.

330

Copyright @ 1977 by Academic Press. Inc. All rights of reproduction in any form reserved. ISSN WO6.2944

ENTRAPMENT OF PHENYLALANINE HYDROXYLASE 331

was selected as carrier from the neutrally charged water-insoluble ma- trices currently available because of the ease of controlling the size of the polymer’s micropores by varying the degree of cross-linking and the mild conditions that can be used to initiate polymerization (6).

If the immobilized Phe H showed sufficient activity, it was then in- tended to coimmobilize the dihydropteridine reductase from rat liver. If these enzymes together proved active in vitro, their effect would then be examined in phenylketonuria. It was hoped that, by placing the entrapped enzymes, encased in a membrane permeable to low molecular weight compounds, directly into the blood stream or peritoneal cavity, sufficient reduced pteridine would be formed from circulating endogenous or added pteridine and pyridine nucleotides, with or without the addition of a reducing agent such as ascorbate, for the hydroxylation of phenylalanine. A three- to sixfold increase in the rate of hydroxylation of phenylalanine has been reported after the administration of 6-methyltetrahydropterin and ascorbate (7).

MATERIALS

L-Phenylalanine, L-tyrosine, Dt-g-chlorophenylalanine, dithiothreitol, L-leucyl- L-alanine, &TPN+-agarose, catalase, and glucose de- hyrogenase were purchased from Sigma. 2-Amino&hydroxy-6, 7-dimethyl-5, 6, 7, &tetrahydropteridine (DMTHP) was obtained from Aldrich and Calbiochem. Acrylamide, N, N’-methylenebisacrylamide, and N, N, N’, N’-tetramethylethylenediamine (TMED) were from Eastman. All other chemicals were of reagent grade from commercial sources. Rat liver Phe H was prepared by the procedure of Kaufman and Fisher (8). The enzyme was purified either through the first ammonium sulfate fractionation or through the adsorption and elution from calcium phosphate gel which was prepared by two procedures (9, 10). The method for the preparation of dihydropteridine reductase from sheep liver (11) was applied with slight modification to rat liver. Purification was carried through the alkaline ammonium sulfate fractionation.

METHODS

Phenylalanine (12) and tyrosine (13) were determined fluorimetrically, and protein was measured calorimetrically (14). With these methods, tyrosine, but not phenylalanine, can be determined in the presence of p-chlorophenylalanine. Unless otherwise stated, the reaction mixture for the assay of Phe H contained the following components, in micromdes, in a final volume of 1.0 ml; phenylalanine, 10; dithiothreitol, 2.0; catalase, 3,060 units (0.2 mg); DMTHP, 0.75; potassium phosphate buffer, pH 6.8, 100. Usually, 0.2 ml of the Phe H preparation containing 2.0 mg of protein, purified through the first ammonium sulfate fractionation, was used. After incubation for 30 min at 25”C, the reaction mixture was

332 WEISS, HUI, AND LAJTHA

deproteinized with an equal volume of 0.6 N trichloroacetic acid. Optimal conditions for enzyme entrapment during acrylamide polymerization were derived from general procedures described previously (15, 16). Solutions of acrylamide and bisacrylamide were prepared in 0. I M potas- sium phosphate buffer, pH 6.8. Reaction mixtures containing polymer were treated with an equal volume of 0.6 N trichloroacetic acid, followed successively by heating in a boiling water bath for 3 min with dispersion of the polymer by means of a spatula, cooling to room temperature, and withdrawing from the supernatant, after centrifugation, 254 samples for tyrosine determinations.

RESULTS AND DISCUSSION

Inhibition by p-Chlorophenylalanine

It has been reported that the origin of the effect of p-chloro- phenylalanine on Phe H is not known and that all attempts to dem- onstrate a direct inactivation of the enzyme by this compound in liver extracts (17-20) or as a partially purified enzyme (21) have been unsuc- cessful. In an effort to clarify this problem, an experiment was repeated as described by Greengard and Delvalle (22) who reported no inhibition by p-chlorophenylalanine of Phe H from the livers of suckling rats. Using their system and their two different levels ofp-chlorophenylalanine as a sonicated suspension in isotonic saline (Table I), we have confirmed their results. Sincep-chlorophenylalanine is highly insoluble as the free acid, it is unlikely that any of the compound would be available for reaction with

