ARCHIVES OF HIOCHEMISTRY AND BIOPHYSICS 14, 261-268 (1971)

Physical Heterogeneity of Hatching Enzyme of the Sea Urchin,

Strongylocentrotus purpuratus’

DE&-NIS BARRETT, BEN F. EDWARDS, DAVID B. WOOD, Ah‘D DOUGLAS .J. LANE

Department of Zoology, University oj California, Davis, Calijorha 95616

Received November 23, 1970; Accept,ed January 4, 1971

Ilatching enzyme from hatching Strongylocentrotue purpuratus blaatulae is physi- cally heterogeneous. (1) Salt fractionation and gel electrophoresis both reveal a dispersion of enzyme act.ivity. (2) Gel filtration in 0.5 M salt gives a mol wt of ea. 150,000 for much of the enzyme activity, with three smaller peaks corresponding to lower molecular weights. (3) DEAE-cellulose chromatography separates three peaks, and further experiments subdivide the pattern into five. (4) In sedimentation equilibrium runs, the activity is associated with two buoyant densities, one at 1.36 g/ml, outside the range of simple proteins. The data give best support to a hypothesis of association between enzyme molecules and varying amount.s and, perhaps, kinds of heterologous molecules.

The sea urchin embryo is enclosed just after fertilization in an ext,racellular coat,, the fertilization membrane or chorion, which persists until the mid-blast,ula stage. At that time, the blastula secretes a pro- teolyt.ic enzyme, the hatching enzyme (EC 3.4.4), which digests the chorion (1).

Hatching enzyme has a perhaps unique potential for studies of genetic control mechanisms, as a stage-specific assayable gene product elaborated while t.he embryo is still cleaving. It has seemed the more at- tractive because it is found in extracellular space, and because a relat,ively easy met,hod for purifying the hatching enzyme of Adhocidaris crassispina to crystal1init.y has been given by Yasumasu (a).

The present communicat,ion reports a variety of results gat,hered while attempting to prepare a single pure hatching enzyme from Strongylocen.trotus purpuratus, the commonly used sea urchin from the Pacific Coast of North America. Taken toget,her they present a consist.ent pattern of hetero-

1 Supported by Grant HD-03479 from the Nat.ional 1nstitut.e of Child Health and Human l)evelopment, U. S. Public Health Service.

geneit,y in the hatching enzyme of Strongy- iocentrotus purpuratus as it is collected. Indeed, the enzyme preparations are sepa- rated into at least. t.wo fractions (each independently able to digest chorions) on the basis of any physical crit,erion we have thus far explored, including solubilit,y, charge, charge distribut.ion, buoyant den- sity, and molecular size.

The action of hatching enzyme is pro- t.eolyt.ic, in S. purpwatus (3, 4) as in other species (1, 5, 6). A second protease, recog- nized by its capacity to activate trypsinogen t,o trypsin, is also present in the hatching supernatant (7). The present. study pro- vides additional evidence that hatching enzyme and trypsinogenasc :tre distinct rnt.it.ies.

MATERIALS AND METHOIX

Enzyme and assays. Hatching enzyme was pre- pared from blastulae hatching at 15’ in 10 vol of Millipore-filtered sea water (pore size 0.45 pm, Millipore Corp., Bedford, Mars.) with 100 ag/ml each of penicillin G and streptomycin (Sigma), a,~ previously described (8). Enzymatic activity in digesting chorions was assayed as previously (8). A concentration of euzyme v.-hich requires 1 hr to

261

262 BARRETT ET AT,.

dissolve half the ethanol-fixed chorions in a st.and- ard assay mixture is defined as 1 unit/ml (1 u/ml). Trypsinogenase activity was assayed as det.ailed previously (9).

Ammonium sulfate fractionation. In the cold, with stirring, dry (NH&SO, (Baker) was added to a crude enzyme preparat.ion to briug it to 2.1 M. After 10 min the precipitate was sediment,ed by 12,000g X 5 min. More (NH&SO4 was added to the supernat.ant fluid to bring it to 2.6 M, and anot.her precipitat.e centrifuged out. Successive addit.ions of salt and centrifugation brought down precipi- tates at 2.9 M, 3.3 M, aud 3.9 M (saturat.ed) (NH&SO,. All precipitates and an aliquot of the final supernatant fraction were dialyzed exhans- t.ively against 1 my CaClr, 1 mM Na-glycylglycine, pJI 8.0, before assrty.

