APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1976, p. 969-976Copyright X) 1976 American Society for Microbiology

Vol. 31, No. 6Printed in U.S.A.

Liberation of Sulfate from Sulfate Esters by SoilsC. HOUGHTON' AND F. A. ROSE*

Department ofBiochemistry, University College, Cathays Park, Cardiff CFI 1XL, U.K.

Received for publication 18 November 1975

When incubated with acid, alkaline, and neutral soils, a variety of syntheticsulfate esters representing the various classes of these compounds was hydro-lyzed by enzymes, probably of microbial origin. The appearance of sulfate in thesoil water occurred immediately after introduction into the soils with someesters, whereas with others it occurred only after lag periods. Heat treatmentdestroyed the hydrolytic activity in the soils. The ester sulfate groups-present inhumic acid extracted from the soil appeared to be resistant to hydrolysis by avariety of sulfohydrolases extracted from bacteria and other organisms.

Sulfuric acid esters of various kinds arethought to play an important part in the metab-olism of sulfur in soils (7, 8, 16, 17, 28) sinceappreciable quantities of sulfur are returned tothe soil in this form in decaying organic matterand natural fertilizers. This arises from thewidespread distribution of sulfate esters asstructural components of plant and animal tis-sues and also as excretion products. The utiliza-tion of sulfate from these sources would involvethe participation of a variety of degradativeenzymes, including sulfohydrolases probably ofmicrobial origin, which could liberate inorganicsulfate.

In support of this notion, several species ofmicroorganisms, some of which are found insoils, have been shown to be capable of hydro-lyzing various types of sulfate esters (31, 32).The latter studies have, however, been con-cerned with culturing microorganisms fromsoils and examining the sulfohydrolases pres-ent in harvested cells or in the culture medium.It has not been possible to deduce from thiswork what the fate of sulfate esters might be inthe soil environment under natural conditions.Furthermore, if a wide variety of sulfohydro-lases are active in the soil environment, it be-comes necessary to explain the existence of es-ter sulfate groups that supposedly occur in thelarge organic soil colloids, humic and fulvicacids (18, 25). These are relatively stable mole-cules that could represent a storage form ofsulfate.The present study was undertaken to exam-

ine the fate of various types of sulfate esterswhen they remained in contact with undis-turbed soil for various lengths of time and to

I Present address: Department of Biological Sciences,Napier College of Science and Technology, Edinburgh EH 105DT, U.K.

determine the stability of the ester sulfategroups of humic acid towards various types ofsulfohydrolases. The implication of the findingsfor better understanding both the soil sulfurcycle and the potential of the soil environmentas a source of sulfohydrolases which may beuseful analytical tools is emphasized.

MATERIALS AND METHODSSoils. A Spodosol orthod, an Inseptisol ochrept,

and a Mollisol rendoll soil were selected for studyand obtained locally from Glamorgan and the Marl-borough Downs, Wiltshire, U.K. Twelve randomsamples of the top 15 cm of the soils were collected,pooled, and sieved (2-mm mesh) while fresh. The pHof the soils was measured immediately after collec-tion in a fresh soil-water mixture (1:2.5, wt/vol).The water content was determined after heatingweighed samples of fresh soil to 110 C for 16 h.Samples were monitored for sulfohydrolase activitywithin 12 h of collection but for other purposes werestored at 2 C for up to 4 weeks.Humic acid. Humic acid was extracted from 1 kg

of fresh Inseptisol ochrept (equivalent to approxi-mately 750 g of oven-dried material) by gently agi-tating with 4 liters of 0.5 M NaOH for 1 h. Theinsoluble material was separated by centrifuging at3,000 x g for 1 h, and the supernatant was thenacidified to pH 2.0 by the careful addition of 5 MHCl. After standing for 30 min, the precipitatedhumic acid was recovered by centrifuging at 3,000 xg for 1 h and dried by lyophilization to give fractionA (ash, 25.9%; water, 4.7%).The acid was then dissolved in alkali as before,

and the solution was clarified by centrifuging at78,000 x g for 1 h. The humic acid was precipitatedby acidification with HCl as before, and the wholeprocedure was repeated on the precipitate twicemore. The final precipitate was then suspended indistilled water, dialyzed overnight, and lyophilized(fraction B: ash, 6.8%; water, 4.4%). A furtherdeashing of fraction B was achieved by treatmentwith a hydrochloric acid-hydrofluoric acid mixtureas described by Lowe (25). This yielded fraction C

