,ANALYTICAL BIOCHEMISTRY 71, 405-414 (1976)

Polarimetry as a General Method for Enzyme Assays

DAVID A . BLASS AND ELIJAH ADAMS

Department of Biological Chemistry, University of Maryland School of Medicine, Baltimore, Maryland 21201

Received August 14, 1975; accepted October 23, 1975

A polarimetric assay has been used for a number of enzymes catalyzing dif- ferent types of reaction including several peptidases, phosphohexose isomerase, glutamate decarboxylase, glutaminase, and glutamate-alanine transaminase. The limitations and requirements of the method for these and certain other enzymes are discussed.

Many enzyme-catalyzed reactions involve changes in optical rotation and in principle are assayable by polarimetry. When practicable, polari- metric assays would seem to offer two principal advantages over the con- ventional assays for certain enzymes: continuous recording, when this is not easily provided by alternative methods; and a more direct assay and simpler reaction mixture, when current methods involve coupling to other reactions. The amino acid decarboxylases represent an example of the first class of assays: while these enzymes are sensitively assayed by release of 14CO2, convenient continuous assays are not available. A number of glycolytic enzymes illustrate the second class of assays: Enzymes such as phosphohexose isomerase are commonly assayed in coupled systems, whereas polarimetry might provide a more direct assay in both reaction directions.

While polarimetry has been used to a limited extent in the assay of racemases (1) and sporadically for certain proteases and miscellaneous reactions (2-4), there seems to have been little interest in exploring its use in assaying the great variety of enzymes which utilize optically active substrates or which introduce a new chiral center in the product. Stimu- lated by the convenience of a polarimetric assay for hydroxyproline-2- epimerase (5-7), we have examined polarimetry as an approach to the as- say of several other reaction types. This method has been effective with peptidases (both purified and crude), phosphohexose isomerase, glutamic decarboxylase, glutaminase, and glutamic-alanine transaminase, as ex- amples of several different types of reaction. We found our methods inade- quate for the assay of certain other enzymes tested (D-amino acid oxidase, histidine decarboxylase).

Our purpose in this report is to call attention to the potential utility of polarimetry for the assay of many enzymes, and to summarize our experience with some of the method's limitations and requirements.

Copyright © 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

405

406 BLASS AND ADAMS

MATERIALS AND METHODS

D-Amino acid oxidase was the crystalline hog kidney enzyme from Sigma Chemical Co.; histidine decarboxylase was the bacterial enzyme (Cl. welchii, Type II) purchased from Sigma as a purified water-soluble powder; leucine aminopeptidase was a purified product of Sigma (hog kidney, Type III CP) and was activated by Mn 2+ treatment according to Smith and Spackman (8); phosphohexose isomerase from yeast was the crystalline Boehringer product; glutamic decarboxylase was the partly- purified E. coli enzyme, obtained as a water-soluble powder from Sigma; glutamic-alanine transaminase was the homogeneous product from pig heart, obtained from Sigma as a suspension in ammonium sulfate; gluta- minase (Sigma) was the lyophilized soluble powder from E. coli, of purity 20-30 units/rag.

For the preparation of a crude source of intestinal peptidases, hog intestinal mucosa was processed as described by Smith and Bergmann (9). Mucosa (20 g), scraped from the upper 3 ft of small intestine, was ground in a mortar at room temperature with 2 g of sand and 20 ml of water. After centrifugation for 20 min at 10,000 rpm (SS-34 head, Sorvall RC2-B refrigerated centrifuge), the supernatant solution was removed, diluted with an equal volume of water, and recentrifuged as above. The super- natant solution was brought to 0.9 saturation with solid ammonium sulfate and kept at 5°C overnight. The precipitate was dissolved in l0 ml of 0.05 M Tris, pH 7.95, and dialyzed overnight at 5°C against 250 vol of the Tris buffer above. The dialyzed solution was centrifuged as above for 20 min. The clear supernatant solution was stored at - 15°C as the enzyme source.

The peptides, L-alanylglycine, glycyl-L-alanine, and L-leucyl-glycine, were purchased from Sigma, as were D-glucose-6-phosphate and o-fruc- tose-6-phosphate. Glycyl-L-proline and L-prolylglycine were commercial products (Sigma) or were made in our laboratory by the N-hydroxysuc- cinimide method (10). Other common chemicals used as buffers or sub- strates were obtained from standard sources as the best quality com- mercially available.

