BIOCHIMICA ET BIOPHYSICA ACTA

BBA 65240

THE ACTIVITY OF CRYSTALLINE RIBONUCLEASE A

JAKE BELLO AND EUGENE F. NOWOSWIAT

Department of Biophysics, Roswell Park Memorial Institute, Buffalo, N.Y. (U.S.A.)

(Received March znd, I965)

SUMMARY

The catalytic action of crystalline ribonuclease A (polyribonucleotide a-oligo­nucleotidotransferase (cyclizing), Ee z.7.7.r6) toward cytidine Z',3'-phosphate wasdetermined in 75% z-methyl-z.a-pentanediol, the medium in which the crystals areequilibrated for crystallographic studies. The crystalline enzyme is active in thismedium. The activity, however, is due not only to the presence of crystalline enzyme,but also to enzyme which is dissolved by the substrate solution. The enzyme is highlyaggregated in this solvent. The activity of a molecule of crystalline ribonuclease isbetween one and ten times that of a molecule of ribonuclease in aqueous solution.

INTRODUCTION

In this laboratory, we are investigating the structure of bovine pancreaticribonuclease (polyribonucleotide z-oligonucleotidotransferase (cyc1izing), EC z.7.7.r6)(RNase) by X-ray diffraction". An obvious and important question that arises iswhether the structure of the protein in the crystal is the same as in aqueous solution.This is of particular interest in this case as the crystals are conditioned in 75 %z-methyl-z.a-pentanediol, rather than the more usual aqueous sulfate or phosphatesolutions.

In the case of an enzyme, the demonstration of its activity in the crystal wouldencourage the idea of similarity of molecular structure in the two states. Such ademonstration is not sufficient, as it is possible that all of the activity may reside in thesurface layer, which may not be representative of the interior. Even if the moleculesin the surface are identical with those of the interior, it is possible that all of themolecules are of non-native but active conformation, or that an active conformationis induced by the substrate. The demonstration of a lack of activity would not besufficient, however, to show that the two states are not identical. It is possible thatthe active site may be normal, but inaccessible because of the nature of the packingin the crystal. Nevertheless, at least one face of the crystal should contain accessiblesites.

Studies of the enzymic activities of two crystalline enzymes have recently been

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j. BELLO, E. F. NOWOSWIAT

reported. The catalytic action of amorphous ribonuclease and crystalline ribonuclease­S toward pyrimidine 2',3'-cyclic phosphates was studied in strong ammonium sulfatesolutions, From the amount of activity observed, it was concluded that, not only isthe crystalline enzyme active, but also the activity is too great to be accounted forsolely by the surface layer.

The activity of crystalline chymotrypsin (EC 3-4-4.5) was investigated in 70%saturated (NH4)2S04 at pH 7 using N-acetyl-L-tyrosine hydrazide as substrates.It was found that when equal amounts of crystalline enzyme and dissolved enzymewere tested for their catalytic action toward the substrate, the crystalline chymo­trypsin was 21 % as active, weight for weight, as the dissolved protein.

A difference in the hydrogen-tritium exchange rates of crystalline and dissolvedinsul'in has recently been reported-. The difference was ascribed to differences infolding in the crystalline and dissolved protein.

The purpose of this study was to determine whether crystals of ribonuclease Aare active in a medium in which they are equilibrated for crystallographic studies.We have not investigated whether the observed reaction is hydrolysis or alcoholysis.

MATERIALS

Ribonuclease crystals of Form II, space group P21 (ref. 5) were used.z-Methyl-a.a-pentanediol was purchased from Eastman Organic Chemicals,

Rochester, N.Y. (U.S.A.). Further purification of this compound was accomplishedby a procedure modified from that of KING et al.". To 1.51 of z-methyl-z.q-pentane­diol mixed with 1.5 1 of water was added approx. 65-70 g of Amberlite MB-3 ion ex­change resin (Mallinckrodt Chemical Works). This mixture was stirred for 3-4 h.After this time, the pH of the solution was checked and was normally 6.0±0.L Theliquid was decanted into a beaker and approx. 30-35 g of fresh resin was added.Stirring was continued for an additional 3 h. The pH was normally found to be 6.z±o.z. In order to minimize acid-catalyzed dehydration of the diol in subsequentoperations, about 4-5 g of K 2HP04 was added. Water was removed by distillationthrough a 70 em "Goodloe" packed column (Scientific Glass Apparatus Company,Inc., Bloomfield, N.J. (U.S.A.)). The temperature in the distilling head was allowedto rise no higher than to !ISo. When all the water was removed, the distilling flaskwas-cooled, and approx. 0.3 g of sodium borohydride was added. The following day,the solution was distilled through the same column in vacuo. The optical density ofthe distillate was measured at 215 mp., and normally found to be 0.2-0.6. Material ofhigher optical density was rejected. The z-methyl-z.a-pentanediol was stored at-20° in 60 ml glass stoppered bottles.

