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A rapid spectrophotofluorometric assay for indoleglycerol phosphate synthase
ANALYTICAL
69, 5 lo-5 17 (1975)
BIOCHEMISTRY
A Rapid
Spectrophotofluorometric
Indoleglycerol CHARLES
N. HANKINS, Department
MICHAEL
Assay
Phosphate LARGEN,
of Biology, University La Jolla, California
for
Synthase AND
of California 92037
STANLEY
E. MILLS
at San Diego,
Received April 10, 1975; accepted June 19, 1975 A rapid and sensitive spectrophotofluorometric assay for indoleglycerol phosphate synthase is presented. The method is based upon the fluorescence of the reaction product indoleglycerol phosphate and has two advantages over previously reported assays. It is more sensitive and is useful for measuring enzyme activities in extracts containing materials that prohibit the use of other methods.
The fourth step specific for L-tryptophan biosynthesis, O-carboxyphenylamino-deoxyribulose-Sphosphate (CDRP)’ --grindoleglycerol phosphate (InGP) + CO, is catalyzed by indoleglycerol phosphate synthase (InGPs, EC 4.1.1.48). Previously, two assays have been utilized to measure this enzymatic activity. The first is an endpoint assay in which the reaction product InGP is dephosphorylated; the cis-hydroxyl groups of the glycerol moiety are then oxidized by periodate and the resulting indolealdehyde extracted into ethyl acetate and the absorbance measured at 290 nm (1). The second assay is a continuous spectrophotometric assay based upon the increase in optical density at 280 nm accompanying the conversion of CDRP to InGP (2). In studies in our laboratory on tryptophan biosynthesis in higher plants we found both assays to be unsuitable. The very low specific activity of InGPs in plant extracts (< 5 nmoles/min/mg protein) made the continuous spectrophotometric assay unsuitable because of background absorbance at 280 nm. The periodate assay was impractical because we found glycerol to be required for the stabilization of several of the plant enzymes, and the periodate oxidation cannot be carried out in the presence of glycerol. A resolution of these unsatisfactory conditions was suggested by the fluorescence properties of InGP. In this communication we describe a continuous spectrophotofluorometric assay for InGPs that is rapid, sensitive, and can be used in the presence of glycerol. ’ Abbreviations: InGPs, indoleglycerol phenylaminok2-deoxyribulose-Sphosphate;
phosphate synthase; CDRP, l-(O-carboxyInGP, indoleglycerol phosphate. 510
Copyright AU rights
@I 1975 by Academic Press, Inc. of reproduction in any form reserved.
TRYPTOPHAN
BIOSYNTHESIS
511
METHODS
Chemicals. InGP was the generous gift of I. P. Crawford, Scripps Clinic and Research Foundation. It was assayed by its absorbance at 280 nm and by periodate oxidation as described below for the InGPs assays. Pure Escherichia coli InGPs was generously provided by C. Yanofsky, Stanford University. CDRP was prepared by chemical synthesis as described by Smith and Yanofsky (2) and assayed by its enzymatic conversion to InGP. Bacterial strains. Wild-type E. coli strain W3 110 was used as the source of repressed and steady-state crude extracts. The tryptophan auxotroph A2/F’A2 was used as the source of the derepressed extract. The designation refers to a chain terminating mutation present both on the chromosome and on an episome in the A gene specifying the (Y subunit of tryptophan synthetase. Corn. Golden Bantam corn seeds were purchased from Burpee Seed Co., Riverside, Calif. Bacterial media and growth. The minimal medium used was VBA medium: Vogel-Bonner salts (3), 1 mg/ml acid hydrolyzed casein, and 2.5 mg/ml D-glucose added separately after sterilization. All cells were grown at 37”C, and growth was monitored using a Klett-Summerson calorimeter with filter No. 66 (660 nm). For the steady state, extract W3 I 10 was grown in VBA medium to late log phase (200 Klett units). For the repressed extract the same procedure was followed except that VBA medium was supplemented with 50 pg/ml L-tryptophan. For the depressed extract A2/F’A2 was grown in VBA medium supplemented with 5 pg/ml indole and the cells harvested 3-4 hr after growth had ceased due to the depletion of indole. Bacterial extract preparation. Extracts were prepared as described previously (4). Corn growth and extraction. Corn seeds were germinated in the dark for 4 days on moist filter paper. The seedlings were harvested by manually removing the endosperm. The fresh radicles were washed with water to remove excess starch and then blended in ice-cold 0.05 M TrisHCl buffer (1 ml/g fresh tissue) at pH 7.5 supplemented with EDTA, 1 mM; 2-mercaptoethanol, 1 mM and glycerol 20% (v/v). The blended tissue was centrifuged at 20,OOOg for 30 min. The supernatant solution was then made 0.1% (w/v) in protamine sulfate by the dropwise addition of a 1% (w/v) solution. The supernatant solution, after centrifuging as before, was used as the source of corn InGPs. ZnGPs assays. The periodate and spectrophotometric assays were performed as described previously (1, 2). Spectrophotojborometric assay. The fluorescence assay was carried out at 37°C with an Aminco-Bowman spectrophotofluorimeter with ex-