Polyacr&mirle gel electrophoresis. Ornstein and Davis’ procedure (10) was used, with the running buffer at, half strength. Samples of 100~1 of enzyme were jelled wit.h 20 ~1 of large-pore gel solution and aubject.ed t,o 5 mA/tube for 1 hr. The bromphenol blue front was permanently marked by inserting a segment of copper wire. The gel was sliced length- wise and i.he halves placed on pieces of black phot.ographic film (exposed to sunlight, before developing, very thoroughly washed) moist,ened with 10 mM CaCI1, 10 rn# glycylglycine, pH 8.0. These were incubated 10 or 20 hr in a moist, box and then gent.ly washed and compared with controls t.o reveal where proteolyt.ic activity had dissolved away the emulsion.

Gel $Zlralion. An enzyme preparation was gen- erally concentrated 20-fold by Diaflow filtration (Ijiaflow membrane UM-10, Amicotl Corp., Lex- ington, Mass.) and then dialyzed against the buffer. 0.5 M NaC1. 20 mM tris(hydroxymethyl)- aminomet.hane (Tris), 10 mM CaC12, 1 mM NaNa. adjusted t.o pH 8.0. It, was then applied to a column equilibrated with the same buffer. Columns 2.5 X 40 cm were packed with Sephadex (.&lo0 (Pharms- aia AB, Uppsala, Sweden) and wit.h Biogel A-15m (Bio Itad Laborat.ories, Richmond, Calif.). Three- milliliter fractions were collected and assayed for absorbance at ‘280 nm, and for enzyme activity. Crude est,imates of molecular weight were calcu- lated from similar runs with purified molecules of known weight: tobacco mosaic virus (4 X IO’), a gift. of 1)r. George Bruening; blue dextran (2 X 10fij, Pharmacia; porcine bhyroglobulin (669,000), hlalul Research Laboratories, Inc.; human gamma globulin (160,000) and hemoglobin (64,500)) Mann; bovine serum albumin (65,000), Pentex, Inc.; bovine pancreatic rihonuclease (13,700) and insulin (5,800), and horse heart cyt,ochrome c (12,360), from Sigma Chemical Co.

Zon-ezchange chromatography. A column 2 X 15

cm WBB packed with diethylaminoethylcellulose (DEAE-cellulose, Whatman Microgranular, DE 52) equilibrated wit,h 10 rnM Tris (pH 8.0), 10 my CaCl2,O.l M NaCl. Sample was applied in the same buffer and eluted with 100 ml or more of the buffer. Then a linear gradient between 0.1 M and 1.0 M

NaCl in the same Tris-Ca buffer was applied. Fractions of 3 ml were collected and assayed for NaCl concentration by refractometry, and for enzyme activity as given above.

To t.est the activity dependence on salt, frac- t.ions were both (a) dialyzed against 10 mrrm Ca- glycylglycine, pH 8.0, assayed with eggs equili- brated with t.he latt,er buffer; and (h) assayed as they emerged from the column, but with eggs in 4.2 M NaCl, so that effective Na+ concent.ration was at least 1.5 M.

In some cases fractions were pooled, recon- centrated by Diaflow filtration, dialyzed against starting buffer, and subjected to chromatography again.

Sedimenlalion equilibrium. Starting solutions (a) 0.35 ml enzyme + 2.3 ml 1.24 M CsCl, and (b) 0.35 ml enzyme -I- 2.3 ml 4.14 M CsCl, were used to generate a 5.3-ml linear density gradient, with enzyme dispersed throughout, as well as 10 mM CaCll and 1 mM glycylglycine at pH 8.2, which sufficed to maint,ain pH. A micule sphere of den- sity 1.306 ZIZ 0.005 g/cm’ (Microspheres Inc., Palo A1t.0, Calif.) was also added. The tubes were spun in a SW39 rotor at 34,000 rpm for 70 hr. A Densi- Flow apparatus, (Buchler Instruments, Ft. Lee, N. .J.), wss used t,o collect 200~~1 fractions. Selected fractions were used for refract.omet,ry ((Goldberg t.emperature-compensat.ed refractometer, Amer- ican Optical Co.) immediately after collecting fractions. Others were dialyzed twice at 4” against 10 rnM glycylglycine, ~1-1 8.0, 26 mM CaC12, for later enzyme assay.