Ribonuclease A was obtained from the Worthington Biochemical Corporationand Mann Research Laboratories. No significant crystallographic differences wereobserved between the several lots that afforded good crystals, although not all lotswere suitable for crystallization. A stock solution was prepared by dissolving 12.5 mgof the enzyme in 25 ml of redistilled water. A I-ml aliquot was pipetted into a IO mlvolumetric flask containing 1.5 ml of redistilled water and the volume adjusted tothe mark with IOO% e-methyl-a.q-pentanediol. The final concentration of the solu­tion was 0.05 mgjml, and I ml was used for an activity determination.

50 mg of the substrate, cytidine-z 'jg vphosphate (Schwarz Bioresearch), was

Bioohim, Biophys. Acta, IOS (Ig65) 325-332

ACTIVITY OF CRYSTALLINE RIBONUCLEASE A 327

dissolved in 12.5 ml of redistilled water and the volume was adjusted to the 50 mlmark with pure z-methyl-a.a-pentanediol. The final concentration was I mgjml,and 5 m1 were used for one determination.

Base, 0.005 N NaOH in 75% z-methyl-z.q-pentanediol was used to neutralizethe acid which was liberated during the hydrolysis of the substrate by the enzyme.This normality was used in all the experiments unless otherwise specified.

Several anionic water soluble polymers were tested for inhibitory effects onribonuclease. Sulfonated polystyrene sodium salts, molecular weights 1.4' 103,

14' lO3, and 70' 103, were gifts from the Monsanto Chemical Corp. Research Labora­tories, Dayton, Ohio (U.S.A.). Polyethylene sulfonic acid, molecular weight 14' 103,

was obtained through the courtesy of the Upjohn Company, Kalamazoo, Mich.(U.S.A.). Heparin (roo International units/mg) was purchased from the ConnaughtMedical Research Laboratories, Toronto (Canada). Each reagent was prepared so thatthe final concentration was 5 mg/ml, and I ml was used in a determination.

The insoluble resins which were tested for inhibitory effects on ribonuclease Awere Dowex AG-5oW-X2 sodium form, 200-325 mesh, purchased from Calbiochem,and Sulfoethyl-Sephadex G-25 from Pharmacia Fine Chemicals, Inc. The amount ofresin used for an activity run was 100 mg contained in I rnl,

The substrate, enzyme and base solutions were prepared fresh each day inorder to obtain consistent results.

METHODS

Enzyme assaysThe titrimetric procedure described by DAVIS AND ALLEN? was used with minor

modifications for the activity measurements. The pH-stat used was the RadiometerTTT-Ia, with recorder SBR, ab, The reaction vessel was a 25-ml water-jacketed cellwhich was supported on a magnetic stirrer. The reaction temperature, 30°, was main­tained by circulating water through the vessel from a constant temperature bath.The cover for the cell was provided with four holes in order to accommodate a combi­nation glass and calomel electrode, inlets for introducing reactants, the delivery tubeof the motor-driven syringe and pure, water-saturated nitrogen.

The catalytic action of the enzyme was studied first in glass-redistilled water.The results were compared with the case in which ribonuclease A was dissolved inredistilled water and diluted with z-methyl-z.q-peritanediol to make a solution con­sisting of enzyme dissolved in 75% z-methyl-z.a-pentanediol. Such preparations ofenzyme will be referred to as dissolved enzyme to distinguish them from crystals ofthe latter.

