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Rapid colorimetric assay for intestinal peptide hydrolases
ANALYTICAL
Rapid
BIOCHEMISTRY
Calorimetric
61, 7285 (1974)
Assay
for Intestinal
Peptide
CHON R. SHOAF, KURT J. ISSELBACHER, WILLIAM D. HEIZERl Departments of Medicine, Chapel Hill, North Carolina,
University
of North
and Harvard
Medical
Received November
Hydrolases AND
Carolina Medical School, School, Boston, Massachusetts
27, 1973; accepted March
19, 1974
A simple two-step method is described for quantitating the release of free L-phenylalanine, L-leucine, L-methionine, or L-isoleucine from di- or polypeptides. The calorimetric assay is based on the ability of L-amino acid oxidase to catalyze the oxidation of free L-amino acid, but not of peptides. The potent metal chelator l,lO-phenanthroline, which is included in the second step of the assay, effectively inhibits peptide hydrolase activity thus permitting the assay to be carried out in two sequential steps in the same test tube with no intervening enzyme-destroying step. Although the assay is indirect and subject to interference by some chemicals, it is not affected by a large number of compounds frequently used in enzyme studies. The method was used to study the subcellular distribution of a number of peptide hydrolase activities in rat intestinal mucosa. For nine substrates, more than 80% of the total recovered activity was present in the cytoplasmic fraction while for two substrates, phenylalanylglycine and phenylalanylglycylglycine, more than 60% of the activity recovered was present in the particulate fraction.
A number of assays for peptide hydrolase activity have made use of the fact that snake venom L-amino acid oxidase catalyzes the oxidative deamination of some free L-amino acids but not of peptides (l-6). Lewis and Harris (6) coupled this reaction with the oxidation of o-dianisidine by hydrogen peroxide for detection of peptide hydrolase activity in starch gels. More recently, quantitative assays which make use of these coupled reactions have been described (3-5). The published methods which consist of an incubation step stopped by heat or perchloric acid, followed by a second step for detection of liberated amino acid, are very analogous to the disaccharidase assay described by Dahlqvist in 1964 (7). This report describes a shorter two-step assay for peptide hydrolase activity. It is analogous to the modified disaccharidase assay described
‘Send reprint requests to, at the Dept. of Medicine, Carolina, Chapel Hill, NC 27514. 72 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
University
of North
PEPTIDE
HYDROLASE
by Dahlqvist in 1968 (8). A brief preliminary has been published previously (9). MATERIALS
AND
73
ASSAY
description
of this assay
METHODS
Materials Peptides, all of L-amino acids, were obtained from Biosynthetika (Oberdorf, Switzerland) and from Sigma Chemical Company. Lyophilized Crotalus adamanteus venom and o-dianisidine were obtained from Sigma Chemical Company, free amino acids from Mann Research Laboratories, l,lO-phenanthroline from Fisher Scientific Company, dithioerythritol from Cycle Chemical Corporation, and horseradish peroxidase from Worthington Biochemical Corporation. All other chemicals used were of reagent grade. Purity of the peptides and amino acids used was determined by high voltage paper electrophoresis by the method previously reported (2). Tissue Preparation Sprague-Dawley rats (175350 g males) were fasted overnight and killed with a blow on the head. Mucosa of the small intestine was obtained by scraping with a glass slide. Cytoplasmic and pellet fractions were effected by homogenizing the mucosa with a Potter-IElvehjem homogenizer in 5-10 vol (5-10 ml/g wet wt) of ice-cold 0.25 M sucrose and centrifuging for 1 hr at 87,000g at 4°C. The supernatant thus obtained was the cytoplasmic fraction. The pellet was washed three times in 0.155 M NaCl containing 1 mM NaHCO.?, pH 7.4, and suspended in one-half the initial volume of the same solution. Centrifugation for 30 min at 87,000g followed each wash. Purified brush borders were prepared as described by Forstner et al. (10) with slight modifications (11). To minimize loss of possible metal cofactors, normal saline was used in place of ethylenediaminetetraacetate (EDTA) solutions for precipitation of nucleic acids, filtration through glass wool, and subsequent washing. Examination by phase contrast microscopy revealed isolated brush borders with little debris. Enzyme
Assay
The standard assay is carried out in two steps at 37°C. A 15-min incubation step during which the peptide substrate is reacted with the enzyme to be assayed is followed by a second step in which released free amino acid is detected. Peptide hydrolase activity is effectively blocked during the second step both by dilution of the enzyme and by the presence of a potent metal chelator, l,lO-phenanthroline.
74
SHOAF,
ISSELBACHER
AND
HEIZER
The standard incubation mixture has a volume of 0.5 ml and contains 5 or 10 pmoles of peptide substrate, 50 pmoles of Tris-HCl buffer, pH 8.0, and enzyme. Buffer and substrate are added first, brought to 37°C in a water bath, and the reaction initiated by adding the enzyme preparation to be assayed. After 15 min, 2.0 ml of a phosphate-amino acid oxidase-phenanthroline reagent is added which stops the peptide hydrolase reaction and starts the free amino acid detection step. Snake venom n-amino acid oxidase in this reagent oxidizes certain L-amino acids to keto acids and hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide oxidizes o-dianisidine to a somewhat unstable brown colored product with maximum absorbance at approximately 420 nm. Two milliliters of 56% H,SO, are added to give a very stable pink colored product with maximum absorbance at 530 nm. Preliminary studies showed that a final H,SO, concn of at least 25% is required to give optimal absorbance readings. Reagents 1. 0.5 M Tris-HCl buffer, pH 8.0. 2. 0.05 or 0.1 M Peptide in water. Made fresh daily to avoid the possibility of spontaneous or bacterial hydrolysis or formation of cyclic compounds, Most peptide solutions can probably be safely stored frozen. 3. Buffered substrate, mixture of equal quantities of reagents 1 and 2. 4. 1.0 mM Amino acid standards, prepared in water and stored frozen in 2-3 ml aliquots. 5. 12 rnx l,lO-Phenanthroline in buffer, 2.38 g of l,lO-phenanthroline dissolved in 1 liter of 0.2 M sodium phosphate buffer, pH 7.0, by stirring 8-12 hr at room temperature or 3-8 hr at 40-8O”C. Stable at least 1 mo in light-protected bottle at room temperature. 6. L-Amino acid oxidase solution, 10 mg lyophylized Crotalus adamanteus venom/ml of water, mixed gently, allowed to stand for 10 min at O”C, centrifuged 10 min at 5OOg, and supernatant saved. Solution prepared fresh just before use. 7. o-Dianisidine solution, 10 mg o-dianisidine/ml of water. Stable for several weeks at 4°C in light-protected bottle. Discarded when dark colored. 8. Peroxidase solution, 10 mg horseradish peroxidase/ml of water. Stable several months frozen in 2-3 ml aliquots. 9. Phosphate-amino acid oxidase-phenanthroline solution. This reagent is prepared no longer than 2 hr before its use as n-amino acid oxidase activity decreases to suboptimal levels in 3 hr. It is kept in a foil-covered bottle at room temperature because a precipitate forms at lower temperatures. The following or any similar proportions are used:
PEPTIDE
HYDROLASE
reagent reagent reagent reagent 10. 56% Sulfuric
ASSAY
5-100 ml 6-1.5 ml 7-1 .O ml 8-O. 1 ml
acid (v/v).