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(ash, 0.3%; water, 2.1%). The yield was approxi-mately 0.75% (wt/vol) with respect to the fresh soil.Attempts to deash the fraction further by treatingagain with hydrochloric acid-hydrofluoric acidyielded a material that had a lower sulfate content(fraction D, see Table 4). Only fractions A, B, and Cwere used for the enzyme studies.

Chemicals. Radioactively labeled sulfate esterswere prepared from 35S-labeled sulfuric acid (98%) orchlorosulfonic acid (Radiochemical Centre, Amer-sham, Bucks., U.K.) by the following methods: cho-line [35S]sulfate, 13.6 ,tCi/mg (29); dipotassium 2-hydroxy-5-nitrophenyl [35S]sulfate, 10.8 ,.Ci/mg(15); potassium dodecyl [35S]sulfate, 18.1 /Ci/mg (2);potassium p-nitrophenyl [U5S]sulfate, 13.1 ,zCi/mg(11); potassium glucose [6-35S]sulfate, 10 ,uCi/mg(24); potassium phenyl [35S]sulfate, 15.8 ,tCi/mg(19); potassium L-tyrosine O- L35S]sulfate, 3.7 ,mCi/mg(6). Potassium tyrosylglycine 0-[V5S]sulfate (3.12,Ci/mg) was prepared by the method employed forL-tyrosine 0-sulfate but substituting equimolarquantities of tyrosylglycine for L-tyrosine. Aqueoussolutions (25 mM) of the esters were prepared, andthe specific radioactivity of each was adjusted to 1.3,uCi/mg by adding the appropriate amount of 25 mMsolution of homologous unlabeled material that hadbeen prepared by the same method from unlabeledacid. Solutions were stored at --20 C until required.

Tyrosylglycine was obtained from Yeda Rehovot,Israel, and Oronite was from the Oronite Division,California Chemical Co., San Francisco, Calif. Allother chemicals were purchased from B.D.H. Ltd.,Poole, Dorset, U.K., and wherever possible were ofAnalaR grade.Enzyme preparations. A sample of alkylsulfohy-

drolase was prepared from Pseudomonas C12B(kindly provided by W. J. Payne, University ofGeorgia); cells were grown, and the sulfohydrolaseswere extracted as previously described (14).

Arylsulfohydrolases were prepared from threesources. A microbial enzyme was obtained from Al-caligenes faecalis NCIB 8734 by the method of Dodg-son et al. (4) by dissolving 20 mg ofthe acetone-driedpowder (powder B) in 20 ml of ice-cold 0.1 M phos-phate buffer, pH 8.75. A crude preparation contain-ing both arylsulfohydrolase and glycosulfohydrolaseactivity was obtained from the limpet Patella vul-gata by the method of Dodgson and Spencer (9). Asoluble extract of the acetone-dried limpet powderwas obtained by homogenizing 1 g in 100 ml of ice-cold 0.5 M sodium acetate-acetic acid buffer, pH 5.5.The insoluble debris was removed by centrifuging at79,000 x g for 30 min at 4 C.The third source of arylsulfohydrolase was the

digestive gland of the snail Helix pomatia. The en-zyme was obtained in a partially purified state(stage 2) by the method of Dodgson and Powell (5).The preparation was diluted by mixing 1 ml of theextract with 200 ml of ice-cold 0.5 M sodium acetate-acetic acid buffer, pH 6.6, before use.A glycosulfohydrolase was extracted from the vis-

ceral sac of the large periwinkle Littorina littoreaand partially purified (stage 5) by the method ofLloyd (23). The enzyme preparation was obtained bydissolving 165 mg of the lyophilized powder in 22 ml

APPL. ENVIRON. MICROBIOL.

of ice-coldc 0.5 M tris(hydroxymethyl)aminometh-ane-acetic acid buffer, pH 5.5.