512
HANKINS,
LARGEN
AND MILLS
citation at 280 nm and emission at 350 nm. An attached strip chart recorder monitored the changes in fluorescence with time. The assay contained buffer (Tris-HCI or potassium phosphate) 50 pmoles, pH 7.5, and CDRP, 50 nmoles in a total volume of 1 ml. An InGP standard was introduced at the end of each assay to obtain an accurate measure of the relative fluorescence of InGP under each specific assay condition. Reaction rates (nanomoles per minute) were calculated from the slopes of the initial linear region of the chart records. Enzyme units are defined as nanomoles per minute. Protein determination. Protein was determined by the method of Lowry et al. (5) with dessicated bovine serum albumin as the standard. RESULTS
The fluorescence spectrum for InGP closely resembles that of indole and other indole derivatives (6). In Fig. 1 are recorded two of the family of curves in the fluorescence scan. When the excitation wavelength was varied and the fluorescence intensity measured at 350 nm (Fig. 1A), maximum intensity occurred at the excitation wave length of 280 nm. Under constant excitation at 280 nm the fluorescence intensity showed a maximum at 350 nm (Fig. 1B). The excitation and emission maxima at
A EXCITATION
WAVELENGTH
(nm)
1. Fluorescence scan of pure InGP. The fluorescence spectra for InGP were performed on a Perkin-Elmer fluorescence spectrophotometer Model MPF-4. (A), Fluorescence intensity at 350 nm when the excitation wavelength was varied. (B), A scan of the fluorescence intensity when exciting at 280 nm. The sample was 4 PM InGP in H,O. FIG.
BIOSYNTHESIS
513
FIG. 2. Linearity of InGP fluorescence. Measurements 0.05 M Tris-HCI, pH 7.5.
were made with pure InGP in
TRYPTOPHAN L
10
20 30 40 $4 InGP
50
280 and 350 nm were therefore chosen for the assay of the enzymatic production of InGP. These spectra were obtained with a Perkin-Elmer (MPF-4) fluorescence spectrophotometer. This instrument was used for the spectra because it has a very narrow band pass and also allows for excitation at constant light energy regardless of wavelength. Fluorescence variation with concentration, pH, and ionic strength. The fluorescence intensity of InGP was linear with concentration to about 40 PM, as shown in Fig. 2. The fluorescence was not significantly altered by varying the pH from 6 to 9. Ionic strength had no significant effect on InGP fluorescence when either phosphate or Tris-HCl buffers were varied from O-O.5 M. Similarly, the addition of NaCl up to 0.5 M to either dilute buffer had no effect. At buffer or salt concentrations greater than 0.5 M the intensity of InGP fluorescence began to diminish and was
TIME
min
3. Linearity of assay with time. The assays were performed as described in Methods. Curves 1, 2, 3, 4, and 5 were obtained by adding approximately I .25, 2.5, 6.25, 12.5, and 20 units of enzyme activity, respectively, from a crude extract of AZ/F’A2. FIG.
514
HANKINS,
LARGEN
1020
AND
MILLS
100
so Enzyme Solutlon(yl)
FIG. 4. Linearity of assay with enzyme added. Assay conditions were as described in Methods. The enzyme used was a crude extract of A2/F’A2. The extract was diluted 1: 100 (0, left scale) and 1: 1000 (A, right scale). Units are nanomoles of InGP per minute.
about 70% of that observed in water when the buffer or salt concentration was increased to 1.OM. Fluorescence variation with time. A time course for the enzyme assay is given in Fig. 3 with a crude extract of E. coli A2/F’A2 serving as the InGPs source. The assay gave linear reaction rates for at least 5 min or until the substrate CDRP became limiting. Variation with enzyme concentration. In Fig. 4 are plotted the data showing that the enzyme assay was linear with enzyme concentration over at least a loo-fold range. With the crystalline enzyme from E. coli (specific activity 4000 U/mg) it was possible to measure as little as 0.2 units of enzyme activity (50 ng of enzyme) in a I-min assay or as little as 0.02 units (5 ng enzyme) in a IO-min assay.