Procedure Step 1. The incubation mixture, placed in 8 ml test tubes, consists of 0.2 ml buffered substrate, 0.01-0.3 ml of enzyme plus any additives such as inhibitors or metals, and distilled water to give a final volume of 0.5 ml. Step 2. After an appropriate time (15 min for the standard assay), 2.0 ml of phosphate-amino acid oxidase-phenanthroline solution (reagent 9) are added, mixed, and the tubes are returned to the water bath. After 45-60 min, 2.0 ml of 56% H?SO, are added, mixed thoroughly, and ahsorbance is read at 530 nm within 4 hr. Blanks are treated in the same manner except that enzyme is not added until immediately following addition of reagent 9. Standards are identical to the blanks except that 0.05-0.2 ml (0.05-0.2 @moles) of amino acid standard is included in the incubation mixture. One unit of activity is defined as that amount of enzyme which catalyzes hydrolysis of 1 pmole of substrate,‘min under standard conditions. In the studies reported here, all assays were performed in duplicate and values recorded are the means of the duplicate readings. Semimicro
Assay
Excellent results were also obtained when each of the volumes was reduced to one-fifth that given above. In this semimicro assay, the incubation mixture contains 1.0 or 2.0 pmoles of substrate, 10 pmoles of Tris-HCl buffer, pH 8.0, and enzyme in a total volume of 0.1 ml. In the second step, 0.4: ml of reagent 9 and finally 0.4 ml of 56% H,SO, are added. RESULTS
Reactivity
of Various Amino Acids
Only 6 of the 20 free L-amino acids investigated yielded significant absorbance readings when tested under standard conditions of t,he assay (Table 1). Three amino acids, phenylalanine, leucine, and methionine, gave similar results while tyrosine, isoleucine, and tryptophan gave lower absorbance readings. After addition of reagent 9, absorbance produced
SHOAF,
Absorbance
Amino
ISSELBACHER
AND
HEIZER
TABLE 1 Produced by 100 nmoles of Various Under Standard Assay Conditions acid
LPhenylalanine LLeucine GMethionine n-Tyrosine n-Isoleucine L-Tryptophan 14 Others
Amino
Acids
Absorbance ,363 ,344 ,322 ,291 ,117 ,031 <.030
by phenylalanine, leucine, methionine, and tyrosine reached a plateau in 45 min while 90 min were required for tryptophan and more than 90 min for isoleucine. Absorbance read at 45 min increased linearly with increasing quantities of phenylalanine, leucine, methionine, or isoleucine present while results for tyrosine and tryptophan were not linear (Fig. 1). The molar extinction coefficient for phenylalanine was determined to be 1.59 x 104 M-l cm-l.
FIG. 1. Absorbance as a function of nanomoles of amino acid present. Assays were performed as described in Methods except that no enzyme was present, and the step 1 incubation was omitted. n-phenylalanine (a), L-methionine (A), L-leucine (0)) L-tyrosine (0)) L-isoleucine ( n ) , n-tryptophan (A).
PEPTIDE
HYDROLASE
ASSAY
77
In preliminary experiments, each component in the second step of the assay (venom, o-dianisidine, and peroxidase) was tested separately and the amount given for the standard assay procedure is slightly more than that amount which gave maximum absorbance at 45 min for phcnylalanine. Although greater absorbance readings for tryptophan, tyrosine, and isoleucine were achieved following a threefold increase in the amount of L-amino acid oxidase (venom) in reagent 9, the values for these three amino acids remained less than those for phenylalanine, leucine! and methionine. To determine whether any amino acids would interfere with the accurate determination of phenylalanine, 0.1 pmole of phenylalanine was assayed in the presence of 0.1 Ilmole of each of the 19 other amino acids. After subtracting the absorbance, if any, attributable to the additional amino acid, the amount of phenylalanine present was accurately determined in the presence of all but three amino acids, namely cysteine, tyrosine, and tryptophan. When these were included in the assay, recovery of phenylalanine was only 65-85%. Suitable
Dipeptides
Based on the above studies, it was evident that this assay could be used to detect hydrolysis of dipeptides which met certain criteria. The dipeptide must contain at least one of the suitable amino acids, phenylalanine, leucine, methionine, or isoleucine, and it should not contain tyrosine, tryptophan, or cysteine. Substrates containing two different suitable amino acids are acceptable for use in the assay. The amount of such substrates split is calculated by using the average absorbance of the two amino acid standards and halving the micromoles of free amino acid detected. Inhibition
of Peptide Hydrolase
Activity
by l,lO-Phenanthroline
Since many peptide hydrolases appear to require a divalent cation for activity, it seemed likely that a potent metal chelator could be used to inhibit peptide hydrolase activity during the second step of the assay. The compound, l,lO-phenanthroline, which was chosen for this purpose, was shown not to interfere with color development in the second stage of the assay. The effectiveness of l,lO-phenanthroline in inhibiting peptide hydrolase during the second step of the assay is shown in Fig. 2. The minimal absorbance which developed in the presence of the chelating agent corresponds to the blank observed in the standard assay. Results shown are for particulate phe-gly hydrolase and are similar to results for other peptide hydrolase activities in the particulate and cytoplasmic fractions of intestinal mucosa. Particulate gly-phe hydrolase activity.
78
SHOAF,
ISSELBACHER
IO 20 MINUTES
AND
HEIZER
30 40 50 60 70 OF INCUBATION
80
FIG. 2. Inhibition of peptide hydrolaae activity by 12 rn~ l,lO-phenaathroline. Immediately after adding enzyme to standard incubation mixtures, phosphate-amino acid oxidase-phenanthroline reagent was added to one mixture (a-@), and the same reagent without l,lO-phenanthroline was added to the other (O-0). Aliquots were removed at intervals, mixed with H,SO, and absorbance read at 530 nm.
which is less completely inhibited by l,lO-phenanthroline, gave slightly higher blank readings in the presence of very active enzyme preparations. Absorbance of blanks in the standard assay varied between 0.02 and 0.05 for all substrates except leucineamide, met-met, and phe-pro where absorbance of the blank was as high as 0.1 because of the presence of free amino acid in the substrate. Reaction Kinetics The quantity of free L-phenylalanine released from phe-gly and glyphe by purified brush borders from rat intestinal mucosa increased linearly with time up to 22.5 min (Fig. 3). The rate of hydrolysis of gly-phe
FIG. 3. Peptide hydrolysis as a function of duration of incubation. borders were incubated with phe-gly (04) and gly-phe (0-O) shown. Otherwise standard conditions were used.
Purified brush for the times
PEPTIDE
,q
FIG. 4. added to standard hydrolysis
BRUSH
HYDROLASE
BORDER
ASSAY
79
PROTEIN
Peptide hydrolysis as a function of the amount of brush border protein the incubation mixture. Purified brush borders were incubated under conditions with phegly (m-0) and gly-phe (0-O). The maximum shown in the graphs represents less than 5% hydrolysis of the substrates.