Paper chromatography and electrophoresis. One-dimensional descending chromatography was per-formed on Whatman no. 1 paper for 16 h at roomtemperature with butan-1-ol-acetic acid-water(4:1:2, by volume). Paper electrophoresis was con-ducted on Whatman no. 1 paper in 0.1 M sodiumacetate-acetic acid buffer, pH 4.5, for 2 h at a poten-tial gradient of 11 V/cm.

Detection and measurement of radioactivity. Ra-dioactive components on paper chromatograms orelectrophoretograms were detected by exposing toX-ray film (Ilford Industrial B) for periods of up to 4weeks and also by scanning with a Packard radi-ochromatogram scanner (model 7200). The relativeamount of radioactivity associated with each areawas measured from the record of the scanner asdescribed by Dodgson and Tudball (12). Total radio-activity in liquid samples was also determined byscintillation counting by the method of Dolly et al.(13).Sulfohydrolase assay procedures. The sulfohy-

drolase activities of the preparations toward theirnormal assay substrates were determined beforetesting their activity toward humic acid. Arylsul-fohydrolase activity was measured spectrophoto-metrically (10) using the colorimetric substrates andconditions described in Table 1. Alkylsulfohydrolaseactivity ofthe Pseudomonas C12B extract was deter-mined by incubating 0.2-ml samples of the enzymeand potassium dodecyl L35S]sulfate under the condi-tions described (Table 1). The reaction was stoppedand deproteinization was achieved by heating at100 C for 2 min, followed by centrifugation at 2,000x g for 10 min. Liberated sulfate was estimated byelectrophoresis and scanning as described by Dodg-son and Tudball (12). The glycosulfohydrolase activ-ity of the periwinkle extract was estimated as de-cribed in Table 1 using the barium chloride-gelatinmethod of Dodgson (3).

Determination of sulfohydrolase activity towardhumic acid. The same conditions were used for theassay of activity of the various enzyme preparationstoward humic acid. Reaction mixtures (20 ml) con-taining equal volumes of fraction C humic acid sus-pension (20 mg/ml) and sulfohydrolase preparationswere incubated for 24 h under the appropriate condi-tions for each enzyme (Table 1). Samples (2.5 ml)were removed at 30-min intervals for up to 2 h andthereafter every 6 h. Organic acid and protein wereprecipitated by adjusting the pH to 1.5 with 5 MHCl. After freezing at -10 C and thawing, the sus-pended material was readily separated by centrifug-ing at 2,00t x g for 30 min. A 1.5-ml sample of thesupernatant was lyophilized, and the solid residuewas taken up in 0.1 ml of water for the determi-nation of its inorganic sulfate content. Controls inwhich the humic acid and enzyme had been incu-bated separately were also prepared. Determina-tions of sulfate by the barium chloride-gelatinmethod of Dodgson (3) were made at 400 nm, wherethe absorption due to pigmented materials not pre-cipitated by acidification did not interfere.