CsCl was from -4merican Potash and Chemical Co. Buoyant densities were calculated from re- fract,ive indices, ignoring the negligible contribll- tion (11) to refrart,ive index by the protein, which in no fraction exceeded 1 mg/ml i II ro!~cent.rat,iotl.

RE:RULTP

Salt j’ractiorLai.iotr artd electrophoresis. The product,s of a fractionation by solubility in (NHI)SSO.I are reported in Table I. Dixon and Webb (12) have argued in detail why a homogeneous protein, depending on its concentration, may begin precipitating out at. various salt, concentrations. Oricc it, begins precipitating, however, 90 % should leave solution with un increment of onI5 0% 11 (R’H.&SOr. The resu1t.v of Table I

HETEROGEXEITY OF SEA URCHIN H.4TCHING ENZYME 203

do not accord with the expectat,ion for a homogeneous prot,ein.

Electrophoretic patterns were variable, with major losses of activit,y, and activit,y generally spread widely over the gel. An atypically discrete patt,ern is diagrammed in Fig. 1. It should be noted t,hat the assay of the electropherograms alone was nonspecific, for protease act,ivity in general rather than for chorion digestion. Elution of the chorion- digesting activity from portions of the gel was attempted without success.

Gel filtration. Figure 2 &o-c\-s that, the enzyme is resolved on Sephadex G-100 into a major peak, a shoulder, and a minor peak of activity, with a scat.tering at even lower molecular weight, which is analyzed for

TABLE I

ACTIVITY OF AMMONIUM SIXX.\TE FR.WTIOXS

Fraction U/Id Ill1 ” _. .-.

O-2.0 M 0.25 1 .(i 0.40 2.1-2.6 M 0.11 2.7 0.38 2.11-2.9 Ivl 0.12 2.2 0.26 2.9-3.3 IX 0.16 2.1 0.34 X.3-3.9 M 0.13 9.7 1.26 X!) M supernaie 0.05 150. 7.50

= 0.0 5 I= u a 0.6

convenience as another peak. The first peak appears t,o be at the upper edge of the separation range of the Sephadex G-100. In an att,empt, to resolve this peak, Hiogel

PIG. 1. Diagram of the patter11 made on black film by a polyacrylamide gel electropherogram of hatching enzyme. Clear areaa reflect dissolution of the gelatin emulsion by proteolytic activity. RF,

ratio of distance to thr hromphenol blue front.

0.2

30 40 50 60 70

FRACTION NUMBER

FIG. 2. Gel-filtration chromatography on dextran. Two-milliliter samples of hatching enzyme or purified molecular weight markers were applied to a column of Sephadex G-100. Buffer: 0.5 M NaCl, 20 mu Tris, 10 mu CaClz, 1 mM NaNa, adjusted to pH 8.0; flow rate: 20 ml/hr; fraction size: 3 ml. Enzyme . . actwlty (0-O); A280 nm CO..-0); molecular weight markers (m): d, Blue Dextran; a, serum albu- min; c, cytochrome e; i, insulin. According to t.he manufacturer, Blue Dextran moves like a protein with mol wt 150,000.

264 BARRETT ET AL.

0.0 3

20 30 40 50

FRACTION NUMBER

FIQ. 3. Gel-filtration chromatography on agarose. Two-milliliter samples of hatching enzyme or purified molecular weight markers were applied to a column of Biogel A-15. Buffer as in Fig. 2. Frac- tion size: 3 ml. Enzyme act,ivity (0-O); Am, nm (O...O); molecular weight markers (m): v, to- bacco mosaic virus; t, thyroglobulin; g, gamma globulin; h, hemoglobin; r, ribonuclease. Note that ac- cording to the manufacturer, the gel separates particles of mol wt 40 X lOa through 20 X 106. Therefore, the virus and ribonucleaee mark these limits, although their weights, in fact, lie beyond this range.

0.0 20 40 60 80

1.345

2 1.340 g

-

W

I.335 2

4 E 1.330 w oz

FRACTION NUMBER

FIG. 4. Chromatography on DEAE-cellulose. A 26-ml sample of hatching enzyme, dialyzed against initial buffer, was applied. One hundred milliliters of buffer were applied, before a 150-ml salt gradient. Fractions were assayed for enzyme activity without further treatment. Enzyme activity (0-e); A 280 nm (o...O); index of refraction (---).