TABLE 1 EFFECT OFP-CHLOROPHENYLALANINE ON LIVER PHENYLALANINE

HYDROXYLASE ACTIVITY IN SUCKLING RATS~

p-ChlorophenylaJanine* Tyrosine” (@mole) (nmole/mg of protein/min)

None 5.41 0.8 (S) 3.42 8.0 (S) 1.80 0.8 (I) 5.41 8.0 (I) 4.45

o Livers from 7-day-old suckling rats were homogenized in 9 vol of 0.15 M KC1 and were centrifuged at 16,OOOg for 15 min. To 0.2 ml of the supemate were added the following components, in micromoles, in a total volume of 1.0 ml: phenylalanine, 10: dithiothreitol, 5: DMTHP, 0.75: potassium phosphate buffer, pH 6.8, 100.

h The p-chlorophenylalanine was used either as a solution (S) previously neutralized to pH 8.0 with I N NH,OH or as an insoluble suspension (I) that was sonicated in isotonic saline. Enzyme and inhibitor were preincubated at 0°C for 5 min before the addition of cofactors. See text for details.

I_ Values are the average of two experiments.

ENTRAPMENT OF PHENYLALANINE HYDROXYLASE 333

the enzyme. If it were made accessible to the enzyme, depending on the conditions of dispersion, results would probably be erratic. Addition of 0.8 and 8.0 kmole of the soluble ammonium salt of the acid under the same conditions produced about 40 and 65% reductions in activity, re- spectively. The ammonium ion alone had no effect. A Lineweaver-Burk plot (Fig. 1) showed unequivocally that the inhibition was competitive. The KI is 3.81 x 10m6 mole. The inhibition produced by p-chloro- phenylalanine was greater in suckling (Table 1) than in mature rats (Fig. 1). As this work was in progress, the same finding of competitive inhibition by p-chlorophenylalanine for human fetal liver Phe H was reported by Woolf (23). Details were not given as to whether the inhibitor was added as a suspension or in solution. Earlier (9), Barranger had shown an inhibition, without indicating the type, by p-chlorophenyl- alanine of the isozymes of rat liver Phe H. In further confirmation of this earlier work (22), it was observed that addition of catalase to the suckling rat liver system had no effect on the hydroxylation, whereas, with purified enzyme preparations, catalase enhanced the for- mation of tyrosine by about 10% (Table 7).

Enzyme isolation

During the purification of Phe H by adsorption and elution from calcium phosphate gel, it was found that enzyme continued to be eluted over a

4-

0.2 0.4 04 0.6

‘/pn~oles Phenylalanine

FIG. 1. Double-reciprocal plot of the effect of phenylaianine concentration on the rate of tyrosine formation in the absence (O---O) and presence (W) of IO pmole of p-chlorophenylalanine.

334 WEISS. HUI, AND I.AJTHA

wide range, up to I.0 M. of phosphate concentrations. Similar results were obtained with samples of gels prepared by different procedures. As much as 55% more Phe H could be removed by treatment with 1.0 M

potassium phosphate buffer, pH 6.8, beyond that eiuted with 0.1 M

phosphate as previously described (8). Since it was not necessary in our work to isolate the purified enzyme, the purification was usually carried through the first ammonium sulfate fractionation. In early studies, we attempted to entrap together these enzymes: Phe H purified through the calcium phosphate adsorption and elution; dihydropteridine reductase, purified through the alkaline ammonium sulfate fractionation; and a TPNH-generating system; glucose dehydrogenase, TPN+, and glucose. Since indications were that too many variables would have been intro- duced, thus complicating our examination of the enzyme, a simple in vitro system (9, 19, 22) employing DMTHP (3) was used.

