dimerization of deoxyribonuclease i, lysozyme and papain : effects of ionic strength on enzymic...

Eur. J. Biochem. 124, 183-189 (1982) (0 FEBS 1982

Dimerization of Deoxyribonuclease I, Lysozyme and Papain Effects of Ionic Strength on Enzymic Activity

Salvatore SORRENTINO, Gennady I. YAKOVLEV, and Massimo LlBONATl

Institute of Organic and Biological Chemistry, Faculty of Sciences, The University of Naples

(Received December 17, 1981)

Transition of bovine ribonuclease A from its monomeric to a dimeric form changes the pattern of enzymic activity response to ionic strength [Sorrentino, S., Carsana, A,, Furia, A,, DoskoCil, J.. and Libonati, M. (19XU) Biocfzinz. Bioplzys. Acta, 609, 40-521. To see whether this phenomenon could be common to other enzyme-substrate systems, the action of various dimeric and monomeric enzymes (ox pancreas deoxyribonuclease. hog spleen acid deoxyribonuclease, bovine seminal ribonuclease, egg-white lysozyme, and papain) on polyelectrolytic substrates has been studied under different conditions of ionic strength.

Dimerization of ox pancreas deoxyribonuclease, lysozyme and papain was obtained by cross-linkage with dimethyl suberimidate.

The main results of the investigation, similar to those obtained with ribonuclease A, are the following. 1. Enzyme monomers and dimers show markedly different patterns of activity response to ionic strength at

given pH values : the reactions catalyzed by monomeric enzymes are highly modulated by salt, whereas those catalyzed by dimeric enzymes are not. In particular, at the reaction optimum the monomeric form of an enzyme is significantly more active than the dimeric one.

2 . The optimum of the reaction catalyzed by a dimeric enzyme is shifted to higher ionic strengths in comparison with that of the reaction catalyzed by a monomeric enzyme.

A model is proposed that could explain these results on the basis of the influence of ionic strength on the intramolecular dynamics of the enzyme molecule and its non-specific interactions with polyelectrolytic substrates.

The interaction between single-stranded or double- stranded RNAs and ribonucleases of the bovine pancreatic type is influenced by several interdependent variables, such as ionic strength and pH of the medium [l], stability of the secondary structure of the nucleic acid duplex [ 2 ] , charge characteristics of the enzyme proteins [3], and the extent of glycosylation of the enzyme molecule [4], all of which affect the efficiency of enzyme action.

Results obtained from the study of the degradation of double-stranded RNA by monomeric and dimeric ribonucleases indicate that the structure of the enzyme protein can be an additional variable in the process. For example, transition of bovine RNAase A from its monomeric to a dimeric form (by cross-linkage with dimethyl suberimidate) strikingly changes the pattern of enzymic activity response to ionic strength at given pH values. The reaction catalyzed by monomeric RNAase A is highly influenced by changes of ionic strength, whereas that catalyzed by the dimeric enzyme is significantly less affected by such variations [I] .

This phenomenon, provided the substrate is a polyelectrolyte, does not seem to be limited to an RNAase/RNA system, as can be demonstrated from the results presented in this work, in which various enzyme/substrate systems have been studied under different conditions of ionic strength and

Enzyme. Deoxyribonuclease from ox pancreas (DNAase I) (EC 3.1.21.1); deoxyribonuclease from hog spleen (DNAase 11) (EC 3.1.22.1); bovine ribonuclease A (RNAase A) (EC.3.1.27.5); bovine seminal ribonuclease (EC 3.1.27.-); egg white lysozyme (EC 3.2.1.17); papain from Curicapupaya (EC 3.4.22.2).

pH, namely (a) two deoxyribonucleases in monomeric or dimeric form, and DNA as substrate. (b) a native dimeric ribonuclease and a monomeric derivative, with polycytidylate as substrate; (c) lysozyme and its cross-linked dimer with Micrococcus luteus cell walls as substrate; (d) papain and its cross-linked dimer with bovine serum albumin as substrate.

