CLIN. CHEM. 24/2, 208-211 (1978)

208 CLINICAL CHEMISTRY, Vol. 24, No. 2, 1978

p-Nitrophenol-a-D-Glucopyranoside as Substrate for Measurement

of Maltase Activity in Human Semen

Pierre Chapdelalne, Roland R. Tremblay, and Jean V. Dube

Hitherto, seminal plasma maltase has been measured withmaltose as substrate; this method is time consuming andlacks specificity. The use of a synthetic substrate, p-ni-trophenol-a-o-glucopyranoside, allows accurate and rapiddetermination of this activity. When maltase is added tothe incubation medium (the substrate and reduced gluta-thione in potassium phosphate buffer, pH 6.8), maintainedat 37 #{176}C,hydrolysis of the original substrate to p-nitro-phenol goes at a constant rate during 4 h. Under optimalconditions of incubation, the Michaelis constant of thereaction, calculated by the Hanes method, was 2.92 0.84 (SD) X iO for six different semen samples. Iso-maltase appeared to be absent from seminal plasma. Theenzyme is stable to freezing and slow thawing and can bestored for at least 26 days at -80 #{176}C.Its molecular weightis 250 000. Tris(hydroxymethyl)aminomethane (pH 6.8)exerts a noncompetitive inhibition on the enzyme activity.In 68 men 23 to 45 years old, whose semen analyses werenormal, the seminal plasma maltase activity was 467 135 (SD) mU/g of protein. It was generally decreased inpatients with infertility disorders.

AddItIonal Keyphrases: infertility disorders and assessmentproperties of the enzyme . isomaltase - normal

values

Human seminal plasma is rich in certain glycosidasesthat act upon degradation products of hyaluronic acid(1, 2). Maltase (a-glucosidase; a-D-glucoside glucohy-drolase; EC 3.2.1.20) is one of these androgen-regulatedenzymes. It might play a significant role, in conjunctionwith amylo-1,6-glucosidase, by degrading seminal gly-cogen and thus providing glucose as an energy sourcefor the motility and the metabolism of sperm (3).

Previously, maltase activity has been evaluated bymeasuring the production of glucose after 24 h of incu-

Department of Endocrinology.Metabolism, Laval Univ. HospitalCenter, 2705, Blvd. Laurier, Ste-Foy, Qu#{233}bec,P. Qu#{233}bec,Canada,G1V4G2.

Address reprint requests to R.R.T., at the Endocrine ResearchLaboratories at the address given.

Received Sept. 12, 1977; accepted Nov. 29, 1977.

bation of seminal plasma with maltose at 37 #{176}C,theglucose concentration being determined by the glucoseoxidase method (4). To circumvent the low specificityof the procedure, we have devised a new assay with p-nitrophenol-a-D-glucopyranoside (PNPG) as substratefor the enzyme. This paper deals with the validity of theassay when seminal plasma is used as the source ofmaltase.

Materials and Methods

PrincipleThe assay is based on the determination of the con-

tinuous hydrolytic release of p-nitrophenol from PNPG;the coloration of this yellow compound further deepensin Na2CO3, 0.1 mol/liter. Absorbance was measuredwith a double-beam spectrophotometer at 400 nm.

ReagentsPotassium phosphate buffer, 67 mmol/liter, pH

6.8.Na2CO3 solution, 0.1 mol/liter, pH 11.3.Reduced glutathi one. Dissolve 2 mg in 2 ml of dis-

tilled water. Prepare daily.Stock solution of substrate, 230 mmol/liter. Dissolve

182mg of PNPG (mol. wt., 301.3) in 2.6 ml of buffer at37 #{176}C(PNPG will precipitate if the solution is kept atroom temperature). Twelve determinations of maltasecan be done with this solution.

Stock solution of p-nitrophenol (PNP), 10 mmol/liter.

Procedures

Sample collection and storage. Ejaculates from fer-tile donors were collected into sterile tubes, examinedfor morphology and motility, and sperm counts ob-tained by use of a Newbower counting chamber. Semi-nal plasma was collected after centrifuging the semenat 385 X g for 30 mm and used immediately or stored at

S7 8pH

7 nmoi

100 300

x(rnoIllitec)1

Fig. 2. Typical example of pure noncompetitive inhibition ofmaltase activity by Tris buffer (1.1 mmol/Iiter) in human seminalplasma (triangles)This curve shares a common (extrapolated) Intersection on the substrate axiswith the cuve that has been obtained with potassium phosphate buffer only (darncircles)

CLINICAL CHEMISTRY, Vol. 24, No. 2, 1978 209

-80 #{176}C.Before assay, the plasma was thawed slowly at4 #{176}C.This plasma was the source of enzyme.