Effect of Metals and Other Compounds

Prior to the entrapment experiments, a study was made of the effects of metal ions and other compounds on Phe H activity to determine the sensitivity to its chemical environment. The almost complete inhibition previously observed for Cu’+ (I I) is extended to other ions including Cu’+, which may contain traces of Cu2+ as a contaminant, Ag+, and Hg2+ (Table 2). More than 65% of the activity was lost with Cd”+, CO”+, and

TABLE 2 EFFECT OF METALS AND OTHER COMPOUNDS ON

PHENYLALANINE HYDROXYLASE ACTIVITY”

Addition

Relative activity

(94) Addition

Relative activity

(76)

None* 100 Ascorbic acid” 136 Glutathione” 117 Iodoacetate (Na)* 106 MgCI, 100 EDTA (Na# 97 Cysteine” 95 FeCI, 91 MnCl, 91 WCI, * 88 AU, 67 ZnCl, 56

FeCI, coa,* NiCI, NaCN” CdCI, p-HO-Hg-benzoate

&NO, CUCI cuso,

H&h HgCN),

53 33 22 21 13 2 4 2

6 5

o The compound, 10 pmole, except for 20 pmole of ascorbic acid, in 0.1 ml of water was added to 0.2 ml of enzyme containing 2.0 mg of protein. After shaking at 25°C for 15 mitt, the cofactors were added for assay.

h In the total assay system. these solutions remained clear, whereas the others developed traces of turbidity.

ENTRAPMENT OF PHENYLALANJNE HYDROXYLASE 335

Ni2+, whereas A13+, Fe*+, Fe3+, Mg*+, Mn*+, Pb*+, and Zn*+ were less inhibitory. Cysteine, EDTA, and iodoacetate had little or no effect whereas glutathione was stimulatory (24). Although CN- produced about an 80% loss in activity at a concentration of 10 mM, the complete abolition of activity could not be effected, with the loss remaining the same, by a lo-fold increase in concentration. The stimulation produced by ascorbic acid is attributed to reduction of the cofactor from the dihydro to the tetrahydro form (25). Since iodoacetate is without effect, it is doubtful that Ag+, Cu+, Cu*+, and Hg*+ exert their action via sulfhydryl groups. It has been found that the native enzyme contains four completely buried and one partially buried sulfhydryl groups per 50,000 molecular weight subunit (26). Since inhibition by these metals is almost as great with an increase (30 pmole) as in the absence of dithiothreitol, it is believed that they compete with the enzyme’s iron atoms (27), thereby interfering with electron transfer, or that they cause inactivation of the pteridine cofactor.

Effect of Ammonium Persulfate

In the early entrapment studies, it was found that hydroxylation was inhibited when (NH,),S,O, was used to catalyze the polymerization (Fig. 2). It had been reported earlier (8) that persulfate gels, when added to the reaction mixture, produced 90% inhibition of enzyme activity. However, the cause of the inhibition was not studied. In the present work, it was

observed that no change in absorption at 260 and 280 nm occurred when

0 1 2 3 4 pmoler (NH4)2S208

FIG. 2. Phenylalanine hydroxylase, 0.3 ml containing 2.0 mg of protein, was preincu- bated with 0.1 ml of ammonium persulfate of the indicated concentration at 0°C for 10 min. Assays were performed immediately after addition of cofactors.

336 WEISS, HUJ, AND LAJTHA

(NH&&O, was added to varying concentrations of enzyme. Under simi- lar conditions, tyrosine and tryptophan showed a decrease in absorption and were presumably oxidized, whereas phenylalanine remained un- changed. A marked increase in absorption resulted when the persulfate was added to solutions of dithiothreitol or cysteine. Observation of the spectrum of I .O ml of 0. I mM DMTHP at 280 and 300 nm (2) indicated that 0.1 ml of 13 mM (NH&S208 oxidized most of the cofactor within 15 min but not as rapidly as did the bubbling of oxygen for 3 min through the solution. In the presence of 4 Fmole of (NH&!&O, (Fig. 2), tyrosine formation was completely abolished. Essentially the same results were obtained when all of the cofactors were pretreated with (NH&&O8 prior to the addition of enzyme (Table 3). Protection of the enzyme system against (NH&&O, is afforded by a fivefold increase in dithiothreitol to 10 pmole. Preincubation of dithiothreitol with (NH&&O, in the presence or absence of cofactors results in no loss of hydroxylating activity. It is concluded that (NH&&O, probably has no effect on Phe H and that the loss in activity of the hydroxylation system is due to the oxidation of DMTHP. As this work was going to press, it was reported (24) that dithiothreitol regenerates the tetrahydropteridine from dihydropteridine and protects the hydroxylating system from peroxide formed during aerobic oxidation of the tetrahydropteridine.