and phe-gly also increased linearly with increasing quantity of purified brush border protein added to the incubation mixture (Fig. 4). Cytoplasmic and particulate preparations of rat intestinal mucosa gave similar zero order kinetics for hydrolysis of gly-phe, phe-gly, met-gly, gly-leu, phe-pro, pro-phe, ser-phe, phe-gly-gly, and leu-gly-gly. The contribution of cytoplasmic and pellet enzymes to the total recovered peptide hydrolase activity against each of these substrates for rat small intestinal mucosa is shown in Table 2. Previous reports (2,3) have documented that aminopeptidase activity of Crotalus adamanteus venom and of purified L-amino acid oxidase may obviate the use of these preparations in assays of amino-peptidase activity because free amino acid released from tripeptides and other polypeptides by the snake venom enzyme cannot be distinguished from that released by the peptide hydrolase being assayed. Auricchio et al. (3) attempted to overcome this problem by using purified L-amino acid oxidase rather than crude snake venom. They were able to accurately determine the amount of free amino acid released from sometri- and tetrapeptides but not from others. A small concentration of venom and a low ratio of venom to polypeptide in the assay mixture should minimize the problem posed by the contaminating aminopeptidase. In the present assay in which only slightly more than the optimal amount of venom for assay of phenylalanine, leucine, and methionine was used, the concentration of crude
80
SHOAF,
ISSELBACHER
AND
TABLE 2 Distribution of Peptide Hydrolsse Activities and Particulate Fractions of Rat Small
HEIZER
Between Intestinal
Cytoplasmic Mucosa
Supernatant
Substrate L-Phenylalanyl-L-proline n-Prolyl-n-phenylalanine Glycyl-Lphenylalanine Glycyl-n-leucine n-Seryl-L-phenylalanine L-Alanyl-n-phenylalanine n-Histidyl-n-phenylalanine n-Leucylglycylglycine n-Methionylglycine LPhenylalanylglycine GPhenylalanylglycylglycine
Units/g mucosa 11.5 32.9 269 307 130 129 25.3 102 72.4 10.6 4.3
Pellet
$& of Total 95.0 92.9 92.8 92.0 90.3 89.3 58.2 87.1 82.0 38.7 30.3
Units/g mucosa .6 2.5 20.9 26.6 14.0 15.4 3.4 15.1 15.9 16.8 9.8
$& of Total 5.0 7.1 7.2 8.0 9.7 10.7 11.8 12.9 18.0 61.3 69.7
venom was 120 pg/ml giving a ratio of 30 pg of venom/pmole of substrate which is a much lower ratio than used in previous assays (2,3). Furthermore, l,lO-phenanthroline decreases the activity of the snake venom aminopeptidase. Thus, as shown in’Fig. 5, the combined effect of dilute venom and inhibition by l,lO-phenanthroline prevented the release of interfering amounts of free leucine from leu-gly-gly. Similar results were obtained with phe-gly-gly and phe-gly-phe-gly.
TIME
(MINUTES)
RG. 5. Effectiveness of low venom concentration and l,lO-phenanthroline in inhibiting release of free leucine from leu-gly-gly in step 2 of the assay. Water (2.4 ml) and buffered leu-gly-gly solution (1.6 ml) were incubated at 37°C with 16 ml of reagent 9 (a-@), reagent 9 from which l,lO-phenanthroline was omitted (O-O), reagent 9 with tenfold excess snake venom (A-A), and reagent 9 without l,lO-phenanthroline but with tenfold excess snake venom (A-A,). At the times indicated, 2.5 ml of each mixture was removed, mixed with 2.0 ml of 56% HzSOs and absorbance read at 530 nm.
PEPTIDE
S,ubstances Which Interfere
HYDROLASE
ASSAY
81
With the Assay
One of the major drawbacks of indirect assays, especially those involving enzymes as components, is that many substances may alter the reactions. Compounds which have been found to interfere with this assay at concentrations ordinarily used in enzyme studies include tryptophan, tyrosine, cysteine, dithioerythritol, mercaptoethanol, ammonium persulfate, S-phenylglycine, /3-phenylpyruvic acid, butanol, CoCl,, and FeCl,. Very small concentrations of CoCl,, butanol, and cysteine do not interfere. Compounds which do not interfere with the assay at concentrations ordinarily encountered include p-hydroxymercuribenzoate, EDTA, l,lOphenanthroline, MgCl,, CaCl,, CuCl,, NaI, LiCl, sodium pyrophosphate, sodium citrate, sodium acetate, sodium bicarbonate, Krebs Ringer phosphate, Ampholine carrier ampholytes, Triton X-100, sodium azide, chloroform, toluene, p-phenyllactic acid, hippuryl-n-phenylalanine, hippuric acid, hippuryl-L-arginine, hippuryl-L-lysine, p-tosyl glycine, phenylmethyl sulfonyl fluoride, N-acetyl-nL-phenylalanine, N-chloroacetyl-nphenylalanine, glycyl-P-alanine, hydrocinnamic acid, n-phenylalanine, T-BOC-n-phenylalanine, p-tosyl-glycine, N-acetyl-L-phenylalanine, and tertiary butyl-threonyl-phenylalanyl-proline. Zinc at’ 0.1 mM in the incubation step resulted in higher than normal blanks due to the inability of l,lO-phenanthroline in the second step of the assay to completely inhibit peptide hydrolase activity in the presence of high concentrations of ZIP+. This effect was very pronounced in the case of brush border leu-gly-gly hydrolase (aminopeptidase) activity which could be accurately measured in the presence of 0.1 mM Zn”+ only by heat-killing the enzyme to terminate the first step of the assay. Freshly prepared MnCl, had an extraordinary effect on the assay. At 0.6 m&r concentration, Mn”+ increased the absorbance produced by tryptophan, tyrosine, isoleucine, leucine, methionine, and phenylalanine by 728, 84, 63, 34, 28, and 28%, respectively. Maximum absorbance for phenylalanine occurred when Mn?+ concentration in the initial incubation step was between 0.5 and 1.0 mM. This color enhancing effect of Mn*+ did not depend on the presence of l,lO-phenanthroline and did not occur when Mn?+ was added just before or just after addition of H&SO,. Furthermore, presence of this cation increased by 26% the absorbance produced by glucose when assayed by the glucose oxidase method. Since the glucose assay, as the peptide hydrolase assay described here, involves oxidation of o-dianisidine by hydrogen peroxide, it seems likely that Mn?+ has its effect on this reaction. This hypothesis is supported by the fact that the absorbance produced by adding free hydrogen peroxide to reagent 9 was increased 410% by the presence of Mn?+. However, when free hydrogen peroxide and Mn2+ were mixed before addition of reagent 9,
82
Absorbance
SHOAF,
ISSELBACHER
AND
HEIZER
TABLE 3 Produced by 100 nmoles of Various Amino was Present in the Step 1 Incubation Amino
acid
L-Tyrosine LPhenylalanine LLeucine LTryptophan LMethionine LIsoleucine LHistidine 13 Others
Acids When Mixture
0.5 mM MnC12
Absorbance ,558 .543 .434 ,427 .423 ,185 .123 <.03
the absorbance was 57% less than when Mn2+ was absent. This decrease probably results from reduction of hydrogen peroxide to water by MI?+. Taken together these data suggest that at least part of the effect of Mn2+ on our assay occurs at the o-dianisidine-hydrogen peroxide step. Additional studies will be required to further elucidate the mechanism of action of Mn2+ in these reactions, especially to determine why the ion increases the absorbance produced by some amino acids more than others. When 0.5 mM Mn2+ was present in the step 1 incubation mixture, it markedly altered the relative intensity of color produced by the amino acids (Table 3, compare with Table 1). In addition, presence of this cation caused histidine to give significant absorbance readings (Table 3). The presence of Mn2+ did not cause absorbance to increase linearly with increasing amounts of tyrosine and tryptophan. Some amino acid derivatives are oxidized by snake venom L-amino acid oxidase and cause color development when they are present in the assay mixture. Those tested include p-amino-m-phenylalanine, p-nitronn-phenylalanine, p-fluoro-m-phenylalanine, and nn-y-amino-phenyl acetic acid. Color Stability
of Assay Products