TABLE A COMPARISON
OF THE PERIODATE FROM
AND
E. coli
1
FLUOROMETRIC AND CORN
ASSAYS
WITH
EXTRACTS
Assay method Periodate
Spectrophotofluorometric
Extract
Volume assayed (~1)
Units/ml
Specific activity
Repressed 64 (b) Steady state Derepressed Corn
500 500 100 1 500
8.5 6.0 45 6600 -
2.8 1.3 6.3 390 -
Volume assayed &l)
Units/ml
Specific activity
10 10 10 0.01 20
6.0 P 43 6300 7.0
2.0 6.1 370 1.0
LIDash (-) indicates that InGPs activity could not be determined.
TRYPTOPHAN
51.5
BIOSYNTHESIS
21)
1.5 T -
1.0
0.5
1 .I
.2
.3
d
5
I/tCDRP)
.6
.7
.8
9
phi-’
FIG. 5. Double reciprocal plot of substrate vs velocity. Dilutions of CDRP were added to assays containing about 2 units of pure Escherichia coli InGPs and rates measured by the fluorescence method. Assay comparison. Table 1 lists the enzymatic activities of several bacterial and plant extracts as determined by the spectrophotofluorometric and periodate assay methods. The bacterial extracts are from E. cofi grown under steady-state, repressed and derepressed conditions. One of the repressed bacterial extracts could not be assayed by the fluorometric method. The other repressed extract for which results are given represents the limit of detection for material of this specific activity and composition. The corn extract could not be measured by the periodate method because of the presence of both glycerol and large quantities of periodate positive substances (mono-, oligo-, and polysaccharides) naturally present in the extract. It can be seen that there is close agreement between the enzyme activities measured by the two assay methods. In addition, the quantities of material required for the fluorometric assay is very small. K, determination. In Fig. 5 is shown a double reciprocal plot of substrate vs velocity obtained fluorometrically for pure E. coli InGPs. Similar results were obtained with enzyme from a crude extract of A2/F’A2. The extrapolated K, for CDRP (5.8 PM) is in good agreement with the value (5 PM) obtained by Creighton and Yanofsky (7).
DISCUSSION
A rapid and continuous spectrophotofluorometric assay for InGPs activity has been developed based upon the fluorescence of its reaction product InGP. With pure InGPs the assay is lo-50 times as sensitive as previously described methods and offers the advantage that glycerol may be incorporated into the assay buffer. Of the two standard assay procedures available, the spectrophotometric method is afso not disturbed by glycerol but is limited in application
516
HANKINS,
LARGEN
AND
MILLS
by the background absorbance of protein at 280 nm, the absorbance maximum of InGP. Only relatively high specific activity material (> 20 units/mg) can be conveniently measured and this method is also reported to give spurious optical density increases that are not enzyme dependent (2).