A typical activity determination was made as follows: One or two crystals wereremoved, blotted and placed in a weighing bottle. The latter was stoppered and theweight of the enzyme was obtained by difference. The crystals and 10 ml of 75%z-methyl-e.a-pentanediol were introduced into the reaction vessel. When dissolvedribonuclease A was used instead of the crystals, I ml of the enzyme solution and 9 mlof 75% z-methyl-z.a-pentanediol were used. Five ml of substrate was then introducedwith a Mohr pipette graduated to tip. One minute was sufficient for the pipette todrain. An additional minute was needed for the adjustment of the reaction mixtureto pH 7.0. Four more minutes were allowed for thermal equilibration to 30°, and to

Biochim. Biopliys. Acta, 105 (1965) 325-332

J. BELLO, E. F. NOWOSWIAT

make the final adjustment to pH 7.0. The rate of reaction was then recorded for thenext 10 min.

After this time, the reaction mixture was subjected to one of three procedures:centrifugation, filtration through a "Millipore" filter, or the addition of an inhibitor.If centrifugation was to be performed, the solution was placed in appropriate tubesand spun for 15 min. During this time the reaction vessel and all parts which came incontact with the reactants were rinsed with distilled water and wiped dry. 12 ml ofthe supernatant were returned to the vessel and after a total of 46 min had elapsed,from time zero, the progress of the reaction was recorded for an additional 10 min.In the case where the effect of an inhibitor was studied or the solution was filtered,20 min and 26 min, respectively, elapsed before the rate of reaction was recorded.

All controls were treated for the first 16 min in the manner previously described.The subsequent treatment of these solutions depended on the operation which wouldbe performed on the experimental solutions. If the latter were centrifuged, the controlsolution was placed in a centrifuge tube and allowed to stay in the centrifuge for ISmin. When the effect of filtration was being studied, the control was passed throughthe fritted glass funnel which supports the "Millipore" filter. When the action of in­hibitors were investigated, I ml of solvent was added to the control to compensatefor the volume of inhibitor solution. All other operations were the same in both cases.

A plot of time versus base consumed was obtained. The slopes which resultedafter the test solutions were treated according to one of the methods described werecompared with those which were obtained in a control experiment.

Cage experimentA stainless steel cage was constructed of So-mesh screen. The inside diameter of

the cage was 4 mm, and the length was 80 mm. The seams were joined with silversolder. Near the top of the cage was a ring of rubber tubing, which was used to adjustthe cage to the desired height. Several activity determinations with dissolved ribo­nuclease without and with the cage immersed in the solution showed that there wasno more than 7% inhibition of activity by this apparatus. Three or four crystalswhich measuredapprox. 0.3-0.4 em on the long edge and about 0.1-0.2 em along theshorter edges were placed into the cage. The combined weight of these was usuallyaround 37 mg. The cage was immersed into the 75% 2-methyl-2,4-pentanediol­substrate solution and the activity was determined in the manner described, exceptthat the base was 2.5' 10-4 N and it was contained in a 3-ml syringe instead of I-mlsyringe which was used in all other experiments. The use of the large syringe obviatedrefilling. After 6 min had elapsed, the progress of the reaction was noted for thefollowing 10 min. The cage was then removed and 6 min were allowed to pass beforethe rate of reaction was recorded for ro min more. The procedure was repeated fourtimes and constituted one run. At the beginning of the experiment and each time thecage was to be immersed into the substrate solution, the crystals contained thereinwere washed six times with r-ml portions of 75% z-methyl-a.a-pentanediol.

RESULTS AND DISCUSSION

It was noted at first that a suspension of ribonuclease crystals in 75%2-methyl-

ACTIVITY OF CRYSTALLINE RIBONUCLEASE A

:4,4-pentanediol-substrate was enzymically active. Filtration or centrifugation toremove the crystals gave filtrates or supernates of reduced but still significantactivity. Similar treatment of dissolved ribonuclease in z-methyl-z,4-pentanediol­substrate gave filtrates or supernates of one-third to one-half the activity of unfilteredor uncentrifuged controls (Table I). This points to aggregation of ribonuclease inz-methyl-z.a-pentanedicl. This is to be expected in any solvent suitable for crystal­lization. The supernates or filtrates from dissolved ribonuclease had activities similarto those of supernates or filtrates of crystal suspensions. In view of this finding, we

TABLE I

FRACTION 011 ACTIVITY REMAINING AFTER CENTRIFUGATION OR FILTRATION OF RIBONUCLEASE

SOLUTIONS

Treatment

Centrifugation at 30000 rev.fminCentrifugation at 20000 rev.jrninClinical centrifuge at full speedFiltration through 5 ftmilliporeFiltration through 1.2 ftmilliporeFiltration through 0.45 ,umillipore

Redistilledwater

0.91 ± 0.04

0.99 ± 0.04

0.97 ± 0.041.00 ± 0.02

0.91 ± 0.02

0.89 ± 0.02

75% z-methyi­2,4-pe'ntanediol

0.50 ± 0.06

0.56 ± 0.05

0.59 ± 0.05

0.30 ± 0.08

0.13 ± 0.07

O.II ± 0.09

were unable to interpret the crystal experiment, as it was possible that all of theactivity arose from dissolved ri.bonuclease, much of which was removed with thecrystals during filtration or centrifugation.