Polarimeters used were the Bendix NPL Automatic Polarimeter Type 143A connected to an external Varian G2000 recorder, and the Jasco J-20 Automatic Recording spectropolarimeter. For Bendix runs, a specially fabricated 5-cm light-path cell of 3-ml vol was used (7). For Jasco runs, the cell had a 2-cm light path and required about 6-ml vol.

At their limit, the sensitivity of both instruments permitted determina- tion of a total reaction span of about 1 millidegree, corresponding to a change of about 1 cm on the chart; thus an initial rate of 0.03 millidegree/ min (Fig. 5) could be determined in about 30 min.

Reaction mixtures were made in a separate tube, the reaction was begun by adding enzyme, and the final assay solution was mixed by inversion and quickly transferred to the polarimeter cell. Runs were made at room

E N Z Y M E ASSAYS BY P O L A R I M E T R Y 4 0 7

temperature (20-25°C) without special efforts to maintain constant temperature. Details of each assay procedure are presented where appropriate.

Our rate data, where displayed in figures, are shown as change in angular rotation (Aa)/min; where equivalent rates in units are cited, the unit is ! ~mol of product formed (or substrate used)/min under the conditions of the assay. Protein concentration in our own enzyme preparations was determined by a nomogram based on absorbance at 280 and 260 nm (11); protein concentration in commercially obtained enzyme samples was based on nominal concentration stated by the vendor.

RESULTS

Intestinal peptidases. For trials of peptidase activity the dipeptides, alanylglycine and glycylalanine, had the attraction of high specific rota- tion at 589 nm (12), while the free amino acid components released by hydrolysis have either no optical activity (glycine) or very low specific rotation (alanine). Our measurement of glycylalanine at 589 nm in Tris buffer (0.5 M, pH 7.9) gave a specific rotation of -41°; the reported value in water is -75 ° (12). These measurements and the enzyme assay were carried out with the Bendix polarimeter.

Figure 1 shows the measured rate of Aa/min when glycyl-L-alanine was hydrolyzed by the crude intestinal mucosa preparation described above. Treatment of the enzyme with MnS 04 (2 mM final concentration) or storage of the enzyme at -15°C led to considerable loss of activity, but proportionality of hydrolytic rate with enzyme, as monitored polari- metrically, was maintained. Chromatography of reaction mixtures showed disappearance of the peptide substrate. Less systematic trials were also

z_ " 5 5

5 O- ~ 4

t 2 5

PROTEIN(rag)

FIG. 1. Initial rate of glycyl-L-alanine hydrolysis by intestinal mucosa extract as a func- tion of enzyme concentration (Bendix instrument). The initial reaction mixture (3.5 ml) con- tained 0.05 M Tris, pH 7.95, 29 mM glycyl-L-alanine and quantities of enzyme shown on the abscissa. Under the conditions used, a change in a of 4.2 × 10 -4 degrees represents 1 p.mol of substrate hydrolyzed.

408 BLASS AND ADAMS

Z

t~ uJ 8 O_

W 6 ~A

._1 _J ~ 2

<1 ~o 2o

PROTEIN (~.g)

FIG. 2. lnitial rate of L-leucylglycine hydrolysis by leucine aminopeptidase as a func- tion of enzyme concentration (Bendix instrument). The initial reaction mixture (3.5 ml) contained 0.05 M Tris, pH 8.3, 29 mM leucylglycine, and quantities of enzyme shown on the abscissa. Under the conditions used, a change in c~ of 1.3 x 10 -3 degrees represents 1 /xmol of substrate hydrolyzed.

made with alanylglycine, prolylglycine, and glycylproline, selected be- cause of the large change in specific rotation between these peptides and their hydrolysis products (12, Vol. 1, p. 96; Vol. 2, p. 1218). These substrates were tested under the conditions described for glycylalanine hydrolysis, and were active substrates, as judged by change in rotation, for the mucosal preparation, either unfortified or after Mn 2+ activation; conditions for activity proportional with enzyme addition were not established.

Leucine aminopeptidase. Assays were carried out at pH 8.5 in 0.5 M Tris using the Bendix polarimeter. Leucylglycine was chosen as substrate

---~ 5 . "5

tl. 4 .