The periodate assay method, besides being unusable when glycerol or other species containing cis-hydroxyl groups are present, is also relatively time-consuming. For low activities the assay may require up to several hours. This is particularly cumbersome when following enzyme activity during chromatographic procedures since several time points must be taken for each fraction assayed. The spectrophotofluorometric assay, while eliminating the difficulties described above, has at least two constraints. The main difficulty encountered is fluorescence quenching by low specific activity extracts. While quenching is variable, depending on the composition of the particular extract, our experience is that the assay is unreliable with extracts that contain less than about 1 unit of enzyme activity per milligram of protein present. Such extracts can be assayed with the periodate method, provided sufficient total enzyme activity is present. Moreover, since quenching varies from extract to extract and with differing amounts of a given extract, a standard sample of InGP must be added to each assay after recording a rate. This enables the accurate determina: tion of rates under the specific conditions of each assay. Such precautions are normal for most fluorometric assays when utilizing crude extracts. The other difficulty with the assay is attributable to background fluorescence. The latter is not a serious limitation but coupled with high quenching, can further decrease the sensitivity two- to threefold. The use of suboptimal wavelengths decreased the background fluorescence but also decreased InGP fluorescence, and no increase in sensitivity could be achieved in this manner. We have observed that CDRP prepared by the method of Smith and Yanofsky (2) contains material that severely quenches InGP fluorescence when the concentration of CDRP exceeds about 100 PM. Little quenching is observed at CDRP concentrations less than or equal to 50 PM, which is saturating for the E. coli enzyme (K, = 5 PM). For an InGPs with a significantly larger K, for CDRP, the use of more highly purified CDRP preparations, such as that described by Doy (8), may be required. Spectrophotofluorometric assays for the first three enzymes specific for L-tryptophan biosynthesis have been described (2). Thus it is now possible to measure rapidly the first four of the five tryptophan enzymes by fluorescence spectrometry. It may be mentioned that in the rare case wherein the preceding en-
TRYPTOPHAN
517
BIOSYNTHESIS
zyme activity, phosphoribosyl anthranilate isomerase (PRAI), and InGPs are associated on a single polypeptide chain, one could follow the purification of the molecule by assaying for PRAI activity (7). However, the results could be confounded by the selective loss of InGPs activity. ACKNOWLEDGMENT This work was supported by U.S. ERDA No. E (04-3)-34
P.A. 133.
REFERENCES 1. Smith, 0. H., and Yanofsky, C. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 5, pp. 794-806, Academic Press, New York. 2. Creighton, T. G., and Yanofsky, C. (1970) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 17A, pp. 365-380, Academic Press. New York. 3. Vogel, H., and Bonner, D. M. (1956) J. Biol. Chem. 218, 97-106. 4. Largen, M., and Belser, W. L. (1975) J. Bncteriol. 121, 239-249. 5. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (I 95 1 )J. Bjo/. Chem. 193, 265-275.
6. Duggan, D. E., Bowman, R. L.. Brodie, B. B., and Udenfriend, S. (1957) Arch. Biochem. Biophys. 68, I-14. 7. Creighton, T. G.. and Yanofsky, C. (1966) .I. Viol. Chem. 241, 4616. 8. Day, C. H. (1966) Nature (London) 211, 736.
69, 5 lo-5 17 (1975)
BIOCHEMISTRY
A Rapid
Spectrophotofluorometric
Indoleglycerol CHARLES
N. HANKINS, Department
MICHAEL
Assay
Phosphate LARGEN,
of Biology, University La Jolla, California
for
Synthase AND
of California 92037
STANLEY
E. MILLS
at San Diego,
Received April 10, 1975; accepted June 19, 1975 A rapid and sensitive spectrophotofluorometric assay for indoleglycerol phosphate synthase is presented. The method is based upon the fluorescence of the reaction product indoleglycerol phosphate and has two advantages over previously reported assays. It is more sensitive and is useful for measuring enzyme activities in extracts containing materials that prohibit the use of other methods.
The fourth step specific for L-tryptophan biosynthesis, O-carboxyphenylamino-deoxyribulose-Sphosphate (CDRP)’ --grindoleglycerol phosphate (InGP) + CO, is catalyzed by indoleglycerol phosphate synthase (InGPs, EC 4.1.1.48). Previously, two assays have been utilized to measure this enzymatic activity. The first is an endpoint assay in which the reaction product InGP is dephosphorylated; the cis-hydroxyl groups of the glycerol moiety are then oxidized by periodate and the resulting indolealdehyde extracted into ethyl acetate and the absorbance measured at 290 nm (1). The second assay is a continuous spectrophotometric assay based upon the increase in optical density at 280 nm accompanying the conversion of CDRP to InGP (2). In studies in our laboratory on tryptophan biosynthesis in higher plants we found both assays to be unsuitable. The very low specific activity of InGPs in plant extracts (< 5 nmoles/min/mg protein) made the continuous spectrophotometric assay unsuitable because of background absorbance at 280 nm. The periodate assay was impractical because we found glycerol to be required for the stabilization of several of the plant enzymes, and the periodate oxidation cannot be carried out in the presence of glycerol. A resolution of these unsatisfactory conditions was suggested by the fluorescence properties of InGP. In this communication we describe a continuous spectrophotofluorometric assay for InGPs that is rapid, sensitive, and can be used in the presence of glycerol. ’ Abbreviations: InGPs, indoleglycerol phenylaminok2-deoxyribulose-Sphosphate;
phosphate synthase; CDRP, l-(O-carboxyInGP, indoleglycerol phosphate. 510
Copyright AU rights
@I 1975 by Academic Press, Inc. of reproduction in any form reserved.