TABLE II

~~WVLVIYONS OF MOLECULAR WEIGHT

1 70 100,0U0 71 150,oXP 2 20 72,030 17 65,OXP 3 7 52,OcW 8 1 3 32,000“ 5

A-15, a molecular sieve of complementary capabilities, was employed, with resulti shown in Fig. 3. This pattern establishes that the high-molecular weight activity does elute as a single peak, with a mol wt about 150,000. The shoulder on Sephadex is well resolved. The peaks of lower molecular weight are too near the lower limit of sepa- ration to yield reliable molecular weight estimates.

The areas under the curves were roughly ” More reliable estimate. integrated, t,o allow est.imation of t:he pro-

HETEROGEh‘EITY OF SK.:9 URCHIN HATCHING ENZYMIS 265

0.5 2 >

0.0

z 5 g 0.5 a

: 0.0 2 El

0.0

0.5

0.0

1.345

t: 1.340 p z

1.335 Y I= 2

1.330 i w

FRACTION NUMBER

FIG. 5. Rechromatography on DEAE-cellulose. Selected fract.ions eluted from the DEAE column of Fig. 4 were coucentrated, dialyzed against st,art- ing buffer (with 46 mg bovine plasma albumin aa a protective ageut), and reapplied. Fract.ions I, II, and III, originating as shown by the bars at the very top of the figure, and expected to emerge iu the same position, in fact were fractionated into patterns shown in graphs I, II, and III, respec- tively. Part of column I (marked with a bar, I’) wan rechromatographed, with results showu in the bottom graph, I’. Elution schemes were uniform for these four runs, so that the refractive index plot in graph I refers to all four graphs. The ab- scissa is also common. Enzyme act.ivity (0-O); refractive index (- - -).

portion of tot.al enzyme units residing in each peak. These and the other result,s are summarized in Table II.

Ion-exchanye chromatography. A typical separation achieved on a DEAE-cellulose column, by a salt, gradient at constant pH, is presented in Fig. 4. Three different peaks have clearly been separated. When the material in each peak w&q concentrated and refractionated on the same column, graphs I, IT, and III of Fig. 5 resulted. In t.he main, most, of a peak which elut.es at a part,icular

salt concent,ration the first. t.ime, will elute at the sume concentration the second time. But in each case a small port,ion of the ac- tivit,y will e1ut.e in a different area of the column! appropriate to a different, peak. The entities separated by the column seem to be interconvertible.

An alternat.ivc to interconversion lay open in t,he case of peak 1. Peak 1 is the run- through, the material which never adsorbs to the column. If the column were over- loaded, excess material of peaks 11 and III would fail to find binding sites on t,lte column and run through. To test that possibility, we fractionated for a third time, second-genera- tion peak I material. The fractions within Ohe bar labeled I’, in Fig. 5, graph I, have run t,hrough n column to which virt,ually nothing was adsorbing; genuine peak II and III material should have adsorbed to the column with no competition. Hut when this clean peak 1 material is now refractionated (Fig. 5, the bott.om graph I’) it does give rise, nonetheless, to small peaks II and 111.

Thus, it. appears that most. of the enzyme activity of a DEAE peak maintains its integrit,y on recycling, but. a small portion changes, and no transition, t.o any other peak, is forbidden. Transitions toward peak I, it, might be not,ed, seem quantit.ativel) favored over the ot,hers.

The various peaks were characterized further by t.heir enzyme uct.ivity in extremes of salt, concentrat.ion. Ratios of activity in 0.01 31 buffer to activity in 1.5 11 salt, (t,he enzyme’s normal medium, seawater, ap- proximates 0.5 31 salt) are plotted for the various DEAE fractions in Fig. 6. The ratios vary considerably, over a ZOO-fold range, and are plotted on a logarithmic scale.

Two new act,ivity peaks were resolved in t.he course of the rechromatography and salt,-dependence experiments. Five DEAE e1uat.e peaks can now be defined, on the basis of characberist.icssummarized in Table III.

Sedimentation. equilibri~um. After 70-hr centrifugation, t.he init,ially dispersed hatch- ing enzyme samples have approached closely enough to equilibrium that, peak positions are reliable, alt,hough peak shapes may not be final.

In two runs at different, times, results

266 BARRETT h’T AL.

40 60 FRACTION NUMBER

FIG. 6. DEAE fractions: dependence of enzyme activity on salt concentration. After separation as in Fig. 4, fractions were assayed twice, in low salt (10 mM Ca-glycylglycine, pH 8.0) and in high salt (same buffer with NaCl at 1.5 M). Ratio of activity in low salt Do high salt, (A-A), on a logarithmic scale. Enzyme activity of each fraction (O-O), as assayed in low salt (fractions l-20), or high salt, (fract,ions 42-92).