O4 O8 ~2

PROTEIN ( ~ g )

FIG. 3. Initial rate of fructose-6-phosphate isomerization by phosphohexose isomerase as a function of enzyme concentration (Bendix instrument). The initial reaction mixture (3.5 ml) contained 0.05 M Tris, pH 7.8, 29 mM fructose-6-phosphate, and quantities of enzyme shown on the abscissa. Under the conditions used, a change in a of 7 × 10 -4 degrees represents 1 /zmol of fructose-6-phosphate isomerized.

ENZYME ASSAYS BY POLARIMETRY 409

(8) because of the large reported change between the substrate (specific rotation at 589 nm in water, +85°; 12, Vol. 1, p. 96) and products (specific rotation of L-leucine at 589 nm in water, - 11°; 12, Vol. 1, p. 86). Our deter- mination of these specific rotations at 589 nm and pH 8.5 (Tris) were: leucyl- glycine, +39°; L-leucine, - 8 °, for a Ao~ of 47 °. Figure 2 shows the rate of change in rotation as a function of enzyme.

Phosphohexose isomerase. Assay of this enzyme was based on the difference between specific rotation for o-glucose-6-phosphate and D- fructose-6-phosphate and utilized the Bendix polarimeter. At the pH of the assay (7.8, 0.05 M Tris), our measurements at 589 nm gave specific rota- tions for glucose-6-phosphate and fructose-6-phosphate of +22.3 ° and +2.1 °, respectively. Figure 3 shows the initial rate of As as a function of enzyme concentration in the direction fructose-6-phosphate ~ glucose- 6-phosphate. By rotation, the equilibrium position of the reaction was consistent with a Keq of 2.2; a recent review (13) cites 3.7 as the value for glucose-6-P/fructose-6-P at 25°C and pH 8.5. Many trials with glucose-6- phosphate as substrate gave variable assay values not consistently linear with enzyme. The reasons for good assay data in one reaction direc- tion and not in the other were not clear and were not investigated systematically,

Glutamate decarboxylase. Figure 4 shows the specific rotation of L- glutamate as a function of wavelength at pH 4.8 (acetate buffer, 0.045 M) using the Jasco instrument. Although use of lower wavelengths would increase the sensitivity of the assay, 350 nm was selected as a compromise

+ t s -

o -

,.o, a - 1 5 - v

- 4 5 - o , 7

- 7 5 -

- 9 o -

550 500 4~0 400 350 300

WAVELENGTH (nm)

FIG. 4. Specific rotation of L-glutamic acid and L-glutamine as a function of wavelength at pH 4.8-5.0. Measurements were made in the Jasco instrument at room temperature. L-Glutamic acid (5.4 mM) was in 0.045 M sodium acetate, pH 4.8; L-glutamine (5.4 mM) was in 0.045 M sodium acetate, pH 4.95.

410 BLASS A N D A D A M S

z i 0O5O

0040-

OO3O -

Q _~1 O02O -

00lO -

08 O't6 O24 O32 O40

PROTEIN ( mg )

FIG. 5. Initial rate of glutamate decarboxylation at 350 nm as a function of enzyme concentration (Jasco instrument). The initial reaction mixture (6.5 ml) contained 0.03 M sodium acetate, pH 4.6, 46 mM L-glutamate, 0.5 mM pyridoxal phosphate, and quantities of enzyme shown on the abscissa. Under the conditions used, a change in c~ of 4.7 × 10 -5 degrees represents 1 /zmol glutamate decarboxylated (Fig. 4).

between sensitivity and the necessity of avoiding wavelengths of high absorbance by pyridoxal phosphate at this pH. Figure 5 shows the rate of change in a with enzyme in this assay.

Glutaminase. A wavelength scan (Fig. 4) of L-glutamate and L-gluta- mine at pH 4.8 (glutamate) and pH 4.95 (glutamine) suggested the use of 350 nm for assays with the Jasco instrument. Figure 6 shows the rate of change in a as a function of enzyme concentration.