Soil analysis. The organic content of the soils was

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VOL. 31, 1976

derived (1) from carbon content estimations per-

formed by the method of Walkley and Black (35).The total sulfur content of the soils was determinedon 100 mg of the sieved, oven-dried (16 h at 110 C)sample by the procedure of Steinbergs et al. (30).The total sulfate (free inorganic sulfate plus estersulfate) was estimated on similar samples by themethod of Johnson and Nishita (20). The free inor-ganic sulfate was determined by the "differencemethod," which consisted of precipitating the sul-fate as lead sulfate by the addition of alcoholic leadnitrate to an aqueous soil extract (22) and estimat-ing the excess lead spectrophotometrically by thedithizonate method (34).The results of the soil analysis are given in Table

2. The total sulfate content of the humic acid frac-tions was determined by the method of Johnson andNishita (20).

Determination of sulfohydrolase activities ofsoils. Sieved, freshly collected soil (2 g) was lightlypacked into each of 15 glass filter funnels (scintered-glass disk, 4 by 1 cm, porosity x 4; A. Gallenkamp &Co., Ltd., London EC2P 2ER, U.K.) at 2 C, and thelabeled sulfate ester solution was then added tomake the total soil water 5 mM with respect to theester. The mixture was stirred thoroughly with aglass rod, and the tops of the filters were pluggedwith cotton wool. A large shallow tray containingwater was placed within the incubator to humidifythe atmosphere. Under these conditions weightchanges of the sample due to water loss were very

slight (1%) over 36 h at 28 C. Three tubes were takenafter standing for 0, 6, 12, 24, and 36 h at 28 C, and

SOIL SULFOHYDROLASES 971

0.2 ml of water was stirred into the mixture. Asample of the soil water was then obtained by centri-fuging the filter funnel within a tapered centrifugetube, fitted with a rubber collar, at 850 x g for 15min. Samples (50 ,ul) of the solution obtained were

analyzed as described. The experiments were re-

peated using samples of soil that were autoclaved at121 C for 15 min and cooled to room temperatureprior to incubation with ester sulfates

RESULTS

Hydrolysis of sulfate esters in soils. Neitherinorganic sulfate nor any of the sulfate esterscould be completely recovered in the soil water,even when the analysis immediately followedmixing. The same effect was observed withsterilized soils. It was not possible to determinethe extent of the adsorption for all possiblemixtures of each of the esters and inorganicsulfate, but this was determined at one concen-

tration. Inorganic sulfate and each of the esterswere added as 25 mM solutions to soil samplesas in the test experiments, followed by immedi-ate analysis of the soil water using the methoddescribed. The experiment was repeated on

sterilized soils. With the exception of the Insep-tisol ochrept sample, both inorganic sulfate andmost of the ester sulfates were adsorbed to thesame extent (Table 3).

Precise determinations of the total hydrolysis

TABLE 1. Conditions used to determine the activities of various sulfohydrolase preparations

Reactants (final Rate of hydrol-concn) ysis (JAg of

Sulfohydrolase prepn Test substrate (potas- Buffer Temp SO42- re-

sium salts of:) Protein Substrate (C) leased/h per

(mg/ml) (mM) ml of reaction

Alkylsulfohydrolase Dodecyl sulfate 0.05 M Tris-hydro- 5.4 5.0 30 62.5Pseudomonas C,.!B chloride, pH 7.5"

Arylsulfohydrolase p-Nitrophenyl sul- 0.1 M phosphate, pH 0.5 1.5 37 57.5Alcaligenes faecalis fate 8.75NCIB no. 8734b

Patella vulgata Nitrocatechol sul- 0.5 M sodium ace- 0.7 10.0 37 172.5fate tate-acetic acid,

pH 5.5Helix pomatia Nitrocatechol sul- 0.5 M sodium ace- 0.1 0.25 37 192.5

fate tate-acetic acid,pH 6.6

Glycosulfohydrolase Glucose 6-sulfate 0.5 M Tris-acetate, 3.8 3.0 37 214.0Littorina littorea pH 5.5

"' Tris, Tris(hydroxymethyl)aminomethane.b NCIB, National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland, U.K.