In accordance with the results previously obtained [I], the rates of degradation of polyelectrolytic substrates by the monomeric forms of these enzymes are markedly influenced by changes in ionic strength, whereas the reactions catalyzed by dimeric enzymes appear to be only slightly affected by the ionic environment.

MATERIALS AND METHODS

Reagents

Dimethyl suberimidate dihydrochloride was a Pierce Chemicals product. Iodoacetamide was purchased from Sigma Chemical Co. NaCI, MgC12, CaC12, as well as 2,4,6- trinitrobenzenesulfonic acid were analytical reagents grade purchased from J . T. Baker. Twice-distilled deionized water was used in all experiments described.

Substrates

Calf thymus DNA was purchased from Worthington Biochem. Co. Polycitidylic acid was a Boehringer (Mannheim) product. Bovine serum albumin was purchased from Sigma Chem. Co. Micrococcus luteus was a Miles product.

184

Enzymes

DNAase I from bovine pancreas (type I, specific activity, 2000 Kunitz units/mg), and bovine pancreatic RNAase A (type XII-A) were purchased from Sigma. DNAase I1 from pig spleen (specific activity, 22736 units/mg) was a Worthing- ton product. Egg-white lysozyme (specific activity, 24000 units/mg) was purchased from Miles. Papain from Curica pupuya (specific activity, about 30 units/mg) was a Boehrin- ger product. Bovine seminal RNAase [5,6] was purified as described [5]; its specific activity was 40 Kunitz units/mg.

Enzyme Derivatives

Monomeric Seminal RNAuse. A monomeric derivative of bovine seminal RNAase was prepared according to Parente et al. [7], by selective reduction of the disulfide bridges (and carboxyamidomethylation of the resulting - SH groups), which link the half-cystine residues at positions 31 and 32 of the two subunits in the native enzyme [8].

Cross-Linked Dimers of Ox Pancreas DNAase. A typical preparation is the following. To 31 mg DNAase I, dissolved in 1 ml 200 mM triethanolamine/HCl buffer, pH 8.5, containing 150 mM NaCI, aliquots of I0 p1 of a 12.5 mM solution of dimethyl suberimidate (dissolved immediately before use) were added every minute under continuous stirring at 25"C, up to a total of 110 p1 (374 pg). The reagent/protein molar ratio was about 1.3. The pH was checked during the reaction, and, if necessary, adjusted to 8.5 with 1 M triethanolamine/HCl buffer. 27 min after starting the procedure, the reaction was stopped by adding 0.5 ml 100 mM ammonium acetate, containing 150 mM NaCI. The incubation mixture (about 1.7 ml) was then applied to a column (1.5 x 97 cm) of Sephadex G-100 superfine, equilibrated with 150 mM NaCI, containing 5 mM MgC12 and 4 mM CaC12. Flow rate, 6 ml/h. 1 -ml fractions were collected, and their absorbance at 280 nm measured. The gel filtration pattern was similar to that shown by Hartman and Wold [9], in the preparation of RNAase A dimers by cross-linkage with dimethyl adipi- midate, and consisted of a small amount of aggregated material followed by two peaks, the first (smaller) corre- sponding to the dimeric, the second (larger) to the monomeric reaction product (indicated, respectively, as eluted monomer or dimer in Table 1). The analysis of free NH2 groups of the amidination reaction products was performed according to Wang et al. [lo] (see Table 1). The relatively low yield (10%) of dimers was probably due to the pH chosen (far from the pI of the protein) and to the low concentration of DNAase used [ I l l .

C,os.s- Linked Dimers of' Egg- White Ljisozyme. Lysozyme dimers were prepared by a procedure similar to that described above, with some modifications: (a) 50 mg protein/ml reaction mixtures were used ; (b) the diimido ester/protein ratio was about 1.2; (c) gel filtration was performed with a column (1 . I x 94 cm) of Sephadex G-75 superfine, and elution carried out with 20 mM ammonium acetate/acetic acid buffer, pH 5.5. The elution pattern was similar to that described for DNAase I, but the yield of dimeric lysozyme was about 18 %. The analytical data of monomers and dimers of egg white lysozyme are shown in Table 1.