Glutamate-alanine transaminase. Figure 7 shows wavelength scans of L-glutamate, L-alanine and e-aspartate in dilute Tris buffer at pH values

0.280 -

i 0240 -

0200"

0.160 -

~Jj 0 120 -

080 "

O4O -

15 30 45 60 75

PROTEIN (p.g)

FIG. 6. Initial rate of L-glutamine deamidation by glutaminase at 350 nm as a function of enzyme concentration (Jasco instrument). The initial reaction mixture contained 0.03 M sodium acetate, pH 4.95, 5.4 mM L-glutamine, and quantities of enzyme shown on the abscissa. Under the conditions used, a change in a of 3.5 × 10 -4 degrees represents 1 /zmol of L-glutamine converted to L-glutamate (Fig. 4).

ENZYME ASSAYS BY POLARIMETRY 41 1

+IO-

UJ W O-

W ~ - 1 0 -

~ -20- - 5 0 -

0 ,'7

-4O -

- 5 0 -

~ I N E ~ J

GLUTAMIC ACID

-60 -

550 500 450 40O 35O 300 WAVELENGTH (nm)

FIG. 7. Specific rotation as a function of wavelength (Jasco instrument) of L-alanine (60 raM, pH 7.8), L-aspartic acid (21 raM, pH 8.1), and L-glutarnic acid (19 raN, pH 7.7). The buffer used in all cases was 0.05 N Tris.

near 8. From these data, alanine transaminase was selected for trials over aspartate transaminase. Although 375 nm was the wavelength used for assays without added pyridoxal phosphate, trials at this wavelength with 0.5 mM pyridoxal phosphate prevented the response of the Jasco instru- ment because of absorbance overload. Figure 8 shows data points for the rate of change in o~ as a function of enzyme. From these data, we obtained a value of 214 units/ml; the sample of enzyme supplied by Sigma had a nomi- nal activity of 275 units/ml, assayed by coupling to lactate dehydrogenase.

12

i 1.0

08

b'~ 0.6

~ 0.A 0.2 , ~ , ,

5 I0 15 20 25 PROTEIN (/zg)

FIG. 8. Initial rate of glutamic-alanine transaminase at 375 nm as a function of enzyme concentration (Jasco instrument). The initial reaction mixture (6.5 ml) contained 0.09 M Tris, pH 7.5, 0.4 M L-alanine, and 0.05 M c~-ketoglutarate. Under the conditions used, a change in ct of 6.4 x 10 5 degrees represents 1 /xmol of pyruvate and L-glutamate formed.

412 BLASS AND ADAMS

DISCUSSION

The data presented for enzymes catalyzing a variety of reaction types indicate the potentially broad, though as-yet little examined, application of polarimetry to enzyme assays. We should note that the method had certain unanticipated limitations which became apparent in trials of other enzymes. Thus, assay trials with D-amino acid oxidase acting on D-proline were based on the availability of pure enzyme preparations of high activity and the expected large change in rotation with D-proline as substrate. However, we failed to obtain consistent assay values using the Bendix instrument at 589 nm. A limitation that soon became obvious was the dependence of the reaction on dissolved oxygen, and the difficulty of continuous oxygenation in a polarimeter cell which must contain an undis- turbed column of liquid during readings. Preoxygenating assay solutions did not solve this problem, and it was apparent that the sensitivity of the instrument was insufficient to record reliable changes in rotation in the short time during which the concentration of dissolved oxygen was not limiting. While a polarimetric assay might be used by measuring optical rotation in samples taken at intervals from shaken vessels (and trials by this method indicated changes in the expected direction), a time-point assay would forfeit one of the principal advantages of the proposed assay method.

Our trials also revealed the significance of two different types of instrument limitation, each associated with one of the instruments used. The Jasco spectropolarimeter, like other spectropolarimeters com- mercially available, has the advantage of variable wavelength over a large span (in the Jasco instrument, from 185 to 700 nm). This advantage became apparent in selecting wavelengths for suitable sensitivity in certain assays described, notably for glutamate decarboxylase, glutaminase, and gluta- mate-alanine transaminase. However, such instruments use the principle of altered optical absorbance as an index of the change in the plane of light polarization, and can tolerate neither high absorbance nor high initial optical rotation in the assay solution. Use of this type of instrument might therefore be limited with solutions of crude enzymes of low activity or solutions containing components of high optical activity. As an ex- ample, we had to omit pyridoxal phosphate from assay solutions monitored at wavelengths near its absorbance maximum.

The Bendix instrument, in contrast, differs from most commercially available, sensitive polarimeters in utilizing the Faraday principle (14) and can tolerate both high initial optical rotation and solutions of rather high absorbance. Its major limitation is dependence on the use of filters and therefore on a limited choice of wavelengths, which would have excluded certain of the assays we carried out with the Jasco.