TABLE 2. Composition of the soils that were assayedSulfur content (Aglg)

Soiltype pH ~~~~~~~~~~~~Watercon- Organic con-Soil type Total sul- Total sul- Inorganic pH tent c tent (%)

fur fate sulfate

Spodosol orthod 777 237 6.5 4.0 38.0 23.0Inseptisol ochrept 613 258 5.4 6.2 28.5 8.4Mollisol rendoll 665 240 0.7 8.4 24.0 6.8

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972 HOUGHTON AND ROSE

TABLE 3. Adsorption of inorganic and organicsulfate by soilsa

Retention Retention ofSoil type of sulfate inorganic

ester (%) sulfate (%)

Spodosol orthod 69 66Inseptisol ochrept 71 93Mollisol rendoll 61 61

a The quantity of [35S]sulfate ester present in 10,ul of the 0-h samples of each soil water used in Fig.la to g was compared with the quantity present in 10,ul of the 25 mM sulfate ester solutions applied to thesoils. Figures for NP35S and NC35S were not ob-tained because of their rapid breakdown, and cho-line sulfate was not tested with the Mollisol rendoll.Identical amounts of a 25 mM K235SO4 solution (0.5,uCi/mg) were added to the soils, and the recovery of[35S]sulfate was similarly determined at zero time.Radioactivity was measured by liquid scintillationcounting. Results are the average of three determi-nations on each of the sulfate esters.

occurring in the sample by measuring the situ-ation in the soil water were, therefore, not pos-sible, and, moreover, complete recovery of theadsorbed materials could not be achieved satis-factorily by elution with solutions of variousions. Measurement of the organic moieties lib-erated was similarly unsatisfactory as a meansof determining the extent of hydrolysis since inmost cases these were metabolized further.Measurements of the amount of ester and inor-ganic sulfate in the soil water at intervals were,however, made since this gave an indication ofthe rate at which sulfate became available fromthe various esters for further utilization as un-der natural circumstances.

It appears that the esters were probably hy-drolyzed by microorganisms or particle-ad-sorbed enzymes in the experimental soils sincesterilization prior to incubation with ester sul-fates destroyed the ability of the soils to hydro-lyze all of the esters during the experimentalperiod of incubation. Rates of sulfate releasediffered markedly, however. Although data arenot presented, potassium dodecyl sulfate washydrolyzed most rapidly in all three soils,nearly all the sulfate being liberated within 1h. The ratio of ester to inorganic sulfate in thesoil water could not be determined in this casesince the ester was completely adsorbed ontothe soil particles immediately. This did not pre-vent the measurement of the rate of inorganicsulfate release into the soil water, however.Potassium p-nitrophenyl sulfate and dipotas-sium nitrocatechol sulfate were also hydrolyzedextremely rapidly in all three soils (Fig. lc andd), some hydrolysis having occurred within thetime taken to separate the soil water withoutpreincubation. In further experiments with

Inseptisol ochrept, hydrolysis was complete in 2h. Other esters were hydrolyzed more slowly,and several were minimally affected whenincubated with Spodosol, pH 4.0 (Fig. la,b,e,f,and g).The initial rate of hydrolysis of phenyl sul-

fate (Fig. lb) appeared slower than that of theother arylsulfates (Fig. lc and d). Hydrolysis ofthe additional esters (Fig. la,b,e,f, and g) ap-peared to require lag periods before hydrolysiscommenced, although lag times were decreasedas soil alkalinity increased. G635S was sub-jected to complex catabolism by soil microorga-nisms and enzymes (Fig. 2A). Within 24 h theparent compound was dissimilated, but foursulfur-bearing intermediates were accumulatedin addition to inorganic sulfate. TG35S under-went apparently simple cleavage before estersulfate was removed. The dipeptide was hydro-lyzed to yield glycine and a component that co-chromatographed with TO35S, and it was fromthe latter monomer that sulfate was removed.