The brown color of oxidized o-dianisidine is somewhat unstable. It decreases slowly with time, and we observed that the color was also affected by light. Absorbance of the brown solution, which was maximal at 450 nm, fell on exposure to incandescent or fluorescent light of moderate intensity for 3 min and slowly increased again over a period of 30-60 min after the solution was placed in the dark. In contrast, absorbance of the pink solution (maximal at 530 nm) which resulted from addition of H&SO, was not affected by light and decreased less than 5% overnight at
PEPTIDE
HYDROLASE
ASSAY
83
room temperature. An aliquot of the brown solution whose absorbance had been reduced 20% by exposure to light gave the same reading at 530 nm as a nonexposed aliquot after addition of H,SO, to both solutions. DISCUSSIOI’G
The fact that L-amino acid oxidase will react with free amino acids but not with peptides was first used as the basis for a peptide hydrolase assay by Zeller and Maritz ( 1). The present assay and several other recently reported methods (2-6) are based on this specificity of Crotahs adamantem venom L-amino acid oxidase. This specificity makes it POSsible to determine the amount of free amino acid released without the necessity of separating it from the peptide substrate. The peptide hydrolase assay reported here is simple and rapid. Enzyme dilution and l,lO-phenanthroline are used to stop peptide hydrolase activity so that the second step of the assay (detection of free amino acid) can be carried out without the necessity of a separate step in which the enzyme is destroyed. Thus, our assay is very analogous t.o the disaccharidase assay of Dahlqvist (8) in which Tris buffer is used to inhibit disaccharidase activity. In addition, the metal chelator also inhibits aminopeptidase activity present in snake venom. This, plus the relatively small amounts of venom used, make this assay suitable for detecting release of free amino acids from the aminoterminus of polypeptides. The number of amino acids which can be accurately determined by the L-amino acid oxidase technique and consequently the number of peptides suitable for this type assay undoubtedly varies with the conditions used. Since we found optical density to vary linearly with the quantity of leucine, methionine, phenylalanine, and isoleucine present, peptides containing one or more of these amino acids should be suitable if the peptide does not also contain cysteine, tyrosine, or tryptophan which interfere with detection of phenylalanine and probably of the other amino acids as well. Fujita, Parsons, and Wojnarowska (5) found that although optical density did not increase linearly with the amounts of phenylalanine, tyrosine, or tryptophan, the curves were reproducible and therefore peptides containing these amino acids were suitable for use with this assay. Therefore, peptides containing tyrosine and possibly tryptophan but not containing other amino acids detectable by this assay may be satisfactory for our method. Fujita, Parsons, and Wojnarowska (5) do not state whether they observed any interference by tyrosine and tryptophan with the detection of other amino acids. Other factors which may affect the number of amino acids that can be accurately detected by the L-amino acid oxidase method and which might 1~8exploited to expand the scope of t.his peptide hydrolase assay
84
SHOAF,
ISSELBACHER
AND
HEIZER
include the type of buffer, the amount of L-amino acid oxidase, the type of n-amino acid oxidase (12)) and the presence of manganese or perhaps other metal ions. Our results show that manganous ions increase the absorbance given by many amino acids and alter the relative absorbance produced by equimolar quantities of the various amino acids. Other metal ions have not been as extensively studied but we observed only minimal changes in the absorbance produced by phenylalanine and methionine when Ca2+, Cuz+, Mg2+, or Zn2+ was added to the assay. This assay compares very favorably with other peptide hydrolase methods that have been published. It is simpler and less time consuming than the other recently described L-amino acid oxidase techniques (35), is more specific and less time consuming than ninhydrin methods (13,14), and does not require the complex equipment used by Lenard et al. (15). The important spectrophotometric method of Josefsson and Lindberg (16) is rapid and requires very little substrate, but we have found it more difficult to perform accurately than the present method. The two methods tend to complement each other as the Josefsson and Lindberg assay is not applicable to the study of dipeptides containing aromatic amino acids but can be used for study of dipeptides which contain amino acids that are not substrates for L-amino acid oxidase. The sensitivity of our method, absorbance of 0.1 corresponds to release of approximately 28 nmoles of free amino acid in the standard assay and 5.6 nmoles in the semimicro assay, is similar to the sensitivity of currently available peptide hydrolase assays. This assay was used to determine peptide hydrolase levels in fractions of rat intestinal mucosa, and it has also been used to determine activities in both the brush border and cytoplasmic fractions of peroral human intestinal biopsy specimens (17). Cytoplasmic activity in the biopsy specimens is easily determined by assaying the supernatants of whole biopsy specimen homogenates. To measure brush border activity, the unwashed pellet obtained by centrifuging the whole homogenate, is assayed following preincubation with p-hydroxymercuribenzoate which inhibits cytoplasmic activity (9,16). The interesting effect of Mn2+ on the intensity of color produced by various amino acids when assayed by the L-amino acid oxidase method must be taken into account. This effect could produce serious errors in studies of metal activation of peptide hydrolases unless appropriate controls are used. Minor differences in the amount of Mn*+ or perhaps other trace metals may account for the differences in relative absorbance produced by various amino acids which have been observed by several investigators (3-5 and the present study), all of whom were using L-amino acid oxidase of Crotalus adamanteus venom.
PEPTIDE
HYDROLASE
ASSAY
85
ACKNOWLEDGMENTS This AM01392
work and
was supported by by USPHS General
USPHS Research
Research Support
Grants Award
1 ROl-AM15119 5 SO1 FR 05406.
and
REFERENCES 1. ZEI,LF.R, E. A., AND MARITZ, A. (1925) Helv. Physiol. Pharmacol. Acta 3, C6-7. HEIZER, W. D., AND LASTER, L. (1969) J. Clin. Invest. 48, 219228. AURICCHIO, S., PIERRO, M., AND ORSATTI, M. (1971) Anal. Biochem. 39, 1528. DONLON, J., AND FOTTRELL, P. F. (1971) Clin. Chim. Acta 33, 345-350. FVIIT.~, M., PARSONS, D. S., AND WOJNAROWSK.~, F. (1972) J. Physiol. 227, 377394. 6. LEWIS, W. H., AND HARRIS, H. (1967) Nature (London) 215, 351-355. 7. DAHI,QVIST, A. (1964) Anal. Biochem. 7, 18-25. 8. DAHLQVIST, A. (1968) Anal. Biochem. 22, 99-107. 9. HEIZER, W. D., KERLEY, R. L., AND ISSELBACHER, K. J. (1972) Biochim. Biophys. Acta 264, 450461. 10. FORSTNER. G., SABESIN, S. M., AND ISSELBACHER, K. J. (1968) Biochem. J. 106, 381390. 11. ALPERS, D. H. (1969) J. Biol. Chem. 244, 1238-1246. 12. ZELLER, E. A., RAMACHANDER, G., FLETSHER, A., ISHIMARU, T., AND ZELLER, V. (1965) Biochem. J. 95, 262-269. 13. ~W~TIIESON, A. T., ASD TATTIRE, B. L. (1964) Can. J. Biochem. 42, 95-103. 14. KIM, Y. S., BIRTHWISTLE, W., .~ND KIM, Y. W. (1972) J. Clin. Invest. 51, 1419 1430. 15. LENARD, J., JOHNSON, S. L., HYMAN, R. W., AND HESS, G. P. (1965) Anal. Biothem. 11, 30-41. 16. JOSEFSSON, L., AND LINDBERG, T. (1965) Biochim. Biophys. Acta 105, 149-161. 17. SHOAF, C. R., AND HEIZER, W. D. (1973) Gastroenterology 64, 742.
2. 3. 4. 5.