We were able to reduce aggregation by the addition of tetramethylammoniumor sodium chloride. This resulted, however, in solubilization of crystals. This is notsurprising, as an effect that decreases aggregation should reduce the stability of thecrystals. This result suggests that electrostatic forces are involved in crystallizationof Form II, which is not the case in the crystallization of ribonuclease-S in strongammonium sulfates, Electrostatic forces are expected to be more important in the lowdielectric constant of 75% z-methyl-e.a-pentanediol than in aqueous media. Theactivity of the aggregated ribonuclease in z-methyl-a.q-pentanediol was 30% greater

TABLE II

FRACTION OF ACTIVITY REMAINING AFTER ADDITION 011 POLYMERIC INHIBITORS

Inhibitors Watey

SolubleSulfonated polystyrene sodium salt, mol. wt. I 400 0.0

Sulfonated polystyrene sodium salt, mol. wt. 10 000 0.0

Sulfonated polystyrene sodium salt, mol. wt. 70 000 0.0

Heparin 0.0

Polyethylene sulfonic acid, sodium salt,mol. wt. 14000 0.0

InsolubleSE-Sephadex G-25 0.0

Dowex AG-50W X 2 0.0

75% 2-methyl­:2,4-pentanediol

0.22 ± 0.02

0.50 ± 0.02

0.52 ± 0.09

0.16 ± 0.06

0.08 ± 0.05

0.0

0.0

Biochim, Biophys, Acta, 105 (1965) 325-332

330 J. BELLO, E. F. NOWOSWIAT

than that of an equal weight of non-aggregated ribonuclease in water. Either theaggregates are very porous or the activity resides chiefly in super-active moleculeson the periphery. The latter appears improbable.

Another approach is the use of polymeric inhibitors to bind and inactivate thedissolved ribonuclease, thereby restricting the enzymic reaction to the crystal. Theresults are shown in Table II. Soluble inhibitors were found not to be entirely effectivein inhibiting the activity of ribonuclease in 75 % z-methyl-a.z-pentanediol at theinhibitor level used. Insoluble inhibitors were completely effective and did not absorbsubstrate. However, this approach was not pressed since there remained the possibilitythat dissolving ribonuclease might react with substrate before encountering an in­hibitor particle. The use of insoluble inhibitors can demonstrate absence of activity.

Since immersion of crystals directly in the stirred reaction medium subjectsthem to breakage, and also makes it very difficult to remove the crystals for investiga­tion of the solution, experiments were done in which the crystals were contained in acage of stainless steel mesh which allowed the solution to flow past the crystals withless danger of breakage. Some breakage may result from rubbing of crystals againsteach other and against the cage. The data obtained from the cage experiment indicatesthat the reaction which results when crystalline ribonuclease is immersed in thesubstrate solution is due to both the crystalline enzyme and soluble enzyme.

TABLE III

ACTIVITY OF CRYSTALLINE RIBONUCLEASE IN CAGE EXPERIMENTS

Time Crystals (37 mg) Crystals out(min) immersed -in of substrate

substrate solution, rate*solution, rate" (flmoleJmin)(umote /11lirt)

16 8.8 ± 2.932 r.z ± 0.2

48 7.8 ± 0.864 I.2 ± 0.380 7.4 ± 0.996 I.6 ± 0.5

lI2 7.0 ± 0·9128 1.6 ± 0.4

• The activities shown are averages of eight determinations performed on the same crystals.