C,nLA

100

75

50

25

t

I- O-OA 5

112 2. 36

Time Ihr)

Time Iv)

FIG. 1. Time courses of sulfate ester hydrolysis insoils of three types. (a) Choline [f'5S]sulfate (C'5S);(b) potassium phenyl [35S]sulfate (P:35S); (c) potas-sium p-nitrophenyl [:I`S]sulfate (NP'5S); (d) dipotas-sium nitrocatechol [:I5S]sulfate (NC:35S); (e) potas-sium L-tyrosine 0-[35S]sulfate (TO:'IS); (f) potassiumtyrosylglycine 0-[:I5S]sulfate (TG:05S); (g) potassiumglucose [6-35S]sulfate (G635S). Symbols: A, Spodosolorthod, pH 4.0; 0, Inseptisol ochrept, pH 6.2; E0,Mollisol rendoll, pH 8.4.

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SOIL SULFOHYDROLASES 973

(i) (ii) (iii) (iv) (v) (vi)

35SO42-

_~~~~~~~~~~~~

..0 .I

36 1~SG6S

A.

(i (ii) (i ii) (iv)

TG35S

To35s

35SO42-

Start - )hB

*I

n-

2~~.

c0

0L-

o)%4--0

0

a)

if)

FIG. 2. Sulfated intermediates of G635S and TG35S degradation in soils. (A) Radioautogram of an

electrophoretogram of the products of G635S catabolism in the Mollisol rendoll soil: (i) authentic G635S plusinorganic [35S]sulfate; (ii) 0 h; (iii) 6 h; (iv) 12 h; (v) 24 h; (vi) (i) plus (v). (B) Radioautogram of a

chromatogram of the products of TG35S catabolism in the Inseptisol ochrept soil; (i) authentic TG35S plusinorganic [35S]sulfate; (ii) 0 h; (iii) 6 h; (iv) 12 h; (v) 24 h; (vi) (i) plus (v).

Sulfur content of humic acid. Although partof the sulfur in humic acid is known to be estersulfate, it was necessary to establish the char-acteristics of the sample used in this work be-fore attempting to detect the sulfate estergroups and characterize them enzymatically.Humic acid was chosen over fulvic acid or othersoil organic fractions because of the relativeease of extracting reasonable yields from soils.Humic acid has a higher sulfate content than

fulvic acid, irrespective of the method of extrac-tion and fractionation (18). The three fractions(A, B, and C) ofhumic acid used in this work allcontained roughly the same amount of sulfur,and approximately 50% of this appeared to bemainly sulfate (Table 4) since it was readilyreducible by the method ofJohnson and Nishita(20). It appeared that the sulfate was relativelystable in acid solution at mild temperatures. Itcan be seen that the procedure employed to

+

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i~Q 1w?

40 4"k

c

0

L.

0)

m

0

0).

_

a)L

(v) (vi)

VOL. 31, 1976

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974 HOUGHTON AND ROSE

diminish the ash content, including incubationat room temperature for 40 h with the stronglyacidic HCl-HF solution, did not result in a nota-ble decrease in the sulfate content of the mate-rial (Table 4). Sulfate release similarly did notoccur in suspensions of the acid kept in 2 M HCIat 4 C for 96 h. A repeat of the deashing proce-dure to give fraction D, however, did result insome loss of sulfate, suggesting that prolongedtreatment with this reagent had a mild labiliz-ing effect. These observations indicate that thesulfate was largely covalently bound and notjust adsorbed.

In agreement with this, sulfate release oc-curred readily at higher temperatures in diluteacid, and all the sulfate could be removed bysuch treatment. This was determined in a fur-ther experiment in which a sample (50 mg) offraction D was suspended in 30 ml of 2 M HCIand heated at 100 C. Samples were withdrawnat 5-min intervals and cooled to 4 C, and theacid was precipitated by centrifuging at 2,000 xg for 1 h before analysis for sulfate by themethod of Johnson and Nishita (20). The sulfurcontent of the humic acid was diminished at therate of 0.9 mg/g per min to give 90% desulfationin 30 min.A liberation of sulfate also occurred when