Rapid
BIOCHEMISTRY
Calorimetric
61, 7285 (1974)
Assay
for Intestinal
Peptide
CHON R. SHOAF, KURT J. ISSELBACHER, WILLIAM D. HEIZERl Departments of Medicine, Chapel Hill, North Carolina,
University
of North
and Harvard
Medical
Received November
Hydrolases AND
Carolina Medical School, School, Boston, Massachusetts
27, 1973; accepted March
19, 1974
A simple two-step method is described for quantitating the release of free L-phenylalanine, L-leucine, L-methionine, or L-isoleucine from di- or polypeptides. The calorimetric assay is based on the ability of L-amino acid oxidase to catalyze the oxidation of free L-amino acid, but not of peptides. The potent metal chelator l,lO-phenanthroline, which is included in the second step of the assay, effectively inhibits peptide hydrolase activity thus permitting the assay to be carried out in two sequential steps in the same test tube with no intervening enzyme-destroying step. Although the assay is indirect and subject to interference by some chemicals, it is not affected by a large number of compounds frequently used in enzyme studies. The method was used to study the subcellular distribution of a number of peptide hydrolase activities in rat intestinal mucosa. For nine substrates, more than 80% of the total recovered activity was present in the cytoplasmic fraction while for two substrates, phenylalanylglycine and phenylalanylglycylglycine, more than 60% of the activity recovered was present in the particulate fraction.
A number of assays for peptide hydrolase activity have made use of the fact that snake venom L-amino acid oxidase catalyzes the oxidative deamination of some free L-amino acids but not of peptides (l-6). Lewis and Harris (6) coupled this reaction with the oxidation of o-dianisidine by hydrogen peroxide for detection of peptide hydrolase activity in starch gels. More recently, quantitative assays which make use of these coupled reactions have been described (3-5). The published methods which consist of an incubation step stopped by heat or perchloric acid, followed by a second step for detection of liberated amino acid, are very analogous to the disaccharidase assay described by Dahlqvist in 1964 (7). This report describes a shorter two-step assay for peptide hydrolase activity. It is analogous to the modified disaccharidase assay described
‘Send reprint requests to, at the Dept. of Medicine, Carolina, Chapel Hill, NC 27514. 72 Copyright @ 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
University
of North
PEPTIDE
HYDROLASE
by Dahlqvist in 1968 (8). A brief preliminary has been published previously (9). MATERIALS
AND
73
ASSAY
description
of this assay
METHODS
Materials Peptides, all of L-amino acids, were obtained from Biosynthetika (Oberdorf, Switzerland) and from Sigma Chemical Company. Lyophilized Crotalus adamanteus venom and o-dianisidine were obtained from Sigma Chemical Company, free amino acids from Mann Research Laboratories, l,lO-phenanthroline from Fisher Scientific Company, dithioerythritol from Cycle Chemical Corporation, and horseradish peroxidase from Worthington Biochemical Corporation. All other chemicals used were of reagent grade. Purity of the peptides and amino acids used was determined by high voltage paper electrophoresis by the method previously reported (2). Tissue Preparation Sprague-Dawley rats (175350 g males) were fasted overnight and killed with a blow on the head. Mucosa of the small intestine was obtained by scraping with a glass slide. Cytoplasmic and pellet fractions were effected by homogenizing the mucosa with a Potter-IElvehjem homogenizer in 5-10 vol (5-10 ml/g wet wt) of ice-cold 0.25 M sucrose and centrifuging for 1 hr at 87,000g at 4°C. The supernatant thus obtained was the cytoplasmic fraction. The pellet was washed three times in 0.155 M NaCl containing 1 mM NaHCO.?, pH 7.4, and suspended in one-half the initial volume of the same solution. Centrifugation for 30 min at 87,000g followed each wash. Purified brush borders were prepared as described by Forstner et al. (10) with slight modifications (11). To minimize loss of possible metal cofactors, normal saline was used in place of ethylenediaminetetraacetate (EDTA) solutions for precipitation of nucleic acids, filtration through glass wool, and subsequent washing. Examination by phase contrast microscopy revealed isolated brush borders with little debris. Enzyme
Assay
The standard assay is carried out in two steps at 37°C. A 15-min incubation step during which the peptide substrate is reacted with the enzyme to be assayed is followed by a second step in which released free amino acid is detected. Peptide hydrolase activity is effectively blocked during the second step both by dilution of the enzyme and by the presence of a potent metal chelator, l,lO-phenanthroline.
74
SHOAF,
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AND
HEIZER
The standard incubation mixture has a volume of 0.5 ml and contains 5 or 10 pmoles of peptide substrate, 50 pmoles of Tris-HCl buffer, pH 8.0, and enzyme. Buffer and substrate are added first, brought to 37°C in a water bath, and the reaction initiated by adding the enzyme preparation to be assayed. After 15 min, 2.0 ml of a phosphate-amino acid oxidase-phenanthroline reagent is added which stops the peptide hydrolase reaction and starts the free amino acid detection step. Snake venom n-amino acid oxidase in this reagent oxidizes certain L-amino acids to keto acids and hydrogen peroxide. In the presence of peroxidase, hydrogen peroxide oxidizes o-dianisidine to a somewhat unstable brown colored product with maximum absorbance at approximately 420 nm. Two milliliters of 56% H,SO, are added to give a very stable pink colored product with maximum absorbance at 530 nm. Preliminary studies showed that a final H,SO, concn of at least 25% is required to give optimal absorbance readings. Reagents 1. 0.5 M Tris-HCl buffer, pH 8.0. 2. 0.05 or 0.1 M Peptide in water. Made fresh daily to avoid the possibility of spontaneous or bacterial hydrolysis or formation of cyclic compounds, Most peptide solutions can probably be safely stored frozen. 3. Buffered substrate, mixture of equal quantities of reagents 1 and 2. 4. 1.0 mM Amino acid standards, prepared in water and stored frozen in 2-3 ml aliquots. 5. 12 rnx l,lO-Phenanthroline in buffer, 2.38 g of l,lO-phenanthroline dissolved in 1 liter of 0.2 M sodium phosphate buffer, pH 7.0, by stirring 8-12 hr at room temperature or 3-8 hr at 40-8O”C. Stable at least 1 mo in light-protected bottle at room temperature. 6. L-Amino acid oxidase solution, 10 mg lyophylized Crotalus adamanteus venom/ml of water, mixed gently, allowed to stand for 10 min at O”C, centrifuged 10 min at 5OOg, and supernatant saved. Solution prepared fresh just before use. 7. o-Dianisidine solution, 10 mg o-dianisidine/ml of water. Stable for several weeks at 4°C in light-protected bottle. Discarded when dark colored. 8. Peroxidase solution, 10 mg horseradish peroxidase/ml of water. Stable several months frozen in 2-3 ml aliquots. 9. Phosphate-amino acid oxidase-phenanthroline solution. This reagent is prepared no longer than 2 hr before its use as n-amino acid oxidase activity decreases to suboptimal levels in 3 hr. It is kept in a foil-covered bottle at room temperature because a precipitate forms at lower temperatures. The following or any similar proportions are used:
PEPTIDE
HYDROLASE
reagent reagent reagent reagent 10. 56% Sulfuric
ASSAY
5-100 ml 6-1.5 ml 7-1 .O ml 8-O. 1 ml
acid (v/v).