T.4BLK III

A&W

l)lq:AE F~~.\~~r~os.\~rroh- --_-..

Salt con- Elution centration

for best activity

Ia Ib

II

III

1v

l< uns t.hrough Adsorbs weakly; din-

placed by starting buf- fer

Klut.es on’y with higher salt. thau 0.1 M NaCl

Rlnt,es wit.h still higher salt

lSlut,es with still higher salt

Lowest Thigh

TIigh

TTigh

Low

-.-~. .-

were obtained like those presented in Fig. T, with two peaks of activity at buoyant densit,ies of about, 1.31 and 1.36 g/ml. Other runs where enzyme was layered on bop of the gradient, instead of dispersed in it, did not reach final po&ions within 70 hr, but t.he bipartite distribut,ion of enzyme ac- t,ivity was already demonstrable. The ac- curacy of the den&y calculations was in all cases confirmed by observing the position in the gradient of micule spheres of known den&y (e.g., Fig. 7).

l’ryps-isogew.ase. Salt) and agarose fract:ions were t,ested for trypsinogen-activating ac- tivity. Some trypsinogenase is precipitated in r:& of t,he (R’H$&301 fract,ions, but the

0.5 th 1 I .45

--

$0.4 -1 - 1.40

: 0. 5 0.3 - %

Y- -

-i

F ‘0 - 1.35 >

: \ \

‘0 \ Q-Y w 0.2- :\ I b\

1.30

0.0 ’ \ I I I\ - - ’ 1.20

5 IO I5 FRACTION NUMBER ’

FIG. 7. Dist.ribution of hatching enzyme ac- tivit.y (0-O) in a density gradient, near equi- librium after sediment,ation for 70 hr at 34,ooO rpm. Buoyant density, g/ml, (O--O). Final position in the gradient of a micule sphere of brloy- ant density 1.300 f 0.005 g/cma (V).

amount is not proportional to hatching enzyme activity. In Biogel h-l.5 fractions, the two enzymes do not, overlap; brypsino- genase elutes at an earlier position whillc reflects much higher molecular weight.

DISCUSSION

The data convincingly show t,he phynkd het.erogeneity of the hnt,ching supernatant

of Stl,o,r!l~locent~otus pulpuratus bub do not lead t,o a satisfactory explanation of the basis of the het.erogeneit,y. Experiments have not yet been carried to cross-fractionation, so that, we are in a position analogous to having five one-dimensional chromutographs and no t.wo-dimensional one. Beyond the f:kct of heterogeneity, however, three hard fwts do emerge from the studies to dat,e: in seawker conditions, enzymat.ic act,ivit\ ia associated v&h species of about, 30,000, 50,000, 65,000, and 150,000 daltons; in similar conditions DIME-cellulose :~llows t.lw separation of five peaks, some differing greatly in catalytic dependence on salt concentrat,ion, but. all interconvert.ible; at 3 \I CsCl, the enzyme is associated with two buoyant densities, one of them 1.36 g/ml.

Alt.hough we recognize that. further ex- lwimentat.ion will prove mow rewarding t Ii:ui extensive speculakion, the following t.hree hypotheses for generating the hetero- gcncity of t,he enzyme nonetheless stem w&h presentation and brief argument: (1) mult,iple forms of one monomer: a single sort of enzyme molecule folds to different conformat,ional variants, or polymerizes with homologs to various sizes; (2) multiple Iwtwoassociat.ions wit,11 ow monomer: a single sort. of enzyme molecule associates I.:tther &ably with various amount.s, and ~wrhnps kinds, of hetorologous molecules; :md (3) mult)iple gene products: the hakh- ing principle is composed of products of two or more gene loci.

1. The- hypothesis of multiple forms deriving from one monomer is indicated b\ t,he interconvertibility experiment., taken at, f:~cc value. Rub simple folding variations wnnot. &isfy the requirement. for different molcculur weights, and, in fact, the p&icu- 1:~ molecular weight,s calculated do not fit. wry well as integral multiples of :L single unit. Xor can any combinations based on a single monomer account for tlw observed two densities.