Incubation. To a constant amount of the syntheticsubstrate (200 il of the stock solution, or 14 mg), weadded 50 l of the reduced glutathione solution and 1.2ml of the buffer. Tubes with this incubation medium,as well as those containing 100 il of seminal plasma or100 tl of water, were incubated at 37 #{176}Cfor 10 mm.Then 50 l of water was added to one tube containingthe incubation medium (blank). This step representedtime 0; at regular intervals of 1 mm, 50 il of seminalplasma was deposited in tubes already containing theincubation medium. After a further 4-h period of in-cubation, at 37 #{176}Cwith gentle agitation, 1 ml of themixture was taken at 1-mm intervals and deposited into50-ml glass tubes already containing 10 ml of Na2CO3(0.1 mol/liter), to stop the reaction. After a 10-s agitationwith a vortex-type mixer, the absorbance of each tubewas determined at 400 nm, and the PNP content of theunknown was estimated according to a standard curveobtained with PNP in Na2CO3. The blank value wassubtracted from each unknown.

Standard curve with PNP. The initial stock solutionof PNP was brought to 0.5 mmol/liter with distilledwater or buffer and a standard curve was prepared forincreasing concentrations of PNP (0, 100, 200, 300,400,and 500 nmol) in 10 ml of Na2CO3, 0.1 mol/liter.

Analytical Variables

Incubation time. To 16.2 ml of potassium phosphatebuffer were added 0.6 ml of the reduced glutathionesolution and 30 mg (0.43 ml) of PNPG. After incubationat 37 #{176}Cduring 10 mm, 0.6 ml of preincubated seminalplasma was deposited in the incubation medium. One-milliliter aliquots were then taken at various time in-tervals during 4 h. The enzymatic reaction was stoppedin Na2CO3 and the absorbance determined as previouslydescribed. The hydrolysis of PNPG was linear for atleast 4 h; values of less than 1% were recorded at thattime. This incubation period affords adequate sensi-tivity for assay of seminal plasma samples. The observedlinearity of the reaction with time indicates that theenzyme is stable under reaction conditions over thistime period. Moreover, the percentage of nonenzymatichydrolysis of PNPG after 4 h is negligible.

Effect of pH and temperature. The velocity of thereaction at substrate saturation (30 mmol/liter) wasplotted as a function of PH; a bell-shaped profile wasobserved (Figure 1), with optimum activity at pH 6.8,the pH used in further experiments.

Incubation media containing the same amount ofenzyme were incubated at low (3 #{176}C)and intermediate(25 and 37 #{176}C)temperatures during 4 h. The highestmaltase activity was recorded at 37 #{176}C.

Kinetics. Six different seminal plasma were incu-bated with increasing concentrations of PNPG (1.1 to14 mmol/liter) in standard conditions, to determine theMichaelis constant (Km) by the Hanes graphicalmethod (4a). The mean Km was 2.92 0.84 (SD) 10

Fig. 1. Relationship between pH of the incubation medium andoptimal maltase activity of human seminal plasma

(n = 6). A 30-mmol concentration of PNPG is sufficientto saturate all the active sites of the enzyme.

Enzyme PropertiesSensibility to freezing and thawing. The seminal

plasma of two normal men was frozen and thawed fourtimes during 26 days. The specific activity of the enzymeremained remarkably constant during this experi-ment.

Inhibitors. Tris(hydroxymethyl)methylamine (Tris)buffer, pH 6.8, decreased the V but did not affect theKm of the reaction, thus showing a classical noncom-petitive inhibition (Figure 2).

By contrast, maltose and D-glucose, two normalconstituents of seminal plasma, acted as competitiveinhibitors of PNPG hydrolysis at the molarity used in

moi/i,ter

0

210 CLINICAL CHEMISTRY, Vol. 24, No. 2, 1978

Fig. 3. Competitive inhibition of human seminal maltase bymaltose (15 mmol/Iiter) (white circles) and D-glucose (60mmol/Iiter) (triangles)The Km of the enzyme was In the expected range, 2.7 X 1O mol/liter (darkcircles), in this experiment

the following experiment (Figure 3). They couldtherefore prevent the access of the synthetic substrateto the catalytic site of maltase, but their concentrationin seminal plasma is so low (0.25 mmol/liter for glucose)that the maltase velocity determination cannot be af-fected in vitro when PNPG is used as a substrate. It wasalso interesting to note that the inhibition constant (K1)for maltose represented half of the Km value of PNPGfor maltase. This observation seems to indicate thatmaltose has a higher affinity for the active site of theenzyme than does the synthetic substrate PNPG.