Cross-Linked Dinzers of Papain. To 32 mg papain in 1.9 ml 200 mM triethanolamine/HCl buffer, pH 8.5, containing 1 niM EDTA and 5 mM 2-mercaptoethanol, aliquots of 10 pl of a 17.5 mM solution of dimethyl suberimidate (dissolved immediately before use) were added every 30 s under continuous stirring at 30 "C, up to a total of 100 p1 (477 pg).

Table 1. Free NH2 groups of the urnidination reaction products Free NHz groups were estimated with 2,4,6-trinitrobenzenesulfonic acid, according to Wang et al. [lo], using bovine RNAase A as a standard. All proteins were gel filtered through columns of Sephadex (3-25 superfine (equilibrated with 20 mM NaCI) before determination of free NH2 groups. Each figure is the average of two determinations performed in duplicate with 100 pg and 150 pg protein

Protein - ~~~~

NH2 groups/protein molecule

calculated found _ _ _ ~ ~ - ~-

~~

Ox pancreas RNAase A 11

Ox pancreas DNAase (eluted dimer") Egg white lysozyine (native monomer) Egg white lysozyme (eluted monomer") Egg white lysozyme (eluted dimer") Papain (native monomeric form) 10 Papain (eluted monomera) -

Papain (eluted dimer") -

Ox pancreas DNAase (native monomer) Ox pancreas DNAase (eluted monomer")

12 -

7 -

- -

~~

11.26 12.60 9.68

22.11 6.92 6.46

11.74 9.68 8.30

15.86

a Eluted monomers or dimers refer to the enzyme species eluted from the columns of Sephadex G-100 or G-75 after the preparation of DNAase I, lysozyme or papain dimers (see Materials and Methods)

18 min after the first addition, the reaction was stopped by adding 35 pl 1 M ammonium acetate. The reaction products were separated at room temperature by gel filtration through a column (1.5 x 85 cm) of Sephadex G-75 superfine equilibrated with 50 mM Tris/HCI buffer, pH 7.5, containing 1 m M EDTA and 5 niM 2-mercaptoethanol. Flow rate, 6 ml/h; fraction volume, 1.5 ml. The elution pattern was similar to that described for DNAase 1. The yield of dimeric papain was 10%. The analytical data of the amidination reaction are shown in Table 1. Enzyme concentrations were estimated spectrophotometrically, using the following absor- bances: A;% = 7.3 for bovine RNAase A [12]; A:% = 4.65 for bovine seminal RNAase [5]; A;& = 12.3 for DNAase I [13]; A t 2 = 12.1 for DNAase I1 1141; A:$ = 26.4 for egg- white lysozyme [15]; A i 2 = 25.0 for papain [16].

Enzyme Activity Assays

Nucleolytic activities were measured spectrophotonietri- cally, using a Varian Cary, model 118, spectrophotometer equipped with a thermostatically controlled water bath. With DNA as substrate, the increase of A260, due to enzyme activity, was measured as a function of time [17]. The absorbance of a buffered solution (see for details legends to Fig. 1 and 2) of DNA was checked at 260 nm for at least 3 min before adding the enzyme. DNAase I or 11, dissolved before use in a mixture of 150 mM NaCl and 5 mM MgCI2, with the addition of 2 mM CaC12 for DNAase I, were added to start the reaction. Specific activity, calculated from the slope of the linear part of the recordings. is expressed as dA260 min-'/ mg protein-' with 47 pg calf thymus DNA/ml at 25 "C. With poly(C) as substrate a similar procedure was used. To the buffered solution (for details, see legend to Fig. 3) of poly(C), the absorbance of which 1181 was checked at 269 nm for 3 min, native seminal RNAase or its monomeric derivative, dissolved before use in 20 mM Tris/HCl buffer, were added. The increase in absorbance at 269 nm, produced by degradation of poly(C), was linear for no more than 4 min. Specific