Procedure Step 1. The incubation mixture, placed in 8 ml test tubes, consists of 0.2 ml buffered substrate, 0.01-0.3 ml of enzyme plus any additives such as inhibitors or metals, and distilled water to give a final volume of 0.5 ml. Step 2. After an appropriate time (15 min for the standard assay), 2.0 ml of phosphate-amino acid oxidase-phenanthroline solution (reagent 9) are added, mixed, and the tubes are returned to the water bath. After 45-60 min, 2.0 ml of 56% H?SO, are added, mixed thoroughly, and ahsorbance is read at 530 nm within 4 hr. Blanks are treated in the same manner except that enzyme is not added until immediately following addition of reagent 9. Standards are identical to the blanks except that 0.05-0.2 ml (0.05-0.2 @moles) of amino acid standard is included in the incubation mixture. One unit of activity is defined as that amount of enzyme which catalyzes hydrolysis of 1 pmole of substrate,‘min under standard conditions. In the studies reported here, all assays were performed in duplicate and values recorded are the means of the duplicate readings. Semimicro
Assay
Excellent results were also obtained when each of the volumes was reduced to one-fifth that given above. In this semimicro assay, the incubation mixture contains 1.0 or 2.0 pmoles of substrate, 10 pmoles of Tris-HCl buffer, pH 8.0, and enzyme in a total volume of 0.1 ml. In the second step, 0.4: ml of reagent 9 and finally 0.4 ml of 56% H,SO, are added. RESULTS
Reactivity
of Various Amino Acids
Only 6 of the 20 free L-amino acids investigated yielded significant absorbance readings when tested under standard conditions of t,he assay (Table 1). Three amino acids, phenylalanine, leucine, and methionine, gave similar results while tyrosine, isoleucine, and tryptophan gave lower absorbance readings. After addition of reagent 9, absorbance produced
SHOAF,
Absorbance
Amino
ISSELBACHER
AND
HEIZER
TABLE 1 Produced by 100 nmoles of Various Under Standard Assay Conditions acid
LPhenylalanine LLeucine GMethionine n-Tyrosine n-Isoleucine L-Tryptophan 14 Others
Amino
Acids
Absorbance ,363 ,344 ,322 ,291 ,117 ,031 <.030
by phenylalanine, leucine, methionine, and tyrosine reached a plateau in 45 min while 90 min were required for tryptophan and more than 90 min for isoleucine. Absorbance read at 45 min increased linearly with increasing quantities of phenylalanine, leucine, methionine, or isoleucine present while results for tyrosine and tryptophan were not linear (Fig. 1). The molar extinction coefficient for phenylalanine was determined to be 1.59 x 104 M-l cm-l.
FIG. 1. Absorbance as a function of nanomoles of amino acid present. Assays were performed as described in Methods except that no enzyme was present, and the step 1 incubation was omitted. n-phenylalanine (a), L-methionine (A), L-leucine (0)) L-tyrosine (0)) L-isoleucine ( n ) , n-tryptophan (A).
PEPTIDE
HYDROLASE
ASSAY
77
In preliminary experiments, each component in the second step of the assay (venom, o-dianisidine, and peroxidase) was tested separately and the amount given for the standard assay procedure is slightly more than that amount which gave maximum absorbance at 45 min for phcnylalanine. Although greater absorbance readings for tryptophan, tyrosine, and isoleucine were achieved following a threefold increase in the amount of L-amino acid oxidase (venom) in reagent 9, the values for these three amino acids remained less than those for phenylalanine, leucine! and methionine. To determine whether any amino acids would interfere with the accurate determination of phenylalanine, 0.1 pmole of phenylalanine was assayed in the presence of 0.1 Ilmole of each of the 19 other amino acids. After subtracting the absorbance, if any, attributable to the additional amino acid, the amount of phenylalanine present was accurately determined in the presence of all but three amino acids, namely cysteine, tyrosine, and tryptophan. When these were included in the assay, recovery of phenylalanine was only 65-85%. Suitable
Dipeptides
Based on the above studies, it was evident that this assay could be used to detect hydrolysis of dipeptides which met certain criteria. The dipeptide must contain at least one of the suitable amino acids, phenylalanine, leucine, methionine, or isoleucine, and it should not contain tyrosine, tryptophan, or cysteine. Substrates containing two different suitable amino acids are acceptable for use in the assay. The amount of such substrates split is calculated by using the average absorbance of the two amino acid standards and halving the micromoles of free amino acid detected. Inhibition
of Peptide Hydrolase
Activity
by l,lO-Phenanthroline
Since many peptide hydrolases appear to require a divalent cation for activity, it seemed likely that a potent metal chelator could be used to inhibit peptide hydrolase activity during the second step of the assay. The compound, l,lO-phenanthroline, which was chosen for this purpose, was shown not to interfere with color development in the second stage of the assay. The effectiveness of l,lO-phenanthroline in inhibiting peptide hydrolase during the second step of the assay is shown in Fig. 2. The minimal absorbance which developed in the presence of the chelating agent corresponds to the blank observed in the standard assay. Results shown are for particulate phe-gly hydrolase and are similar to results for other peptide hydrolase activities in the particulate and cytoplasmic fractions of intestinal mucosa. Particulate gly-phe hydrolase activity.
78
SHOAF,
ISSELBACHER
IO 20 MINUTES
AND
HEIZER
30 40 50 60 70 OF INCUBATION
80
FIG. 2. Inhibition of peptide hydrolaae activity by 12 rn~ l,lO-phenaathroline. Immediately after adding enzyme to standard incubation mixtures, phosphate-amino acid oxidase-phenanthroline reagent was added to one mixture (a-@), and the same reagent without l,lO-phenanthroline was added to the other (O-0). Aliquots were removed at intervals, mixed with H,SO, and absorbance read at 530 nm.
which is less completely inhibited by l,lO-phenanthroline, gave slightly higher blank readings in the presence of very active enzyme preparations. Absorbance of blanks in the standard assay varied between 0.02 and 0.05 for all substrates except leucineamide, met-met, and phe-pro where absorbance of the blank was as high as 0.1 because of the presence of free amino acid in the substrate. Reaction Kinetics The quantity of free L-phenylalanine released from phe-gly and glyphe by purified brush borders from rat intestinal mucosa increased linearly with time up to 22.5 min (Fig. 3). The rate of hydrolysis of gly-phe
FIG. 3. Peptide hydrolysis as a function of duration of incubation. borders were incubated with phe-gly (04) and gly-phe (0-O) shown. Otherwise standard conditions were used.
Purified brush for the times
PEPTIDE
,q
FIG. 4. added to standard hydrolysis
BRUSH
HYDROLASE
BORDER
ASSAY
79
PROTEIN
Peptide hydrolysis as a function of the amount of brush border protein the incubation mixture. Purified brush borders were incubated under conditions with phegly (m-0) and gly-phe (0-O). The maximum shown in the graphs represents less than 5% hydrolysis of the substrates.