TRYPTOPHAN
BIOSYNTHESIS
511
METHODS
Chemicals. InGP was the generous gift of I. P. Crawford, Scripps Clinic and Research Foundation. It was assayed by its absorbance at 280 nm and by periodate oxidation as described below for the InGPs assays. Pure Escherichia coli InGPs was generously provided by C. Yanofsky, Stanford University. CDRP was prepared by chemical synthesis as described by Smith and Yanofsky (2) and assayed by its enzymatic conversion to InGP. Bacterial strains. Wild-type E. coli strain W3 110 was used as the source of repressed and steady-state crude extracts. The tryptophan auxotroph A2/F’A2 was used as the source of the derepressed extract. The designation refers to a chain terminating mutation present both on the chromosome and on an episome in the A gene specifying the (Y subunit of tryptophan synthetase. Corn. Golden Bantam corn seeds were purchased from Burpee Seed Co., Riverside, Calif. Bacterial media and growth. The minimal medium used was VBA medium: Vogel-Bonner salts (3), 1 mg/ml acid hydrolyzed casein, and 2.5 mg/ml D-glucose added separately after sterilization. All cells were grown at 37”C, and growth was monitored using a Klett-Summerson calorimeter with filter No. 66 (660 nm). For the steady state, extract W3 I 10 was grown in VBA medium to late log phase (200 Klett units). For the repressed extract the same procedure was followed except that VBA medium was supplemented with 50 pg/ml L-tryptophan. For the depressed extract A2/F’A2 was grown in VBA medium supplemented with 5 pg/ml indole and the cells harvested 3-4 hr after growth had ceased due to the depletion of indole. Bacterial extract preparation. Extracts were prepared as described previously (4). Corn growth and extraction. Corn seeds were germinated in the dark for 4 days on moist filter paper. The seedlings were harvested by manually removing the endosperm. The fresh radicles were washed with water to remove excess starch and then blended in ice-cold 0.05 M TrisHCl buffer (1 ml/g fresh tissue) at pH 7.5 supplemented with EDTA, 1 mM; 2-mercaptoethanol, 1 mM and glycerol 20% (v/v). The blended tissue was centrifuged at 20,OOOg for 30 min. The supernatant solution was then made 0.1% (w/v) in protamine sulfate by the dropwise addition of a 1% (w/v) solution. The supernatant solution, after centrifuging as before, was used as the source of corn InGPs. ZnGPs assays. The periodate and spectrophotometric assays were performed as described previously (1, 2). Spectrophotojborometric assay. The fluorescence assay was carried out at 37°C with an Aminco-Bowman spectrophotofluorimeter with ex-
512
HANKINS,
LARGEN
AND MILLS
citation at 280 nm and emission at 350 nm. An attached strip chart recorder monitored the changes in fluorescence with time. The assay contained buffer (Tris-HCI or potassium phosphate) 50 pmoles, pH 7.5, and CDRP, 50 nmoles in a total volume of 1 ml. An InGP standard was introduced at the end of each assay to obtain an accurate measure of the relative fluorescence of InGP under each specific assay condition. Reaction rates (nanomoles per minute) were calculated from the slopes of the initial linear region of the chart records. Enzyme units are defined as nanomoles per minute. Protein determination. Protein was determined by the method of Lowry et al. (5) with dessicated bovine serum albumin as the standard. RESULTS
The fluorescence spectrum for InGP closely resembles that of indole and other indole derivatives (6). In Fig. 1 are recorded two of the family of curves in the fluorescence scan. When the excitation wavelength was varied and the fluorescence intensity measured at 350 nm (Fig. 1A), maximum intensity occurred at the excitation wave length of 280 nm. Under constant excitation at 280 nm the fluorescence intensity showed a maximum at 350 nm (Fig. 1B). The excitation and emission maxima at
A EXCITATION
WAVELENGTH
(nm)
1. Fluorescence scan of pure InGP. The fluorescence spectra for InGP were performed on a Perkin-Elmer fluorescence spectrophotometer Model MPF-4. (A), Fluorescence intensity at 350 nm when the excitation wavelength was varied. (B), A scan of the fluorescence intensity when exciting at 280 nm. The sample was 4 PM InGP in H,O. FIG.