1x5

N P 5 40 =-. c > c 0

.- .- 2 0 - ._

r ._ 0 0 (1

500 - 'm E 7 400 .- E

PN 300 a

f 200

0

L 100

0

- h

._ c

0

.- 0 m (1 cn

0

-

000

800

600

400

200

I

"0 50 100 150 NaCl (rnM)

Fig. 1. Action qf'monomeric or dimeric DNAase I on D N A under various ionic strc,ngtlis andpH conditions. (A) pH 6.2; (B) pH 7.0; (C) pH 7.7. Incuba- tion mixture: 47 pg calf thymus D N A and 62 ng monomeric (0) or dimeric (0) ox pancreas DNAase weremixed in 1 mI20 ~ J M jmjdazole/HC'l buffer, containing 5 m M MgC12 and 2 mM CaC12. NaCl as indicated on the abscissa. Temperature, 25 "C. Assays (in duplicate) perlormed as described under Materials and Methods

activity is expressed as dA269 min-' mg protein-', with 80 pg poly(C)/inl at 25 'C.

Lysozyme activity toward M . luteus cell walls was measured with the classical turbidimetric method described by Shugar [19]. The decrease of the absorbance of a buffered suspension of M . luteus, due to enzyme action, was recorded at 450 nm. Specific activity (dA450 min-' mg protein-', with 240 pg M . luteus/ml, at 25 "C) was calculated from the slope of the linear part of the curve, taken 1 min after starting the reaction.

Papain activity was measured with a precipitation assay similar to that described by Arnon and Shapira [20]. Bovine serum albumin and enzyme were mixed in 1 ml of the appro- priate buffer (see legend to Fig. 5). After 10 min at 37 "C, 1 ml cold 10% trichloroacetic acid was added to the rapidly chilled reaction mixture, which was then kept at 0 ° C for 30 min. Precipitates were discarded by centrifugation (7000 xg, 30 min), and the absorbance of supernatants was mea- surcd at 280 nin versus enzyme-free blanks. Specific activity is expressed as A280 min-' mg protein-', using 8 mg albumin/ ml at 37 'C.

In the experiments which follow, comparisons between dimeric and monomeric enzymes were made using the monomeric species that were recovered after, and therefore went through, a complete dimerization procedure. However, no differences could be detected when native monomers were used in control experiments.

RESULTS A N D DISCUSSION

Experiments tcitlz DNAaselDNA Systems

The DNAase I/DNA system was chosen because ox pancreas DNAase, a monomeric acidic protein [13,21], could be covalently dimerized, allowing one to test the same enzyme as either the monomer or the dimer. Furthermore, the information obtained could be checked and corroborated by studying the reaction catalyzed by native spleen acid DNAase 11, a dimeric, basic protein [14,22]. Fig. I shows the action of monomeric and dimeric DNAase I on calf thymus DNA as a function of ionic strength and pH. Whereas the activity of the monomeric form of the enzyme is strongly influenced, at any pH values, by salt concentration of the medium, the activity of dimeric DNAase I seems to be significantly less dependent on ionic strength. Similarly, the reaction catalyzed by native spleen acid DNAase (Fig.2), a dimer in its native

80 1 I

'-5 60

(0 O L I

0 50 100 150 Nat (rnM)

Fig. 2. Action of native DNAase I 1 on DNA under clilferenf ionic .strmgt/z and p l f condirions. Incubation mixture: 47 11% calf thymus D N A and 344 ng porcine spleen acid DNAase were mixed in 1 ml 20 mM sodium acetate/acetic acid buffer for pH 4.2 (O), 4.8 (W), 5.2 (A); 5 mM MgC12 was always present. NaCI, as indicated on the abscissa. Temperature, 25 "C. Assays (in duplicate) performed as described under Materials and Methods

form, also appears to be relatively independent of ionic strength. From the data presented in Fig.1 and 2, further information can be gained, namely that the low salt dependence of DNA degradation by a dimeric enzyme does not seem to be influenced by the acidic or basic nature of the enzyme protein.