Table III shows that some activity remains in the solution after removal of thecage of crystals. Some of this may arise from enzyme in solution, and some from smallcrystal chips that may have passed through the mesh. If all the residual activity inthe solution resulted from the latter cause, then the activity of crystals is the totalactivity observed when the cage of crystals is immersed in the reaction mixture. Thusthe upper limit of activity of the crystals is given by the middle column of Table III.Since filtration and centrifugation experiments showed that some crystalline ribo­nuclease dissolves in the reaction medium, it appears probable that the differencebetween the middle and right-hand columns of Table III fairly well represents the

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ACTIVITY OF CRYSTALLINE RIBONUCLEASE A 331

activity of the crystalline enzyme. The decrease in activity with time when the cageis in the substrate solution is probably due to inhibition by products.".

The activity shown in Table III is equal to that shown by 0.003-0.005 mg ofdissolved ribonuclease. The 37 mg of crystals were present as three or four crystals ofabout 1.2 em- total surface area. Assuming the molecules in all faces to be equallyreactive, a layer I.2 em? in area and containing 0.004 mg ribonuclease (2.8· 10-6 ern",assuming a density of 1.4) is about 240 .f,. thick. Correcting by the factor 2 for thecrystal being about 50% solvent, a value of 480 A is obtained. This must be correctedfor imperfections in the surface which result in a larger effective area, With a moderatefactor of 2, the effective thickness of the crystal is 240 A, or about 7 molecules thick,taking an average diameter of 33 A. The dimensions of the molecule as derived fromX-ray diffraction are approx. 30 X 30 X 38 A (see ref. 10). If half of the activity isin the first layer, then the activity of the molecules in this layer is 3-4 times that ofribonuclease in solution. Considering the errors from various sources, it appears thatthe activity of a molecule of ribonuclease in the crystal is between one and ten timesthat of a molecule in solution.

We must also consider the possibility that all of the observed activity arosefrom dissolving enzyme, which was quickly inhibited after removal of the crystal cage.This was shown not to be the case by the observation that the rate of decay of theactivity of 0.004 mg of dissolved ribonuclease was not more than 5%of that resultingfrom removal of the crystal cage. Two other possibilities remain. One is that the activi­ty per ribonuclease molecule is small, but that many layers are involved. This appearsimprobable as the dissolved enzyme in z-methyl-a.a-pentenediol is more active thanin water. An unfavorable conformational change is possible. A second possibility isthat the ribonuclease molecules in the crystal are super-active, but that active sitesin only one or two faces are accessible to substrate. The super-active sites would beabout 10 times as active as calculated above. In the absence of evidence as to thevalidity of these alternate ideas, we propose that ribonuclease in crystal Form IIexhibits enzymic activity of the same order of magnitude as in aqueous solution.

It will be necessary to investigate also a number of chemical properties of thecrystalline enzyme before any definite conclusions can be drawn as to the similarityor dissimilarity of the enzyme in the two states.

ACKNOWLEDGEMENTS

We express our grateful appreciation to Mrs. T. FALZONE for purifying the sol­vents and for growing the crystals of the enzyme used in this study, and to MissE. SWYERS who did some of the early experiments.

This investigation was supported by the following grants: GMog826 from theNational Institute of General Medical Sciences, National Institutes of Health, DRG­70I from the Damon Runyon Fund, NSF-GB-429 from the National Science Founda­tion, and NIH-A-3942 from the National Institutes of Health.

REFERENCES

I G. KARTHA, J. BELLO, D. HARKER AND F. E, DEJARNETTE, in G. N. RAMACIiANDRAN,

Aspects of Protein Structure, Academic Press, 1963. p. 13·

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332 J. BELLO, E. F. NOWOSWIAT

2 M. S. DOSCHER AND F. M. RICHARDS, J. Bioi. Chem., 238 (I963) 2399.3 J. KALLas, Biochim. Biopbys. Acta, 89 (I964) 364-4 M. PRAISSMAN AND J. A. RUPLEY, J. Am. Chern, Soc., 86 (I964) 3584.5 M. V. KING, B. S. MAGPOFF, M. B. ADELMAN AND D. HARKER, Acta Cryst., 9 (I956) 460.6 M. V. KING, Biochim. Biophys, Acta, 79 (I964) 388.7 F. F. DAVIS AND F. W. ALLEN, J. BioI. Chem., 217 (I955) 13.8 C. A. ZITTLE,]. Bioi. Chem., 163 (I946) III.g C. A. lITTLE AND E. H. READING, J. Biol. cs-«, 160 (I945) 519.

IO D. HARKER, Brookhaven Symp. Biol., 13 (1960) 86.

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