and phe-gly also increased linearly with increasing quantity of purified brush border protein added to the incubation mixture (Fig. 4). Cytoplasmic and particulate preparations of rat intestinal mucosa gave similar zero order kinetics for hydrolysis of gly-phe, phe-gly, met-gly, gly-leu, phe-pro, pro-phe, ser-phe, phe-gly-gly, and leu-gly-gly. The contribution of cytoplasmic and pellet enzymes to the total recovered peptide hydrolase activity against each of these substrates for rat small intestinal mucosa is shown in Table 2. Previous reports (2,3) have documented that aminopeptidase activity of Crotalus adamanteus venom and of purified L-amino acid oxidase may obviate the use of these preparations in assays of amino-peptidase activity because free amino acid released from tripeptides and other polypeptides by the snake venom enzyme cannot be distinguished from that released by the peptide hydrolase being assayed. Auricchio et al. (3) attempted to overcome this problem by using purified L-amino acid oxidase rather than crude snake venom. They were able to accurately determine the amount of free amino acid released from sometri- and tetrapeptides but not from others. A small concentration of venom and a low ratio of venom to polypeptide in the assay mixture should minimize the problem posed by the contaminating aminopeptidase. In the present assay in which only slightly more than the optimal amount of venom for assay of phenylalanine, leucine, and methionine was used, the concentration of crude
80
SHOAF,
ISSELBACHER
AND
TABLE 2 Distribution of Peptide Hydrolsse Activities and Particulate Fractions of Rat Small
HEIZER
Between Intestinal
Cytoplasmic Mucosa
Supernatant
Substrate L-Phenylalanyl-L-proline n-Prolyl-n-phenylalanine Glycyl-Lphenylalanine Glycyl-n-leucine n-Seryl-L-phenylalanine L-Alanyl-n-phenylalanine n-Histidyl-n-phenylalanine n-Leucylglycylglycine n-Methionylglycine LPhenylalanylglycine GPhenylalanylglycylglycine
Units/g mucosa 11.5 32.9 269 307 130 129 25.3 102 72.4 10.6 4.3
Pellet
$& of Total 95.0 92.9 92.8 92.0 90.3 89.3 58.2 87.1 82.0 38.7 30.3
Units/g mucosa .6 2.5 20.9 26.6 14.0 15.4 3.4 15.1 15.9 16.8 9.8
$& of Total 5.0 7.1 7.2 8.0 9.7 10.7 11.8 12.9 18.0 61.3 69.7
venom was 120 pg/ml giving a ratio of 30 pg of venom/pmole of substrate which is a much lower ratio than used in previous assays (2,3). Furthermore, l,lO-phenanthroline decreases the activity of the snake venom aminopeptidase. Thus, as shown in’Fig. 5, the combined effect of dilute venom and inhibition by l,lO-phenanthroline prevented the release of interfering amounts of free leucine from leu-gly-gly. Similar results were obtained with phe-gly-gly and phe-gly-phe-gly.
TIME
(MINUTES)
RG. 5. Effectiveness of low venom concentration and l,lO-phenanthroline in inhibiting release of free leucine from leu-gly-gly in step 2 of the assay. Water (2.4 ml) and buffered leu-gly-gly solution (1.6 ml) were incubated at 37°C with 16 ml of reagent 9 (a-@), reagent 9 from which l,lO-phenanthroline was omitted (O-O), reagent 9 with tenfold excess snake venom (A-A), and reagent 9 without l,lO-phenanthroline but with tenfold excess snake venom (A-A,). At the times indicated, 2.5 ml of each mixture was removed, mixed with 2.0 ml of 56% HzSOs and absorbance read at 530 nm.
PEPTIDE
S,ubstances Which Interfere
HYDROLASE
ASSAY
81
With the Assay
One of the major drawbacks of indirect assays, especially those involving enzymes as components, is that many substances may alter the reactions. Compounds which have been found to interfere with this assay at concentrations ordinarily used in enzyme studies include tryptophan, tyrosine, cysteine, dithioerythritol, mercaptoethanol, ammonium persulfate, S-phenylglycine, /3-phenylpyruvic acid, butanol, CoCl,, and FeCl,. Very small concentrations of CoCl,, butanol, and cysteine do not interfere. Compounds which do not interfere with the assay at concentrations ordinarily encountered include p-hydroxymercuribenzoate, EDTA, l,lOphenanthroline, MgCl,, CaCl,, CuCl,, NaI, LiCl, sodium pyrophosphate, sodium citrate, sodium acetate, sodium bicarbonate, Krebs Ringer phosphate, Ampholine carrier ampholytes, Triton X-100, sodium azide, chloroform, toluene, p-phenyllactic acid, hippuryl-n-phenylalanine, hippuric acid, hippuryl-L-arginine, hippuryl-L-lysine, p-tosyl glycine, phenylmethyl sulfonyl fluoride, N-acetyl-nL-phenylalanine, N-chloroacetyl-nphenylalanine, glycyl-P-alanine, hydrocinnamic acid, n-phenylalanine, T-BOC-n-phenylalanine, p-tosyl-glycine, N-acetyl-L-phenylalanine, and tertiary butyl-threonyl-phenylalanyl-proline. Zinc at’ 0.1 mM in the incubation step resulted in higher than normal blanks due to the inability of l,lO-phenanthroline in the second step of the assay to completely inhibit peptide hydrolase activity in the presence of high concentrations of ZIP+. This effect was very pronounced in the case of brush border leu-gly-gly hydrolase (aminopeptidase) activity which could be accurately measured in the presence of 0.1 mM Zn”+ only by heat-killing the enzyme to terminate the first step of the assay. Freshly prepared MnCl, had an extraordinary effect on the assay. At 0.6 m&r concentration, Mn”+ increased the absorbance produced by tryptophan, tyrosine, isoleucine, leucine, methionine, and phenylalanine by 728, 84, 63, 34, 28, and 28%, respectively. Maximum absorbance for phenylalanine occurred when Mn?+ concentration in the initial incubation step was between 0.5 and 1.0 mM. This color enhancing effect of Mn*+ did not depend on the presence of l,lO-phenanthroline and did not occur when Mn?+ was added just before or just after addition of H&SO,. Furthermore, presence of this cation increased by 26% the absorbance produced by glucose when assayed by the glucose oxidase method. Since the glucose assay, as the peptide hydrolase assay described here, involves oxidation of o-dianisidine by hydrogen peroxide, it seems likely that Mn?+ has its effect on this reaction. This hypothesis is supported by the fact that the absorbance produced by adding free hydrogen peroxide to reagent 9 was increased 410% by the presence of Mn?+. However, when free hydrogen peroxide and Mn2+ were mixed before addition of reagent 9,
82
Absorbance
SHOAF,
ISSELBACHER
AND
HEIZER
TABLE 3 Produced by 100 nmoles of Various Amino was Present in the Step 1 Incubation Amino
acid
L-Tyrosine LPhenylalanine LLeucine LTryptophan LMethionine LIsoleucine LHistidine 13 Others
Acids When Mixture
0.5 mM MnC12
Absorbance ,558 .543 .434 ,427 .423 ,185 .123 <.03
the absorbance was 57% less than when Mn2+ was absent. This decrease probably results from reduction of hydrogen peroxide to water by MI?+. Taken together these data suggest that at least part of the effect of Mn2+ on our assay occurs at the o-dianisidine-hydrogen peroxide step. Additional studies will be required to further elucidate the mechanism of action of Mn2+ in these reactions, especially to determine why the ion increases the absorbance produced by some amino acids more than others. When 0.5 mM Mn2+ was present in the step 1 incubation mixture, it markedly altered the relative intensity of color produced by the amino acids (Table 3, compare with Table 1). In addition, presence of this cation caused histidine to give significant absorbance readings (Table 3). The presence of Mn2+ did not cause absorbance to increase linearly with increasing amounts of tyrosine and tryptophan. Some amino acid derivatives are oxidized by snake venom L-amino acid oxidase and cause color development when they are present in the assay mixture. Those tested include p-amino-m-phenylalanine, p-nitronn-phenylalanine, p-fluoro-m-phenylalanine, and nn-y-amino-phenyl acetic acid. Color Stability
of Assay Products