However, one could argue that the artificial procedure of dimerization could damage DNAase I in some way, and therefore be responsible [or the marked change in activity of the enzyme in passing from a monomeric to a dimeric form. The results obtained with DNAase I1 (Fig. 2) do not support this view. However, the following experiments were devised to clarify the problem.

Experiments with a RNAaselRNA Systcw

Bovine seminal RNAase is a basic (PI, 10.3) dimeric enzyme, consisting of two identical polypeptide chains linked by two interchain disulfide bridges [5 ,6 , X I . By selective reduction of the double -S-S- bond, catalytically active monomers are obtained, which can be stabilized by alkylation of the

186

- Y,, 3000 E

._ 2500 E

c

: 2 0 0 0 P

1500

z 1000

2 x c ,- .E

c 'C 500

0

G, Q rn

N a C l (mM)

Fig. 3. Action of dimeric or monomeric bovine seminal RNAase on poly(C) under diffhrent ionic strengths and p H conditions. (A) pH 1.7; (B) pH 8.7. Incubation mixture: 8 0 pg poly(C) and 14 ng native, dimeric seminal RNAase (0) or 13 ng of its carboxyamidomethylated monomeric derivative (0) were mixed in 1 ml of 20 mM Tris/HCI buffer. NaCl, as indicated on the abscissa. Temperature, 25 "C. Assays (in duplicate) performed as described under Materials and Methods

-0 5 0 100 150 200 "0 50 100 150 200 NaCl (mM)

Fig.4. Action of native lysozyme or its cross-linked dimers on M. luteus cell ~vulls, as afunction of ionic .strength and p H . (A) pH 1 . 5 ; (B) pH 8.5. Incubation mixture: in 1 ml 20 mM Tris/HCl buffer 250 ng native, monomeric (0) or dimeric (0) egg-white lysozyme were mixed with 240 ~g M . luteus cell walls. NaCI, as indicated on the abscissa. Temperature, 25 "C. Assays (in duplicate) performed according to Shugar [19]

- SH groups at positions 31 and 32 of the polypeptide chain [7]. Therefore, the system formed by native seminal RNAase and its artificial monomeric derivative(s) is, in a way, the reverse of that consisting of native DNAase I and its artificial dimeric derivative.

Fig.3 shows the pattern of degradation of poly(C) by native seminal RNAase and its carboxyamidomethylated monomeric derivative under different conditions of ionic strength. The activity responses of the dimeric enzyme to changes in salt concentration are low, the curves obtained being reminiscent of those observed with dimeric DNAase I, or DNAase 11. Instead, the reaction catalyzed by the monomeric form of seminal RNAase acquires a significant suscep- tibility to variations in ionic strength similar to that observed with monomeric DNAase I. Therefore, whether the structural change of an enzyme occurs from the monomeric to the dimeric form, or vice versa, the different patterns of activity response of the two enzyme species to changes in ionic strength are essentially unchanged, and cannot be ascribed

to the artificiality of the dimerization or monomerization procedures.

A question could be raised here. Are the phenomena thus far described limited to the action of nucleases, or could they be observed with other enzyme/polyelectrolytic substrate systems? To verify this point, we studied the activity (a) of lysozyme on Micrococcus luteus cell walls, and (b) of papain on bovine serum albumin.

Experiments with the LysozymelM. luteus Cell Walls System

Fig. 4 shows the results obtained studying, under different ionic strength conditions, the lytic action of monomeric egg- white lysozyme and its cross-linked dimers on M . luteus cell walls, which develop a strong electrostatic potential above neutral pH [23]. Marked changes in specific activity of monomeric lysozyme are produced by relatively small differences in salt concentration, whereas the reaction catalyzed by the

187

-7 3 F - c .- E 0

-? 4 2 - ZI

>

0 m

c .- .- I

u 1 .- - .- " m Q

In

7 B

0 50 100 150 300 500- 0 50 100 150 300 500 NaCl (mM)

Fig.5. Action of monomericpapain or its cross-linked dimrrs on albumin us a function ojionic strenxth a n d p H . (A) pH 7.5; (B) pH 8.5. Incubation mixture: I0 pg monomeric (0) or dimeric (0) papain were mixed with 8 mg bovine serum albumin in 1 ml 50 m M Tris/HCI buffer, containing 1 mM EDTA and 5 m M 2-mercaptoethanol. NaCI. as indicated on the abscissa. Temperature, 37 "C. Assays (in duplicate) performed as described under Materials and Methods

dimeric enzyme is scarcely affected by changes in ionic strength.