Expression of Maltase Activity

In this study, the catalytic activity of the enzyme wasinitially determined in milli-IUB units (mU) per literof seminal plasma: one unit being 1 pmole of PNP re-leased per minute per milliliter of incubation medium.The protein content of the seminal plasma was alsodetermined by the method of Lowry et al. (5) and theresults were finally expressed in terms of specific ac-tivity of the enzyme, namely mU/g of protein.

Results

The intra-assay coefficient of variation of the methodwas determined with seminal plasma pools possessinglow (225 mU/g of protein) or high (1225 mU/g of pro-tein) maltase activity. It was, respectively, 5.1 and 3.1%for 12 measurements. The inter-assay coefficient ofvariation of the method was 2.4% after 12 determina-tions in duplicate at daily intervals.

Fig. 4. Human seminal plasma maltase activity in normal adultsubjects and in infertile patientsNumber of observations is indicated in parentheses

Sixty-eight intact men whose ages varied between 23and 45 years were selected as the control group in thisstudy; their semen was collected after two or three daysof abstinence from sexual intercourse. The number ofspermatozoa per milliliter, and the in vitro motility andmorphology of the spermatozoa were found to be nor-mal. Maltase activity was 467 135 (SD) mU/g of pro-tein in this limited population. In infertile patients witha well-documented varicocele or in azoospermia, malt-ase activity was significantly decreased (P < 0.05).Vasectomy was associated with a drastic decrease inmaltase activity (Figure 4).

DiscussionIt is well known since the original work of MacLeod

in 1941 (6) that mammalian spermatozoa must utilizevarious sugars for maintenance of sperm motility andits metabolism. In order to be utilized by spermatozoa,disaccharides must be converted to monosaccharides,and various enzymes for doing this are present in thesemen. After a careful examination of this enzymaticmilieu, maltase was selected as a biological marker ofpotential value in male infertility, because it appearedto be androgen-dependent in rat and mice (7). This ledus to develop a sensitive and rapid assay with p-nitro-phenol-a-D-glucopyranoside as substrate for the en-zyme. The method has been properly adapted forhuman semen. Evidence has been obtained that theenzyme is highly stable in conditions of freezing andthawing; however, the catalytic activity is inhibited byweak complexing agents such as Tris buffer. We haveshown that maltase activity is generally decreased inpatients with various infertility disorders but the exactsignificance of this finding remains to be elucidated.Nevertheless, on a short-term basis, the method will beused to determine the localization of maltase in the

In the process of purification and in the study of the propertiesof an a-glucosidase of Saccharomyces italicus Y 1225, this substratehas been used extensively with success (8).

CLINICAL CHEMISTRY, Vol. 24, No. 2, 1978 211

accessory organs of reproduction in men and to assess,in particular, the results of surgical cure of varicocele.

This work was supported by a grant (MA-5848) from the Medical

Research Council of Canada.

References1. Sheth, A. R., and Rao, S. S., Maltase activity in human semen.Experientia, 18, 370 (1962).2. Sheth, A. R., and Rao, S. S., Purification and properties of humanseminal maltase. J. Reprod. Fertil. 4, 267 (1962).3. Freund, M., and MacLeod, J., Effect of addition of fructose andglucose on the fructolysis and motility of human semen. J. Appi.Physiol. 13, 506 (1958).

4. Dahiquist, A., Determination of maltese and isomaltase activitieswith a glucose-oxidase reagent. Biochem. J. 80, 547 (1961).4a. Plowman, K. M. (Ed.), Enzyme Kinetics (Series in AdvancedChemistry), 11, McGraw-Hill, New York, N.Y., 1972.5. Lowry, 0. H., Rosenbrough, N. H., Farr, A. L., and Randall, R. J.,Protein measurement with the Folin phenol reagent. J. Biol. Chem.,193, 265 (1951).6. MacLeod, J., The effect of glycolysis inhibitors and of certainsubstrates on the metabolism and motility of human spermatozoa.Endocrinology, 29, 583 (1941).7. Gunaga, K. P., Sheth, A. R., and Rao, S. S., Maltose activity in theaccessory reproductive organs of rats and mice. Indian J. Exp. Biol.5, 141 (1967).8. Halvorson, H., and Ellias, L., The purification and properties ofan a-glucosidase of Saccharomyces italicus Y 1225. Biochim. Bio-phys. Acta 30, 28 (1958).