The brown color of oxidized o-dianisidine is somewhat unstable. It decreases slowly with time, and we observed that the color was also affected by light. Absorbance of the brown solution, which was maximal at 450 nm, fell on exposure to incandescent or fluorescent light of moderate intensity for 3 min and slowly increased again over a period of 30-60 min after the solution was placed in the dark. In contrast, absorbance of the pink solution (maximal at 530 nm) which resulted from addition of H&SO, was not affected by light and decreased less than 5% overnight at
PEPTIDE
HYDROLASE
ASSAY
83
room temperature. An aliquot of the brown solution whose absorbance had been reduced 20% by exposure to light gave the same reading at 530 nm as a nonexposed aliquot after addition of H,SO, to both solutions. DISCUSSIOI’G
The fact that L-amino acid oxidase will react with free amino acids but not with peptides was first used as the basis for a peptide hydrolase assay by Zeller and Maritz ( 1). The present assay and several other recently reported methods (2-6) are based on this specificity of Crotahs adamantem venom L-amino acid oxidase. This specificity makes it POSsible to determine the amount of free amino acid released without the necessity of separating it from the peptide substrate. The peptide hydrolase assay reported here is simple and rapid. Enzyme dilution and l,lO-phenanthroline are used to stop peptide hydrolase activity so that the second step of the assay (detection of free amino acid) can be carried out without the necessity of a separate step in which the enzyme is destroyed. Thus, our assay is very analogous t.o the disaccharidase assay of Dahlqvist (8) in which Tris buffer is used to inhibit disaccharidase activity. In addition, the metal chelator also inhibits aminopeptidase activity present in snake venom. This, plus the relatively small amounts of venom used, make this assay suitable for detecting release of free amino acids from the aminoterminus of polypeptides. The number of amino acids which can be accurately determined by the L-amino acid oxidase technique and consequently the number of peptides suitable for this type assay undoubtedly varies with the conditions used. Since we found optical density to vary linearly with the quantity of leucine, methionine, phenylalanine, and isoleucine present, peptides containing one or more of these amino acids should be suitable if the peptide does not also contain cysteine, tyrosine, or tryptophan which interfere with detection of phenylalanine and probably of the other amino acids as well. Fujita, Parsons, and Wojnarowska (5) found that although optical density did not increase linearly with the amounts of phenylalanine, tyrosine, or tryptophan, the curves were reproducible and therefore peptides containing these amino acids were suitable for use with this assay. Therefore, peptides containing tyrosine and possibly tryptophan but not containing other amino acids detectable by this assay may be satisfactory for our method. Fujita, Parsons, and Wojnarowska (5) do not state whether they observed any interference by tyrosine and tryptophan with the detection of other amino acids. Other factors which may affect the number of amino acids that can be accurately detected by the L-amino acid oxidase method and which might 1~8exploited to expand the scope of t.his peptide hydrolase assay
84
SHOAF,
ISSELBACHER
AND
HEIZER
include the type of buffer, the amount of L-amino acid oxidase, the type of n-amino acid oxidase (12)) and the presence of manganese or perhaps other metal ions. Our results show that manganous ions increase the absorbance given by many amino acids and alter the relative absorbance produced by equimolar quantities of the various amino acids. Other metal ions have not been as extensively studied but we observed only minimal changes in the absorbance produced by phenylalanine and methionine when Ca2+, Cuz+, Mg2+, or Zn2+ was added to the assay. This assay compares very favorably with other peptide hydrolase methods that have been published. It is simpler and less time consuming than the other recently described L-amino acid oxidase techniques (35), is more specific and less time consuming than ninhydrin methods (13,14), and does not require the complex equipment used by Lenard et al. (15). The important spectrophotometric method of Josefsson and Lindberg (16) is rapid and requires very little substrate, but we have found it more difficult to perform accurately than the present method. The two methods tend to complement each other as the Josefsson and Lindberg assay is not applicable to the study of dipeptides containing aromatic amino acids but can be used for study of dipeptides which contain amino acids that are not substrates for L-amino acid oxidase. The sensitivity of our method, absorbance of 0.1 corresponds to release of approximately 28 nmoles of free amino acid in the standard assay and 5.6 nmoles in the semimicro assay, is similar to the sensitivity of currently available peptide hydrolase assays. This assay was used to determine peptide hydrolase levels in fractions of rat intestinal mucosa, and it has also been used to determine activities in both the brush border and cytoplasmic fractions of peroral human intestinal biopsy specimens (17). Cytoplasmic activity in the biopsy specimens is easily determined by assaying the supernatants of whole biopsy specimen homogenates. To measure brush border activity, the unwashed pellet obtained by centrifuging the whole homogenate, is assayed following preincubation with p-hydroxymercuribenzoate which inhibits cytoplasmic activity (9,16). The interesting effect of Mn2+ on the intensity of color produced by various amino acids when assayed by the L-amino acid oxidase method must be taken into account. This effect could produce serious errors in studies of metal activation of peptide hydrolases unless appropriate controls are used. Minor differences in the amount of Mn*+ or perhaps other trace metals may account for the differences in relative absorbance produced by various amino acids which have been observed by several investigators (3-5 and the present study), all of whom were using L-amino acid oxidase of Crotalus adamanteus venom.
PEPTIDE
HYDROLASE
ASSAY
85
ACKNOWLEDGMENTS This AM01392
work and
was supported by by USPHS General
USPHS Research
Research Support
Grants Award
1 ROl-AM15119 5 SO1 FR 05406.
and
REFERENCES 1. ZEI,LF.R, E. A., AND MARITZ, A. (1925) Helv. Physiol. Pharmacol. Acta 3, C6-7. HEIZER, W. D., AND LASTER, L. (1969) J. Clin. Invest. 48, 219228. AURICCHIO, S., PIERRO, M., AND ORSATTI, M. (1971) Anal. Biochem. 39, 1528. DONLON, J., AND FOTTRELL, P. F. (1971) Clin. Chim. Acta 33, 345-350. FVIIT.~, M., PARSONS, D. S., AND WOJNAROWSK.~, F. (1972) J. Physiol. 227, 377394. 6. LEWIS, W. H., AND HARRIS, H. (1967) Nature (London) 215, 351-355. 7. DAHI,QVIST, A. (1964) Anal. Biochem. 7, 18-25. 8. DAHLQVIST, A. (1968) Anal. Biochem. 22, 99-107. 9. HEIZER, W. D., KERLEY, R. L., AND ISSELBACHER, K. J. (1972) Biochim. Biophys. Acta 264, 450461. 10. FORSTNER. G., SABESIN, S. M., AND ISSELBACHER, K. J. (1968) Biochem. J. 106, 381390. 11. ALPERS, D. H. (1969) J. Biol. Chem. 244, 1238-1246. 12. ZELLER, E. A., RAMACHANDER, G., FLETSHER, A., ISHIMARU, T., AND ZELLER, V. (1965) Biochem. J. 95, 262-269. 13. ~W~TIIESON, A. T., ASD TATTIRE, B. L. (1964) Can. J. Biochem. 42, 95-103. 14. KIM, Y. S., BIRTHWISTLE, W., .~ND KIM, Y. W. (1972) J. Clin. Invest. 51, 1419 1430. 15. LENARD, J., JOHNSON, S. L., HYMAN, R. W., AND HESS, G. P. (1965) Anal. Biothem. 11, 30-41. 16. JOSEFSSON, L., AND LINDBERG, T. (1965) Biochim. Biophys. Acta 105, 149-161. 17. SHOAF, C. R., AND HEIZER, W. D. (1973) Gastroenterology 64, 742.
2. 3. 4. 5.