humic acid was subjected to treatment withmethanolic HCl by the method of Kantor andSchubert (21), a procedure used to release sul-fate ester groups from macromolecules withouttheir degradation. Isolated humic acid wasdried in vacuo over P2O, at 40 C for 8 daysbefore adding o.i g to 70 ml of the dry metha-nol-acetyl chloride mixture. The suspensionwas shaken in a stoppered flask at room tem-perture for 24 h before decanting the spent re-agent. The suspension was again shaken withtwo further portions of reagent and then driedin vacuo (yield, 115 mg). Estimates of the sul-

TABLE 4. Sulfur content of humic acid"

Sulfur content

Humic acid prepn Total (mg/ Total sul- fate (o ofg) fate (mg/g) ftotal S)

Fraction A 10.2 5.0 49Fraction B 10.3 5.2 50.5Fraction C 10.1 5.0 39.5Fraction D 8.2 2.8 34

" The total sulfur and sulfate content of the acidwas determined by substituting lyophilized organicmaterial (10 mg) for soil in the systems described inthe text. Results are the average of three determina-tions and are corrected for ash and water content.Fractions A, B, C, and D were successive samplesobtained in the deashing process described in thetext.

fate content of the dried product are presentedin Table 5.These observations were again consistent

with the existence of covalently bound (i.e.,ester) sulfate in the isolated humic acid. Thealternative possibility that the inorganic sul-fate was simply adsorbed onto the organic ma-terial was also checked by an isotope-exchangeexperiment. An aqueous solution of humic acidcontaining 17 mg of fraction A was mixed withan equal volume of water or 0.2 M phosphatebuffer, pH 7.0, containing a quantity of 3S-labeled sodium sulfate representing eight timesthe amount of sulfate ion present in the organicacid. The solutions were incubated at roomtemperature for 1 h before adjusting the pH ofthe solution to 2 with 5 M HCl. After centrifug-ing at 2,000 x g for 1 h the amount of radioac-tivity in a sample of the supernatant was deter-mined and compared with that ofcorrespondingcontrols not containing humic acid. No adsorp-tion or exchange of sulfate onto the humic acidhad apparently occurred. To determine theamount of isotope in the humic acid sulfate, theprecipitated organic acid was washed five timesby successively resuspending in 10 ml of waterand centrifuging. The residue was then di-gested with 2 ml of fuming HNO3 for 4 h at360 C as described by Young et al. (36). Afterdiluting the digest to 15 ml with water, inor-ganic sulfate was precipitated by adding 5 ml of4 M HCI, 3 ml of 0. 15 M K2 S04, and 4 ml of 10%BaCl2. The precipitated BaSO4 was washedthree times with water and once with acetoneand was dried by heating at 100 C for 30 min.Assay of the radioactivity of the sample indi-cated that no significant quantity of the labeledsulfate had attached to the sample.

Activity of sulfohydrolases toward humicacid. Although the sulfohydrolases were activetoward their assay substrates, no sulfate wasliberated from humic acid, even after incuba-tion for up to 24 h under a variety of conditions.

DISCUSSIONSeveral types of sulfohydrolase, probably of

microbial origin, were detected within the soil

TABLE 5. Effect of methanolic HCI on the sulfurcontent of humic acid"

Material Sulfur content

Total (mg/ Total sul- Total sul-g) fate (mg/g) fate (% of

total S)

Untreated acid 10.4 4.9 47Treated acid 8.9 3.2 36

1' Results are the average of three determinationsand are corrected for ash and water content.