BIOSYNTHESIS
513
FIG. 2. Linearity of InGP fluorescence. Measurements 0.05 M Tris-HCI, pH 7.5.
were made with pure InGP in
TRYPTOPHAN L
10
20 30 40 $4 InGP
50
280 and 350 nm were therefore chosen for the assay of the enzymatic production of InGP. These spectra were obtained with a Perkin-Elmer (MPF-4) fluorescence spectrophotometer. This instrument was used for the spectra because it has a very narrow band pass and also allows for excitation at constant light energy regardless of wavelength. Fluorescence variation with concentration, pH, and ionic strength. The fluorescence intensity of InGP was linear with concentration to about 40 PM, as shown in Fig. 2. The fluorescence was not significantly altered by varying the pH from 6 to 9. Ionic strength had no significant effect on InGP fluorescence when either phosphate or Tris-HCl buffers were varied from O-O.5 M. Similarly, the addition of NaCl up to 0.5 M to either dilute buffer had no effect. At buffer or salt concentrations greater than 0.5 M the intensity of InGP fluorescence began to diminish and was
TIME
min
3. Linearity of assay with time. The assays were performed as described in Methods. Curves 1, 2, 3, 4, and 5 were obtained by adding approximately I .25, 2.5, 6.25, 12.5, and 20 units of enzyme activity, respectively, from a crude extract of AZ/F’A2. FIG.
514
HANKINS,
LARGEN
1020
AND
MILLS
100
so Enzyme Solutlon(yl)
FIG. 4. Linearity of assay with enzyme added. Assay conditions were as described in Methods. The enzyme used was a crude extract of A2/F’A2. The extract was diluted 1: 100 (0, left scale) and 1: 1000 (A, right scale). Units are nanomoles of InGP per minute.
about 70% of that observed in water when the buffer or salt concentration was increased to 1.OM. Fluorescence variation with time. A time course for the enzyme assay is given in Fig. 3 with a crude extract of E. coli A2/F’A2 serving as the InGPs source. The assay gave linear reaction rates for at least 5 min or until the substrate CDRP became limiting. Variation with enzyme concentration. In Fig. 4 are plotted the data showing that the enzyme assay was linear with enzyme concentration over at least a loo-fold range. With the crystalline enzyme from E. coli (specific activity 4000 U/mg) it was possible to measure as little as 0.2 units of enzyme activity (50 ng of enzyme) in a I-min assay or as little as 0.02 units (5 ng enzyme) in a IO-min assay.
TABLE A COMPARISON
OF THE PERIODATE FROM
AND
E. coli
1
FLUOROMETRIC AND CORN
ASSAYS
WITH
EXTRACTS
Assay method Periodate
Spectrophotofluorometric
Extract
Volume assayed (~1)
Units/ml
Specific activity
Repressed 64 (b) Steady state Derepressed Corn
500 500 100 1 500
8.5 6.0 45 6600 -
2.8 1.3 6.3 390 -
Volume assayed &l)
Units/ml
Specific activity
10 10 10 0.01 20
6.0 P 43 6300 7.0
2.0 6.1 370 1.0
LIDash (-) indicates that InGPs activity could not be determined.
TRYPTOPHAN
51.5
BIOSYNTHESIS
21)
1.5 T -
1.0
0.5
1 .I
.2
.3
d
5
I/tCDRP)
.6
.7
.8
9
phi-’
FIG. 5. Double reciprocal plot of substrate vs velocity. Dilutions of CDRP were added to assays containing about 2 units of pure Escherichia coli InGPs and rates measured by the fluorescence method. Assay comparison. Table 1 lists the enzymatic activities of several bacterial and plant extracts as determined by the spectrophotofluorometric and periodate assay methods. The bacterial extracts are from E. cofi grown under steady-state, repressed and derepressed conditions. One of the repressed bacterial extracts could not be assayed by the fluorometric method. The other repressed extract for which results are given represents the limit of detection for material of this specific activity and composition. The corn extract could not be measured by the periodate method because of the presence of both glycerol and large quantities of periodate positive substances (mono-, oligo-, and polysaccharides) naturally present in the extract. It can be seen that there is close agreement between the enzyme activities measured by the two assay methods. In addition, the quantities of material required for the fluorometric assay is very small. K, determination. In Fig. 5 is shown a double reciprocal plot of substrate vs velocity obtained fluorometrically for pure E. coli InGPs. Similar results were obtained with enzyme from a crude extract of A2/F’A2. The extrapolated K, for CDRP (5.8 PM) is in good agreement with the value (5 PM) obtained by Creighton and Yanofsky (7).
