On the mechanism of metal activation of deoxyribonuclease I

.~RCIIIVES

Oh’

BIOCHE11lSTRY

ASI)

BIOPHYSICS

73,

337-385

(195x)

On the Mechanism of Metal Activation Deoxyribonuclease I’s”

of

I>Fosyri~)oiluclense 1 (I)Xase I) is t’he “pai~~rcas dcosyril)onu~le:~se” studied clxt,eusivrly by Kunitz (l), among ot’hers, and first c*rystnllizcd tq him (2). This enzyme is :L phosphodiest,erasc, specific for verbin int,crlruc~leotidc linkages of deosyril)onucltic acid (DKA), has a pEI optimum from 0 to 7.3, \-arying tvith the ass:ly met,hod used, aud recjuirts c.ertain bivalellt met:lls for ac:ti\ity. IIN:isc I is found in large :tmounts ill t ht p:u~~eas :llld ilr p:rllcwatic juivc, and its prcscnw has IW~YIilrdicxt)cd in liver, kidney, splcc11, lung, :~nd placelltn (:‘,, -1). 1t \v:w early rcwguizcd t,h:Lt, rclat i\-cly high ~on~tiltl.:lt,iolls of the :\vt ivat ilrg metal \verc needed for maximum wtivity (1, 5-I 0). M&arty (,!I) llotcd t,h:rtm MgSO4 “in thr ordrr of 0.003 AI” IVM required in his sgstcm; Iiullit,z (I) fwnld t,hnt. t,hc opt,imal wnwtltratiol~ of MgSC)4 IV:W 0.01-0.02 ~11, varying ill the samt direction :IS the c’ollwntr;Ltion of I )X;l; I,a;ikowki :uld Scidtl (7, 8) found maximum rates with (YIIIcurt r:ttiolls of -\IgS04 of O.OlbO.02 Al. Se\w:~l ~vorkrrs (1, 10) were l)rompt,tvl t.o point, out, that’ the xti~xting capwity of the magllcsiunl xppeared to Ix: :L funct~ioll of the suhstrat~e rather t.h:tn of’ t.he enxymc c,ollc,clrtr:ltioll, illdicat~ing, according to Weissman nnd Fishw (lo), that “the function of Jig . . . is to alter the suhstrat,e so that the enzyme

337

system may function.” It was evident that by making certain allowances for the different ionic strengths pertaining in the afore-mentioned experimen&, these “optimal concentrations” of Mg++ corresponded very closely with the Mg++ concentrations shown in a previous study (11) to he just saturating the phosphodiester sites of DNA. To verify this apparent correlation, a study was made of the metal activation and t,he effect of certain inhibitors of DNase I, under conditions of pH, ionic strength, and temperature closely approximating those in the DN& mctnl-binding studies (11). The results indicate a close correlation bctwccn t,hc Mg-binding of l>KA and it,s rate of hydrolysis by DNase I. &In++, also shown tarlicr (9) to be a potent activator of t’his enzyme, also exhibit,s such a correlation ; however, Mn++ gives a minimum of 335 times the rate given by any equivalent concentration of Mg++. Ca++ is shown to activat,c weakly, but this act,ivution fails to shorn the correlation with DN:1 binding that is seen with Mg++ and Mn++. However Ca++ was found to be a pot’ent synergist in the Mg-activated react&, increasing t)he maximum rate seen with Mg++ more than threefold, to give rat’cs comparable tlo those seen with Mn++ alont. ~thylenedianlinet,ctr:lncrt,ic arid (EDTA) was demonstrated t)o bc a complet,ely effective inhihit,or of DKL4se I, while citrate \vas show1 to bc :a poor one; as part of this latter study, the dissociation csonst’allt of Olin citrate \vas detcrmincd. bhTERI.?LS

1)Nasn I was t,l~c “I X crystallized” prcparat,ion from Worthingt 011 I~iorlrclriian:~lyxetl” JwtgrJlt. I’:iLStm:J.II gr:tdo cal (‘orp. Sodium cit,r:tte was the “Baker p-nitrophcnol \vas orlce-rccr).stnllizcd from water. All othrr ma(rrinls, irlclutliuy 1)x:\ (~‘C~JI. c?), have hccJ1 th!ScJ’i~Jcd pJ’CViOUdy (I I). Melxous

AI cmwcmerrt

of DNase

I ActiGty

‘~}1(, Illcthd of (‘avalieri and Hatch (12) was used, whicll iuvolvw mo:wuriug the lil,er:ll,iou of acid as the erJzyme hydrolyzes phosphodiester bonds it1 IINIZ. The acid decreases the optical density at 440 mp of a p-nitrophenol buffer; this decrease is measured in l-cm. Corex cells in a Beckman DU quartz spectrophotomcter. The temperature was maintained at 27.0 f O.l”(:. by circulating wat,cr from a constant-temperature bath through a Beckman cell-housing attachments. The ionic strength w-as preadjusted to 0.15 with appropriat,c amounts of Na(‘1. The use of gelat.in (9) to protect DNase 1 from denaturatiou was :hHJdOJlC’t~ w ZLJI unnecessary variable. That no significant drnat,ur:rtion occurred was sho1~11, first, l,y t,he fact that control rates determined at the start of a given series of