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SOIL SULFOHYDROLASES 975

environment. The absolute activity of these en-zymes could not be determined since the situa-tion was undoubtedly influenced by complexchanges in the adsorption pattern of the un-changed esters and liberated inorganic sulfateas the incubation proceeded. However, changesin the appearance of sulfate and the concomi-tant disappearance of sulfate ester in the soilwater could be observed. The relative rates ofthese changes for the various esters studied canbe compared and can be considered to be due inpart at least to the rate of sulfate hydrolysis,which in turn might be related in certain casesto enzyme induction in soil microorganisms.The rapidity with which they were degradedindicated that KD35S, NC35S, and NP35S werehydrolyzed by enzymes already present in themicrobial cells or adsorbed onto the colloids ofeach ofthe soil types. Other esters (C35S, G6 35S,TG35S, and TO35S) were not hydrolyzed imme-diately, indicating the possible necessity for in-duction of dissimilatory enzyme synthesis inthe soil microorganisms. The existence of en-zyme systems other than sulfohydrolases wasalso detected. It appeared that G635S was de-graded to at least four sulfated products andfurther that TG35S was first slowly convertedinto another metabolite (probably into TO35S)by peptidase action before desulfation (Fig 2Aand B). It is thus not surprising that naturallyoccurring low-molecular-weight sulfate estersdo not accumulate in soils, although the possi-bility remains that traces have not been ob-served due to the lack of suitable means ofextraction and detection.

Ester sulfate, often generally referred to asreducible sulfate (16, 18, 25), was, however,shown to be present in the humic acid extractedfrom one of the soils used in this study, thusconfirming the observations of earlier workers.Unfortunately, there is no specific test for theester sulfate group, and its nature can only beinferred from the hydrolytic effect of variousreagents, including 2 M HC1 (100 C), metha-nolic HCI (room temperature), and HI (underreflux). The action of hot dilute HC1 producessulfate ions from sulfate esters (and moreslowly from some sulfonates) but not from or-ganic sulfides, sulfones, or sulfoxides. Underrelatively mild conditions, sulfonates can gen-erally be considered to be completely stable(28). Methanolic HCl will not attack sulfonategroups (33) but will attack the C-O-S linkage ofsulfate esters. Reducing mixtures containingHI have been shown to produce H2S only fromester and inorganic sulfate and organic sulfites(e. g., dimethyl sulfite; see T. H. Arkley, Ph.D.thesis, Univ. of California, Berkley, 1961, and

unpublished data, our laboratory). Thus, theeffect of these reagents on the isolated humicacid observed in the present study are consist-ent with the existence of sulfate ester groups inthe material.We were not able to determine whether the

sulfate groups in the humic acid was alkyl-,aryl-, or carbohydrate esters because of theirrefractiveness to the various sulfohydrolasepreparations. Indeed, the inactivity of the sul-fohydrolases toward humic acid might suggestat first glance that sulfate ester groups are notpresent in the material, but other sulfated mac-romolecules (e. g., chondroitin sulfates, hepa-rin; see references 7, 8) are resistant to enzy-matic desulfation and require breakdown intolow-molecular-weight subunits before desulfa-tion occurs. The degradation of humic acid orother soil organic polymers apparently does oc-cur slowly and is almost certainly carried outby microbial, multienzyme catalyzed sequences,producing low-molecular-weight intermediatessuch as benzoquinone, 2-methyl 1,4-naphtho-quinone, salicyl alcohol, and salicylaldehyde(26, 27). Desulfation of humic acid couldresult from a sequential attack by depoly-merizing and desulfating enzymes. Furtherstudies along these lines would seem worth-while since the existence of such a mechanismcould indicate that the sulfohydrolases have animportant role in releasing bound sulfate fromstorage in soil colloids for microbial and plantutilization.

In any event, the existence of a wide varietyof interesting and useful sulphohydrolases inthe soil environment is indicated. It can besafely concluded that any naturally occurringsulfate ester returned to the soil will yield inor-ganic sulfate in the soil water within a rela-tively short time. Moreover, it seems that thesesources of enzymes could yield a number ofvaluable analytical tools for structural studieson sulfate esters.

ACKNOWLEDGMENTSWe are grateful to the Agricultural Research Council,

U.K., for financial support.

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