TABLE 3 EFFECT OF DITHIOTHREITOL ON PHENYLALANINE HYDROXYLASE ACTIVITY

IN THE PRESENCE OF AMMONIUM PERSULFATE

Conditions Relative activity

(%)

Enzyme” 100 Enzyme + (NH&&O,” 38 Cofactors* 117 Cofactors + (NH,),S,O~* 50 Cofactors + dithiothreitol’ 145 Cofactors + dithiothreitol + (NH&&O, 147 Dithiothreitol + (NH,),S,O,d 142

” Enzyme, 0.2 ml containing 2.0 mg of protein, was preincubated at 0°C for 10 min without and with 0.1 ml of 1.76 rmole of (NH,)&Os prior to assay for hydroxylase activity.

b The cofactors phenylalanine, dithiothreitol, catalase, and DMTHP in 0.4 ml of 0.1 M potassium phosphate buffer, pH 6.8, were preincubated at 0°C for 10 min without and with 0.1 ml of 1.76 pmole of (NH&.SoOs prior to the addition of enzyme for assay.

r The concentration of cofactors was the same except that dithiothreitol was increased fivefold to JO PmoJe.

d The 10 pmole of dithiothreitol and 0. J ml of 1.76 PmoJe of (NH,),S,O, were preincu- bated at 0°C for JO min before addition of remaining cofactors and enzyme.

ENTRAPMENT OF PHENYLALANINE HYDROXYLASE 337

Photocatalytic Polymerization

When enzyme, in the presence or absence of catalase, is entrapped by photocatalytic polymerization, a progressive loss in activity occurs with increasing concentrations of acrylamide and bisacrylamide with their ratios held constant (Table 4). Similar losses occur with varying amounts of bisacrylamide with acrylamide held constant (Table 5). No potentiation of effect by bisacrylamide upon acrylamide was observed. TEMED and riboflavin, at the concentrations used, had no effect on hydroxylation. The slightly greater loss in activity when entrapment of enzyme is effected in the absence of catalase (Tables 4 and 5) is probably due to diffusional barriers imposed by the polymer pore size. Since the presence of catalase

TABLE 4 EFFECT OF VARIATIONS OF ACRYLAMIDE AND BISACRYLAMIDE CONCENTRATIONS WITH THEIRRATIOS HELD CONSTANT ON

PHENYLALANINE HYDROXYLASE ACTIVITY"

Relative activity (%)

Polymer concentration acrylamide:

bisacrylamide (%)

Catalase No catalaae

Light* Light

No Yes No Yes

None 100 100 None’ 71 80 153.75 46 50 30 41 30:7.50 46 45 24 22 60: 15.0 34 35 26 33 90:22.5 16 33 14 20 w-15* 13 13 w-30 11 4 W-60 5 3 w-90 5 1

(1 To enzyme (containing 2.0 mg of protein) and 0.2 mg of catalase in 0.3 ml of 0.1 M potassium phosphate buffer, pH 6.8, were added 0.3 ml of the indicated polymer concentra- tion, 0.025 ml of 5% TEMED, and 0.1 ml of 0.4% riboflavin. AAer chilling at 0°C for 10 min, the mixture was irradiated with a fluorescent light for 10 mitt at room temperature. After polymerization, the tubes were rechilled, and hydroxylase activity was assayed following the addition of cofactors.

* Mixtures not subjected to illumination did not polymerize. c Enzyme, in the presence and absence of catalaae, was held at room temperature for 10

min before addition of cofactors. * After entrapment, the gels were washed twice with 0.2~ml portions of 0.1 M potassium

phosphate buffer, pH 6.8, and the combined washings were assayed for hydroxylase activity after the addition of cofactors.

