An automated assay for glycogen phosphorylase

BIOCHEMISTRY

47, 451456

An Automated

Assay

ANALYTICAL

RICHARD

H. HASCHKE

Physiologisch-Chemiches

(1972)

for

Glycogen

AND I,~~D\\iI(‘; Irrstitut der Koellikerstr. Received

Phosphorylase

$1. CT. HEXMEYER,

Cniversitiit 2. Germrrny

August

Wikzburg,

JR.

SYOO Wiirzbwg,

27, 1971

The physiological role of glycogen phosphorylase is the degradation of glycogen, even though the concentration ratio of glucose-l-P/Pi at equilibrium is ca. 0.28 (equation a) (1) : (glycogen),

+ Pi =

glucose-l-P

+

(glycogen),-l

(a)

Therefore, the release of inorganic phosphate from glucose-l-P, easily determined according to Fiske and SubbaRow (2)) provides a convenient method for assaying this enzyme (3). Based on the same phosphorylase assay principle, the activity of phosphorylase kinase can also be determined. The latter enzyme phosphorylates one specific serine residue of each phosphorylase subunit, thereby converting the b form of this enzyme (inactive in absence of AMP) to the active a state. The amount of phosphorylase a formed is a measure for the kinase activity. The reconversion of phosphorylase a to 6, catalyzed by phosphorylase phosphatase, may be assayed by the disappearance of phosphorylase a (4). The determination of both kinase and phosphatase which necessarily resu1t.s in many phosphorylase (1 assays led to the development of a convenient automated phosphorylase test. Several reports may be found concerning improvements (5) and automation (Technicon IV-4b methodj of the Fiske-SubbaRow test. for inorganic phosphate. However, this report describes not just the detect.ion of released inorganic phosphate but a completely automated assay for glycogen phosphorylase. A complication of this automated procedure is due to precipitated protein formed upon addition of the acidic molybdate reagent of the Fiske-SubhaRow method (2) to the phosphorylase assay mixture. The need for a dialysis step to free the inorganic phosphate of denatured protein is eliminated by employing sodium dodecyl sulfate (SDS) as a solubilizcr to prevent this precipitation. The automated phosphorylase assay also has a lower standard error than the test performed by hand. 451 @ 1972 by

Academic

Press,

Inc.

452

HASCHKE

Ah-D

HEILMEYER

SAMPLER 0

Q

COLORIMET-

FIG.

1. Flow

diagram

for

automated

phosphorylase Tubing

1. 2. 3. 4. 5. 6. 7. 8.

Sample Substrate SIB ANSA Acid molybdate Air H.20 Waste (a) tb) (c) (d) (e) (f) (g) (h)

reagent

determination:

id.

Flow

0.015” 0.015” 0.020” 0.040” 0.110” 0.065” 0.056” 0.110”

“T”-fitting Cactus-fitting Single glass mixing coil (3 mm i.d.) 1)ouble glass mixing coil (3 mm i.d.) React.ion coil (0.8 mm i.d. polyvinyl chloride Platinum nipple side-arm fitt)ing Tygon tubing (0.065” i.d.) Polyvinyl chloride tubing (0.8 mm i.d.)

rate,

ml/min

0.10 0.10 0.16 0.60 3.90 1.60 1.20 3.90

length

3 m)

AUTOMATED

PHOSPHORTLASE

453

ASSAY

METHODS

The apparatus employed is a Technicon AutoAnalyzer system composed of the following: sampler II, proportioning pump, calorimeter and recorder. The manual phosphorylase test is performed according to the method of Hedrick and Fischer (61. ill1 reagents for the automated procedure are the same as used in the manual test with the exceptions that. the substrat,e contains 0.01% Triton S-100 and the stoplGng reagent contains 0.01% SDS. The AutoAnalyzer system is assembled as show~l in Fig. 1. All tubing is the standard Tygon variety supl)lied by Technicon. Samples are taken at a rate of l/min with an actual sampling time of 18 set spaced by a 42 set water wash. Sample and substrate are mixed in eqal flow rates and the reaction takes place for ca. 6 min while passing through the rtaction coil immersed in a 30°C water bath. The reaction is stopped by successive addit,ion of 10% SDS and the acid molybdate reagent containing 1-amino-2-l~ydroxynaphtl~alene-4-sulfonic acid (ANSA) Color development occurs at room temperature for ap1~roximately 2 min in two double mixing coils and the absorbancy of this solution is then measured in the calorimeter using a red filter (660-18-28 1. Results are recorded on logarithmic paper. RESUI,TS

Figure

2 shows

AND

the relationship

DISCI‘SSIOS

between

absorbancy

ant1 the conccn-

0.6

0.6

jd moles

FIG. 2. Standard calibration conditions are described in

the

curve text.

for

/ml

determination

Phosphate

of inorganic

phosphate.