Experiments with the PapainlAlbumin System

Fig.5 shows the results obtained analyzing the action of monomers and cross-linked dimers of papain toward bovine serum albumin, which is a polyanion under the experimental conditions chosen. A different pattern of the influence of salt on the reaction catalyzed by a monomeric or a dimeric enzyme can also be observed with the papainlalbumin system, although the clear salt dependence of albumin digestion by monomeric papain is not expressed by bell-shaped curves, but by monotonic decreases.

In conclusion, the role that the enzyme structure plays in the interaction between nucleases and polyanionic substrates appear to be strengthened, and possibly extended to different enzyme/substrate systems, by the data presented in Fig. 4 and 5.

We attempted to consider our results in the light of the theory of the catalytic implications of electrostatic potentials [23,24]. The theory predicts that within the enzyme-substrate complex a polyanionic substrate may change the pK values of the catalytically active groups of the enzyme. Any disso- ciable group, under the influence of the polyanionic electrostatic potential $, has an apparent pK, pK($), given by the equation :

pK($) = pK($ = 0) - 0.43 e$/KT, (1 ) where pK($ = 0) is the 'normal' pK of the group in the absence of an electrostatic potential and e is the unit of charge. The polyelectrolyte theory states that $ depends on ionic strength through the electrostatic screening of the mobile charges :

(2) $ ( I ) = $ ( I = 0) - A log I ,

where I is ionic strength, and A a positive constant. The electrostatic potential will also affect the apparent Michaelis constant according to the following equation [23] :

K, = Kk exp (2 e $ / K T ) , (3) Z being either the substrate or enzyme charge (negative or positive). As the ionic strength increases, $ as well as pK($)

decrease according to Eqn (2), and the pH optimum of the reaction is shifted to lower values. At constant pH, the vari- ation of enzyme activity as a function of increasing ionic strength is at least in part the consequcnce of this pH profile shift [24].

The patterns of activity responses (bell-shaped curves or monotonic decreases) to ionic strength at given pH values, obtained in this work, are qualitatively in line with the theory proposed by Douzou and Maurel [23,24]. However, it remains unclear why the modulation by the mobile charges is markedly different for reactions in which a monomeric or a dimetic enzyme, and a polyelectrolytic substrate are involved.

It is worth considering that the intramolecular dynamics of an enzyme may depend on ionic strength. For example, it was shown that the intramolecular mobility of RNAase A depends both on salt concentration and on the type of anions present in solution [25]. It may be assumed that the most rigid conformation of an enzyme occurs when, in the absence of solvation by salt o r solute ions, the electrostatic interactions between its charged groups are at maximum. In this case the enzyme could have a low activity, since the formation of a productive enzyme-substrate complex needs the possibility of definite conformational transitions of the enzyme molecule [26-321. As the ionic strength increases, the enzyme activity connected with enzyme 'rigidity' also increases, reaching its maximum at a given salt concentration (Fig. 6 A , curve 1). The higher the local concentration of charges in the enzyme molecule (dimeric versus monomeric enzymes), the higher will be the concentration of salt at which maximal activity occurs (Fig. 6A, curve 2). However, solvation of the charged groups of both enzyme and substrate reduces the non- specific electrostatic interactions between enzyme and substrate [33], and consequently, the K,,, increases as Eqn (3) predicts. The influence of ionic strength on enzymic activity controlled by non-specific enzyme-substrate interactions is shown in Fig. 6B, curve 1. For dimeric enzymes, endowed with higher local concentrations of charges, non-specific interactions are stronger; as a consequence, the activity curve is shifted to higher ionic strength values (Fig.6B, curve 2). The real dependence of the Michaelis constant and accord- ingly, that of enzyme activity, expressed as kcat/Krn, on ionic

188

I I !