A final comment on the strategy of approaching an assay by this method concerns the choice o f p H , wavelength, and substrate. In selecting appro-

ENZYME ASSAYS BY POLARIMETRY 413

priate conditions, we found it necessary to obtain our own optical rotatory dispersion curves (Figs. 4, 7) for substrates and products in the appro- priate pH range of the assay, since such data are either not in the literature or are difficult to find. For example, while many data are recorded for specific rotations of major amino acids as a function of wavelength (12, Vol. 1, pp. 114-116), pH as a variable does not appear to be thoroughly documented; in the case of compounds with ionizable groups sensitive to pH changes near the assay pH, recorded data at a few pH extremes may well be useless in planning an assay. As an example, our plan to test the histidine decarboxylase assay failed because the specific rotation of histidine at 589 nm is very low (<2 °) over the pH range 3.5 to 5.0 in which the bacterial enzyme is significantly active (15); while the specific rotation of histidine at pH 3.7 rises rapidly at decreasing wavelengths (about + 28 ° at 300 nm), our enzyme sample was too crude to permit the use of the Jasco instrument at low wavelengths, for the reasons noted above. In this case, use of smaller aliquots of enzyme and a longer recording period failed because of increasing turbidity of the assay solution with time.

When an enzyme to be assayed utilizes several substrates, the choice among these will depend both on the magnitude of the Aa for the reaction and the relative activity of the substrate. Thus our trials of leucine amino- peptidase used I.-leucylglycine since it has substantial substrate activity (86% that of L-leucinamide (8)), while the Aa between substrate and hydrolytic products is much higher than for leucinamide hydrolysis (12, Vol. 1, pp. 114-116).

A final caution is one applicable to many types of assay: A change in optical rotation may reflect a side reaction rather than the desired reaction. In attempts to assay crude preparations of dopa decarboxylase, a rather rapidly changing rotation was attributable to nonemzymatic dopa polymerization rather than dopa decarboxylation. Our calculations indi- cate that the polarimetric method should be applicable to the assay of dopa decarboxylase if sufficient enzyme were present to complete an assay before the relatively slow polymerization reaction interfered.

ACKNOWLEDGMENT

This work was supported by Public Health Service Research Grant GM-11105.

REFERENCES

1. Adams, E. (1972) in The Enzymes (Boyer, P. D., ed.) 3rd ed., Vol. 6, pp. 479-507, Academic Press, New York.

2. Abramowitz, N., Schechter, I,, and Berger, A. (1967) Biochem. Biophys. Res. Commun. 29, 862-867.

3. Mason, M., and Fasella, P. (1971)Anal. Biochem. 43, 57-65. 4. Lowry, W. T., and Vercellotti, J. R. (1972) Fed. Proc. 31,882. 5. Adams, E., and Norton, I. L. (1964)J. Biol. Chem. 239, 1525-1535. 6. Finlay, T. H., and Adams, E. (1970) J. Biol. Chem. 245, 5248-5260.

414 BLASS AND ADAMS

7. Zervos, C., and Adams, E. (1975) Mol. Cell. Biochem. 8, 113-121. 8. Smith, E. L., and Spackman, D. H. (1955)J. Biol. Chem. 212, 271-299. 9. Smith, E. L., and Bergmann, M. (1944) J. Biol. Chem. 153, 627-651.

10. Anderson, G. W., Zimmerman, J. E., and Callahan, F. M. (1964)J. Amer. Chem. Soc. 86, 1839-1842.

11. Adams, E. (1967) in Laboratory Experiments in Biochemistry (Daniel, L. J., and Neal, A. L., eds.), p. 255, Academic Press, New York.

12. Greenstein, J. P., and Winitz, M. (1961) Chemistry of the Amino Acids, Vols. 1, 2, John Wiley and Sons, Inc., New York.

13. Noltmann, E. A. (1972) in The Enzymes (Boyer, P. D., ed.) 3rd ed., Vol. 6, p. 292, Academic Press, New York.

14. Instruction Manual, No. M5911/1, NPL Automatic Polarimeter, Type 143A, Cincin- nati Division, Bendix Corporation.

15. Epps, H. M. R. (1945) Biochem. J. 39, 42-46.