338 WEISS. HUI. AND LAJTHA

TABLE 5 EFFECT OF VARIATIONS OF BISACRYLAMIDE WITH ACRYLAMIDE HELD CONSTANT

ON PHENYLALANINE HYDROXYLASE ACIVITY”

Relative activity (%)

Polymer concentration acrylamide :

bisacrylamide (%)

Cataiase No catalase

light” light

No Yes No Yes

None 100 100 None’ 67 91 3O:lSO 25 25 24 24 30:4.50 22 27 23 30 3oz7.5 37 35 26 31 30: 10.5 18 29 18 16 W-19 3 0 w-4.5 2 0 w-7.5 2 0 w-10.5 2 0

(I Conditions and footnotes are the same as described in the legend to Table 4.

in the complete system enhances the activity by about 10% (Table 7), the effect of its absence in these studies would not be great as can be seen by increasing the cross-linking (Table 5). No difference is observed in the presence or absence of illumination; the latter results in no polymeriza- tion. Less than 15% of the Phe H escapes entrapment, but generally more than 95% of the activity is held by the polymer matrix (Table 5). Standing of the enzyme at room temperature for 10 min, either in the presence or absence of catalase, before the addition of cofactors resulted in a loss of about 10% or more of activity.

Entrapment with Cofactors

Entrapment of enzyme and catalase in a 90% acrylamide:22.5% bis- acrylamide matrix did not protect the enzyme against inactivation (Fig. 3). Although approximately 65% of the initial hydroxylating activity is lost upon entrapment, after 5 hr at 0°C less than 20% of the initial activity remains in either the trapped or untrapped preparation. Entrapment of the enzyme with individual cofactors in 30% acrylamide:7.5% bisacrylamide resulted in a more than 90% loss in activity (Table 6). The omission of cofactors in the presence or absence of the polymer constitutents, without polymerization, yielded similar results (Table 7).

ENTRAPMENT OF PHENYLALANINE HYDROXYLASE 339

0 2 4 6 18

Hours

FIG. 3. The effect of standing on phenylalanine hydroxylase activity was studied after entrapment was effected in 90% acrylamide:22.5% bisacrylamide under the same conditions described in the footnote to Table 4. All activities were calculated on the basis of untrapped ( O-O ) enzyme at zero time. The untrapped and trapped ( 0-O ) enzymes were held in an ice bath for the indicated period and then were promptly assayed after the addition of cofactors.

TABLE 6 EFFECT OF ENTRAPMENT OF ENZYME AND COFACTORS ON

PHENYLALANINE HYDROXYLASE ACTIVITY

Cofactor entrapped

No entrapment” PhenylaIanineh Dithiothreitol Catalase DMTHP’ Phenylalanine + dithiothreitol Phenylalanine + catalase Phenylalanine + catalase + dithiothreitol Phenylalanine + DMTHP

Relative activity (%)

100 5 2 3 4 6

10 11 9

u In the absence of polymer, 106% activity represents the formation of 13 nmole of tyrosine/mg of proteimmin.

b In all instances, the solid cofactor was added to 0.2 ml of the enzyme solution containing 2.0 mg of protein, followed by addition of 0.2 ml of 30% acrylamide:7.5% bisacrylamide, 0.025 ml of 5% TEMED, and 0.1 ml of 0.4% riboflavin. After chilling at 0°C for 10 min, the reaction mixture was polymerized, rechilled, and assayed after the addition of the remaining cofactors in a total volume of 1.0 ml at the concentrations described in the text.

r Polymerization was inhibited by DMTHP at the level used in the assay.

340 WEISS. HUJ, AND LAJTHA

TABL>E I EFFECT OF COFACTORS ON PHENYLALANINE HYDROXYLASE ACIIVIT\

IN THE PRESENCE OF POLYMER COMPONENTS”

Activity (‘%‘c)

Omission No polymer Polymer”

None 100 100 Phenylalanine 0 0 Dithiothreitol 24 42 Catalase 89 89 DMTHP 0 0

0 To 0.2 ml of enzyme containing 2.0 mg of protein was added 0.3 ml of 30% ac- ryJamide:7.5% bisacrylamide, 0.025 ml of 5% TEMED, and 0.1 ml of 0.4% riboflavin. After standing at 0°C for 10 min, cofactors were added, and hydroxylase activity was assayed.

b Activity is calculated on the basis of complete polymer-containing system.