I I o n i c s t r e n g t h -

Fig. 6. A model for the control of enzyme activity by ionic-strmgth-dependent specific and non-specific enzyme-substrate interactions. (A) Influence of enzyme ‘rigidity’ on the ionic-strength-dependent activity of monomeric ( I ) and dimeric (2) enzymes. (B) Influence of the solvation-dependent strength of non-specific ionic interactions between polyanionic substrates and monomeric (1) or dimeric (2) enzymes. (C) The activity of monomeric ( I ) or dimeric (2) enzymes on polyanionic substrates depends on the combination of the effects shown in (A) and (B)

strength, observed in the experiments presented here (Fig. 1 - 5) and elsewhere [I], can be considered as the resul- tant of both of these effects: at low salt concentrations enzyme activity is controlled by the specific enzyme-substrate interactions, while at high salt concentrations such control is effected by non-specific enzyme-substrate interactions (Fig. 6C).

From these arguments, which are supported by experimental evidence presented in this work and elsewhere [l], the following conclusions emerge. (a) When the number of positively charged groups of an enzyme molecule increases (dimeric versus monomeric seminal RNAase, dimeric lysozyme and papain, but also bovine RNAase A dimers [10,34] or derivatives like polysperminc-ribonuclease [35] versus native RNA- ase A, as well as very basic RNAases [3]), the optimum of enzyme activity on a polyanionic substrate is shifted to higher ionic strength values (Fig. 3,4, and [l]). It is worth mentioning that with monomeric and dimeric DNAase I, which is an acidic protein, such shift can hardly be seen (Fig.1). More- over, the optimum activity of monomeric and dimeric papain occurs at salt concentrations close to zero (Fig. 5), probably due to the fact that in the region of pH values where experiments were performed the positively charged groups of the enzyme are solvated by the negative solute ions. (b) At the reaction optimum, the monomeric form of the enzyme is more active than the dimeric one (Fig. 6C). This could explain the apparently lower modulation by ionic strength of thc reactions catalyzed by the dimeric enzymes studied here

(Fig. 1 -5 ) and elsewhere [I]. (c) A particular case is that of enzymes acting on double-stranded nucleic acids: the activity curves of monomeric and dimeric enzymes controlled by non- specific enzyme-substrate interactions (Fig. 6B) can be shifted to lower ionic strengths in comparison to those occurring with single-stranded polynucleotides as substrates if a de- stabilization of the nucleic acid duplex is necessary for enzymic attack [36,37]. As a consequence, the optimum for enzyme activity is also shifted to lower ionic strengths [I].

On the basis of the ideas discussed above, all the present and many of the past observations can be explained, in particular those concerning ribonucleases or ribonuclease derivatives having a higher number of basic charges than bovine RNAase A, all of which appear to be more efficient than RNAase A at degrading double-stranded RNAs at high ionic strengths [l, 3,10,35,38].

The authors are very grateful to Prof. A. Colosimo (The University of Rome) for helpful discussions. They also acknowledge the help of Dr Rocco De Prisco in the purification of bovine seminal RNAase. This work was supported by Consiglio Nazionale delle Ricerche grant no. CT 79.01 9.17.04.

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S. Sorrentino and M. Libonati, Istituto di Chimica Orgdnica e Biologica, Facolth di Scienze. UniversitB degli Studi, Via Mezzocannone 16. 1-80134 Napoli, Italy

G. 1. Yakovlev. Laboratoriya Enzymologii, lnstitut Molekulyarnoj Biologii, Akademiya Nauk S.S.S.R., Vavilova ulitsa 32, Moskva, 117312 USSR

dimerization of deoxyribonuclease i, lysozyme and papain : effects of ionic strength on enzymic...

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