2. The heteronrsociation hypothesis ac- cords natt,ly with the spread of molecular weights. suggesting that t,lle enzyme has a molecular weight of about 30,000 and, under t,he various condit.ions used, associates rat.hcr st:tbly with other particles to pield the v:wi-

ous heavier size classes. We do not reproduce here the many calibration curves for exclu- sion chromatography but have inspect,ed them to ascertain that hatching enzyme elutes in peaks of about the same shape its peaks of purified proteins, not appreciabl) wider. This somewhat unexpected result suggests the conclusion that the enzyme is bound, not. to :L variable class of particles, but to :I few sorts only, of discret.e sizes.

The interconvertibilit~ of peaks need not be incompatible with this hypot,hesis. Thcrch is :L predilection for the wvious peaks to revert to I, so that the simplest scheme makes I pure enzyme. Then II, III, and IV are enzyme bound rather stably to various other molecules. Het,ween fractionations (perhaps especially when ionic strength is changed) the complexes slowlg dissociate? so that. scrambling can go on between enzyme molecules and various contaminants. Xonr of the peaks is pure as oluted, so that some contaminating material will be uwtilnble. Iii the conversion experiment shown in l’ig. 5, bovine serum albumin was nddcd to Al samples aft,er fractionation, but in other t.rials t,he same effect.a occurred without the addition of exogenous protein.

Finally, the density result is compatible only with this hypothesis. Ifft has surveyed the literat.ure and concluded (11) that. rc- gnrdless of varying hydration, ion binding, etc., all noncoqugated soluble proteins thus far investigated shorn buoyant densities in the range 1.24 to 1.34 g/ml. Thus, at least some of the heterogeneity of hatching en- zymr must derive from associat.ion of part, of the enzyme wit.11 moieties denser than protein, most probably saccharidc. At, 3 ar salt concentration. the proportion so bound is about half.

3. The hypothwis t,hat multiple gene products are involved receives no support. from the data. It cannot, explain the densit\, of 1.X g/ml. It can explain tlw molecul:u weights only if it is accepted that one of the enzymes, the major one, is an extracellular hydrolase of mol wt 150,000, which seems unlikely (13) though not impossible. The failure to find distinct I)EAE peaks (which could not interconvert) dso militates ag;lillst tlir existencr of more tli:ul one enzymt!.

268 BARRETT ET AL.

It must be remembered that each of the assays reported here requires the enzyme to digest. away a whole chorion, so that if we were resolving different enzymes, each would have to suffice t,o do t.he whole job alone. Even so, if they were proteases with different specificities of att,ack, one would expect to find synergism between them-or, in reverse, t,hat when t,hey were separated, the sum of bhe part,s would be less than the whole. That expectation is not: met. When individual peaks are chromatographed, unst.able peaks sometimes lose activity (generally there is 100 % recovery), but when whole enzyme is fractionated, there is generally a gain in total activit,y.

In sum, the data in hand strongly support association with heterologous molecules as a basis for the observed heberogeneity. We specu1at.e bhat. the association is quit.e likely to be with fragments of the enzyme’s natural substrate, for which it does have a strong afiinit,y (14). The dat,a give no support, to models based on more than one enzyme, or on homopolymerizat~ion of a single enzyme. They do not rigorously exclude the latter posslbilit,ies, however, and nothing save Occam’s razor excludes that two or &en all three of the postulated mechanisms are operating.

Finally, the relationship between hatch- ing enzyme and t,rypsinogenase remains clouded. The one does not digest chorions; the other cannot act,ivate trypsinogen; the present. study shows them separable by phys- ical means. Yet t,hev arc both proteases active at pH S, distmct from the class of serine proteases, and secreted cont.empora- neously by the embryo. The data exclude that they cm be one and the same enzyme. But the molecular weight, determinat,ions hint that, perhaps t,he same process of com-

plexing a single enzyme with heterologous molecules, which diversifies the hatching enzyme, may extend to even higher molecu- lar weight.s and modify catalytic character- istics, and so account for t.rypsinogenase. Clearly the best test of the hypothesis lies in fractionating enzyme prepatrations which have been freed of all associated molecules by denaturing so1vent.s. Research is pro- ceeding in this direction, hampered at pres- ent by irreversible losses of activit,y.

ACKNOWLEDGAMl+%T

We thank Gerald M. Angelo, Donald J. Kling- borg, and Selma K. Sholes for skillful help during various phrases of t.he work.

1. 2.

3. 4. 5.

6.

7. 8.

9. 10.

11.

12.

13.

14.

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