Effect of Polymer

Almost all hydroxylating activity was abolished when the enzyme was shaken prior to assay at 25°C for 10 min with acrylamide at a final concentration of 30% (Fig. 4). At 0°C for 10 min and at the same concen- tration of acrylamide, about 75% of the initial activity was retained. The progressive loss of Phe H activity is attributed to denaturation of the

0 20 40 60 80

Per cent Acrylamide

FIG. 4. Phenylalanine hydroxylase activity was examined in the presence of varying concentrations of acrylamide. Enzyme, 0.2 ml containing 2.0 mg of protein, and acrylamide, 0.2 ml of the indicated concentrations, were chilled at 0°C for 10 min ( O-O ) or were shaken at 27°C for 10 min ( 0-O 1. After all samples were chilled, hydroxylase activity was assayed following the addition of cofactors.

ENTRAPMENT OF PHENYLALANINE HYDROXYLASE 341

enzyme by acrylamide. That the denaturing effect is not exerted on catalase is evident from the similar activities obtained in the presence and absence of polymer when catalase is omitted from the system (Table 7). The denaturing effect of acrylamide is consistent with its similarity in structure to other amidic compounds such as urea. It is probable that the dimer, bisacrylamide, would exert the same effect, but this could not be demonstrated because of its limited solubility. Although the enzyme was present as a complex, any action by acrylamide on the reductase would not be apparent in the current assay system. Since raising the temperature to 30°C would cause dissociation of the dimeric isozyme, MW 110,000, to the monomeric form, 55,000 (8), it is possible that the combined effects of temperature and acrylamide act on the low molecular weight units, whereas, under the same conditions at 0°C the dimer is relatively stable. A similar denaturing effect has been attributed to the action of acrylamide on horse serum butyrylcholinesterase (28).

Effect of Various Conditions

Since attempts are currently being made to modify the enzyme, its activity was studied under a variety of conditions. Preincubation at 0°C for 5 min with ethanol and n-butanol at levels of 33%, followed by addition of cofactors, depressed the hydroxylase activity by 19 and 13%, respec- tively. At 50% concentrations of ethanol and n-butanol, the remaining activity was 0 and 14%, respectively. Little or no loss in activity occurs when the final concentration of glycerol is 50%. Previous saturation of the enzyme solution with O2 or Nz at 0°C had little or no effect on the storage stability in the refrigerator or freezer. The loss in activity with prolonged blending during preparation of the enzyme (8) may be due to the release of intracellular proteinases. When the TPNH-generating system was used for assay, it was found that /I-TPN+-agarose substituted equally well on a molar basis as the unbound nucleotide. Although a marked stimulation by surfactants has been reported with Phe H preparations of varying purity (29-31), no effect was found at concentrations of 0.02 and 0.2% with Tween 20, Tween 80, Brij 30, sodium deoxycholate, and Dow Corning antifoams, C, H-10, and Q. The failure to obtain a stimulatory effect with these surfactants is consistent with earlier findings (23) for other surfac- tams.

CONCLUSION

Our efforts have been focused on obtaining a stable Phe H preparation which can be used in the treatment of phenyiketonuria, a subject which has been of considerable biochemical and clinical interest (1, 9, 32, 33). Although hydroxylation can proceed in a polyacrylamide matrix obtained by photocatalytic polymerization, no protection was conferred upon the

342 WEISS, HUI, AND LAJTHA

enzyme by the gel. Thus, other methods are being sought for stabilization. The immobilization of Phe H by covalent linkage to an insoluble support will be the subject of a forthcoming report.

SUMMARY

Rat liver phenylalanine hydroxylase has been shown to be competi- tively inhibited by p-chlorophenylalanine. More than 90% inhibition was produced by Ag+, Cu+, Cu2+, and Hg2+. It is assumed that these metal ions do not exert their action via sulfhydryl groups since iodoacetate is without effect. The observed inhibition might be due to competition with the enzyme’s iron atoms by interfering with electron transport or to inactivation of the pteridine cofactor. Inhibition of hydroxylation by am- monium persulfate was caused by oxidation of the cofactor, 2-amino-4- hydroxy-6, 7-dimethyl-5, 6, 7, 8-tetrahydropteridine: no inhibition oc- curred in the presence of an excess of dithiothreitol. Approximately 35% of the hydroxylating activity was retained in a polyacrylamide matrix after photocatalytic polymerization. The progressive loss of phenylalanine hy- droxylase activity with increasing concentrations of acrylamide was at- tributed to denaturation.

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ENTRAPMENTOFPHENYLALANINEHYDROXYLASE 343

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