The

454

HASCHKE

AND

HElLMEYER

tration of orthophosphatc. The curve is linear between 1 and 10 mM; t,herefore, the sensitivity range of this automated assay is the same as found with the Fiske-SubhaRow procedure (2). Since the remaining substrate, glucose-l-P, is slowly hydrolyzed by the acid molybdate reagent, the color development time is minimized. Under this restriction, the ratio of flow rates from the reaction coil and Fiske-SubbaRow reagents is adjusted so that color development is complete before measuring absorbancy. The standard curve for orthophosphate bet,ween 1 and 10 mM will become nonlinear if this ratio is increased more than ca. 2-fold. The ANSA reagent is maintained fresh by storage at 4°C in the dark. If the reducing power of the ANSA solution becomes too weak, again a nonlinear standard curve will result. Also, a linear increase in absorbancy is obtained as a function of phosphorylase b concentration (Fig. 3), and for phosphorylase a (not shown). In Fig. 3, the units of phosphorylase b (abscissa) per milliliter are determined from the specific activity of phosphorylase measured according to Hedrick and Fischer (6). In this manner, the absorbancy obtained from the AutoAnalyzer chart is directly convertible to the enzyme activity unit employed in the hand test. If increased sensitivity of the phosphorylase assay is desired, the reaction coil should be lengthened to increase the incubation time of enzyme with substrate. The alternative of increasing the flow rate of products of the phosphorylase reaction relative to t,he Fiske-SubbaRow reagents should be avoided because of the above stated reasons. With the 3 meter reaction coil employed in this system, the incubat,ion time is ca. 6 min, which requires an amount of

units/ml

FIG.

conditions units/mg.

3.

Standard calibration are described in the

curve text.

phosphorylase

b

for determination of phosphorylase Phosphorylase b has a specific activity

b. The of 80

At’TOMATED

PHOSPHORTLASE

ASSAI

455

phosphorylase between 2 and 20 pg/ml in order to produce an absorbancy in the range of the standard curve. The automated test for orthophosphate is reproducible within 1.2% (S.E., n = 51, the automated phosphorylase test wit’hin 3.370 (SE., n = 5), and the hand test within 4.570 (SE., n = 5). The number of tests per hour is selected as a compromise between two factors: first, this rate should be as fast as possible and, second, the washout between samples must be sufficient to avoid unacceptable error when a solution with low activity follows one with high activity. Optimum conditions are obtained with a rate of 1 assay/min. By using an actual sampling time of 18 set, the interceding 42 set water wash is sufficient to reduce the error to only ca. 3% when a sample containing 0.2 mM phosphate follows one with 5 mM. When an extreme situation is encountered, i.e., a 1 mM phosphate solution immediately following one of 10 mM, an error of 10% is introduced into the lower value. By arranging assays in order of increasing concentration, no detectable error due to insufficient washout is found. Alternatively, insertion of a buffer blank between test solutions of high and low concentration eliminates any carryover of material. When using shorter sampling times, while holding the assay rate at l/min, the height of the peaks are appreciably decreased and the washout does not appear to be more effective. Precautions must. be t.aken to prevent clogging of the tubing by the substrate containing 2% glycogen. Inclusion of 0.01% Triton X-100 in the substrate maintains a clean reaction coil. Phosphorylasc activity is not influenced by addition of 0.01% Triton S-100 to the assay system. The final concentration of Triton is not high enough to affect the phosphate determination as reported by Eibl and Lands (7). A major problem of the automated phosphorylase test is the precipitate formed upon addition of t.he acidic molybdate reagent to the phosphorylase dilution buffer containing 1 mg bovine serum albumin/ml. The need for dialysis to free the inorganic phosphate from denatured protein is eliminated by addition of 10% SDS to the effluent from the reaction coil before mixing with the stopping reagent. At the protein concentration used in the phosphorylase assay buffer (final, 0.5 mg/ml), no precipitation occurs; the system has had no problems over approximately six rnonths of operation. It has been determined that SDS at this concentration has no effect on the Fiske-SubbaRow phosphate assay (7). In a different test, when a prot.ein concentration of ca. 5 mg/ml is used, a precipitate forms that is not entirely prevented by the SDS. The addition of 0.01% SDS to the acid molybdate reagent maintains clean tubing and thus assures a more constant bubble pattern. Even though the system is designed especially for phosphorylase tests,

456

HASCHKE

AND

HEILMEYER

any enzyme liberating inorganic phosphate may be easily assayed with the same procedure. For example, the system was used to measure ATPase activities and was found to be entirely satisfactory. SUMMARY

An automated assay of glycogen phosphorylase based on t,he method of Hedrick and Fischer (6) was developed using the Technicon AutoAnalyzer system. Since a protein precipitate is formed when measuring the product (inorganic phosphate), a dialysis step is commonly employed. Dialysis is unnecessary because SDS is used to prevent, any protein precipitation. The method, which can measure any enzyme releasing orthophosphate, has a smaller standard error than the test performed by hand. ACKNOWLEDGMENTS This work One of the Foundation.

was supported authors (R.

by H.)

a grant from was a. fellow

Deutsche of the

Forschungsgemeinschaft. Alexander van Humbolt

REFERENCES 1. LEMIR, L. F.. in “Control Cameron, M. Y., eds.) London, 1964. 2. FISKE, C. H., AND ~UBR.&OW.

of Glycogen Metabolism” (Whelan, (Ciba Foundation Symposium). p.

3. ILLINGWORTH, B.. AND CORI. G. 4. HURD, S. S., TELLER. II. C., AND

24, 79 (1966). 5. BARTLETT, G. R., J. Biol. 6. HEDRICK, 7. EIBL, H.,

J. I,., AND AWD LAXDS.

J. Biol. C’hem. 66, 375 (1925). T.! Biochem. Prep. 3, 1 (1953). FISCHER. E. H.. Biochem. Biophus.

T.,

Chem. 234, 466 (1959). E. H., Biochemistry 4, 1337 (1965). W. E., Anal. B&hem. 30, 51 (1969).

FISCHER,

W. T., and 68. Churchill,

Res.

Commun.