DISCUSSION
A rapid and continuous spectrophotofluorometric assay for InGPs activity has been developed based upon the fluorescence of its reaction product InGP. With pure InGPs the assay is lo-50 times as sensitive as previously described methods and offers the advantage that glycerol may be incorporated into the assay buffer. Of the two standard assay procedures available, the spectrophotometric method is afso not disturbed by glycerol but is limited in application
516
HANKINS,
LARGEN
AND
MILLS
by the background absorbance of protein at 280 nm, the absorbance maximum of InGP. Only relatively high specific activity material (> 20 units/mg) can be conveniently measured and this method is also reported to give spurious optical density increases that are not enzyme dependent (2).
The periodate assay method, besides being unusable when glycerol or other species containing cis-hydroxyl groups are present, is also relatively time-consuming. For low activities the assay may require up to several hours. This is particularly cumbersome when following enzyme activity during chromatographic procedures since several time points must be taken for each fraction assayed. The spectrophotofluorometric assay, while eliminating the difficulties described above, has at least two constraints. The main difficulty encountered is fluorescence quenching by low specific activity extracts. While quenching is variable, depending on the composition of the particular extract, our experience is that the assay is unreliable with extracts that contain less than about 1 unit of enzyme activity per milligram of protein present. Such extracts can be assayed with the periodate method, provided sufficient total enzyme activity is present. Moreover, since quenching varies from extract to extract and with differing amounts of a given extract, a standard sample of InGP must be added to each assay after recording a rate. This enables the accurate determina: tion of rates under the specific conditions of each assay. Such precautions are normal for most fluorometric assays when utilizing crude extracts. The other difficulty with the assay is attributable to background fluorescence. The latter is not a serious limitation but coupled with high quenching, can further decrease the sensitivity two- to threefold. The use of suboptimal wavelengths decreased the background fluorescence but also decreased InGP fluorescence, and no increase in sensitivity could be achieved in this manner. We have observed that CDRP prepared by the method of Smith and Yanofsky (2) contains material that severely quenches InGP fluorescence when the concentration of CDRP exceeds about 100 PM. Little quenching is observed at CDRP concentrations less than or equal to 50 PM, which is saturating for the E. coli enzyme (K, = 5 PM). For an InGPs with a significantly larger K, for CDRP, the use of more highly purified CDRP preparations, such as that described by Doy (8), may be required. Spectrophotofluorometric assays for the first three enzymes specific for L-tryptophan biosynthesis have been described (2). Thus it is now possible to measure rapidly the first four of the five tryptophan enzymes by fluorescence spectrometry. It may be mentioned that in the rare case wherein the preceding en-
TRYPTOPHAN
517
BIOSYNTHESIS
zyme activity, phosphoribosyl anthranilate isomerase (PRAI), and InGPs are associated on a single polypeptide chain, one could follow the purification of the molecule by assaying for PRAI activity (7). However, the results could be confounded by the selective loss of InGPs activity. ACKNOWLEDGMENT This work was supported by U.S. ERDA No. E (04-3)-34
P.A. 133.
REFERENCES 1. Smith, 0. H., and Yanofsky, C. (1962) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 5, pp. 794-806, Academic Press, New York. 2. Creighton, T. G., and Yanofsky, C. (1970) in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 17A, pp. 365-380, Academic Press. New York. 3. Vogel, H., and Bonner, D. M. (1956) J. Biol. Chem. 218, 97-106. 4. Largen, M., and Belser, W. L. (1975) J. Bncteriol. 121, 239-249. 5. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (I 95 1 )J. Bjo/. Chem. 193, 265-275.
6. Duggan, D. E., Bowman, R. L.. Brodie, B. B., and Udenfriend, S. (1957) Arch. Biochem. Biophys. 68, I-14. 7. Creighton, T. G.. and Yanofsky, C. (1966) .I. Viol. Chem. 241, 4616. 8. Day, C. H. (1966) Nature (London) 211, 736.