~LIIIS wcrc not significantly different from those determined at t,hc end (up to S hr. later), indicating that the stock solution of enzyme had not attenuated. Sccondly, the linearity of the rates during a single run (5-15 min.) can be taken as strong evidence that no denaturation was occurring in the reaction wssel. DlYasc was dissolved in distilled water at room temperature, and stock NILItions of from 0.05 t,o 0.5 mg./ml. were stored at 2°C. until used-always within 2 days. Frequently a minute amount of a filmy precipitate was visible in thcsc solutions, and it was thercforc necessary to centrifuge them and pipet off thr supernatant to obtain rcproducihlc rates. The I)?jA was dissolved in (‘O-frctr distilled water by placing a stoppered flask on a vigorous shaker for at Icast 5 hr. at room t~cmpcrat~urc, then kept at 2°C. unl,il used-always 1vithiri 3 days. Five milliliters of the appropriate salt, solution was placed in each of two 15-ml. cew trifuge tubes, and 1 ml. of 0.0020 XT p-nitrophcnol (adjusted with KaOH t,o give a final pII of 7.4--7.5) was added. Four milliliters of 0.064’% DiV‘1 (I .D X 10m3dl in I’) was then added to each tube. These Tvcre covered with l’:~rafilm, misctl thoroughly, and suspended in the constant-temperat,urc bat,11 for at least, 45 min. Iwforc starting a run. h volume of 0.2 ml. or less of the stock cnz)-me solut,ion (kcl)t rcatlily availnhlc in an ice bath) was added from a calibrated delivny t,utx :Lt zero time. Mixing was effected by gently inverting the tuhc ten times. ‘l‘hc tmounts of ad&xl rriz,vme varied, of necessity, because of the large range of rak studied; however, in 0.05 .?I LIg ++ the rate was drmorrst,rnt,ed to ho propori.ionnl 1o the amourrt~ of enzyme added. The first readings were obtained in 1’ d--2 tnin ,: and the rrartion was followed for IO-15 min. t,aking readings every 30 or 60 SW. ‘I’hc rates wcrc linear for at lcast 5 min., and in the slower rcxctions rcwnnirrrtl lirlc,ar for f he entire I5 min. Over the period for lvhich linrarit,y obtaittc(l, tto IIJO~C 1h:cn 5y0 of t,he hydrol!-zat)lc Iwr~tls hat1 lwcn I~rol~cw [wsutning 25~0 of a11 I,hospliotlicstcr I)onds arc susceptible (1, O)], and t.his atnouttt of lil)ctxtctl :xi(l v~:ts cquivnlcnt to a drop in optical drttsit.y of the buffer of at~~~roxitn:ti.cly 0.1 unit. Since the cottccnl raliott of I)ull’cr was low, (‘0, :tl)soq)tiott Itad to lw tnittimizr(I Iry tnakitig 111)all solutions in (‘02-fret xalcr, IiWI)irlg a11 iulws sfop~wrc~l, (>1(*. I~‘:tirl~. wl)roducil)lc inilinl ol)l,ic:d tl(snsit,ics (:tt~tl t,hus pH’s) WVL’V t l~(~r(~l)>- 01) I ,Lincd. :I11 rates reporkd hcrc xclre oI)t.aincd wil~hirt f he 1)H rattgc 7.5--7.2. ‘1‘1~: s!:mtlartl compatxt~or cuvett.e ins fillrtl \I-ith pnitrol)hcrrol in 0.01 .I/ K:t2(‘O: , tlilul,cd lo give :L~Lopt,ical dcneity of almuf, 0.50. The K:L~(Y):~ prrwrlfs :I s~n:~lI, gradu:d fall in ol)t ical tlettsit,y xvhiah ol,hcrwisc owurrcd over the G-8 hr. rcqttiwtl for :I given series, al)pnt.ettt,l>~ from (!Oz alworp~~iott. This fall was not sigttific:tt1( , h:3u-cwt., during 1,he lO- 1.5mitl. wquiret 1 for :i giwn ralr ni(~:Lsurcmcn~. Wit Ii t I](% comparxlor cuvrt,i,c set at, 0.000, the react iotas w\cre followed frotn ahout, O.:( t 0 0.2 ol)iical dcni;it,y. Control titrations with H(‘1 of various MgC’ly , 1\ln(‘l? , ant1 Sac’1 solutions showed that these salts did not significantly ittt(lrfcrc with ([t(s change in optical density of the p-ttitrol)hrttolnte per unit of acid :tcl&d. With rach new series, standard rat,cs in 0.05 .II Mg(‘12 vvw tletertninrtl, antI t IlC other rntc‘s arc expressed rrlative to thrsr. This wnccttt ration of ^\Ig(‘lu givps a nxtsinxrl activity (for ;\lg’~+; we Fig. I), attd 7vith a final cottrcttt,r:tl iott of cttZ,! tnr cclunl t,0 0.5 ,ug./nll., g:tvc :t tlccremrnt of :tl)[)roxim:1tply (J.007 Ol)f.ic:~l ,J(:ll. rily unils:‘min. lfkrch point plot trtl is an :tvcrage of duljlic:tt(: (l[.((,t,tttilt:tt,iotti;.

340

WIBEIIG

In Figs. l-3, the concentration small amount bound to DNA.

Standardization

of metal

ion has been corrected

[Kq. (S)] for the

of Xolutions of Biualent Metals

Uccause of t,he difficulty involved in accurat,cly weighing deliquescent salts of bivalent metals, the danger of dccomposit,ion upon drying their hydrates at, high temperatures, and the undesirability of a substitute anion (such as the sulfate, or the carbonate -which must be dissolved in acid), a simple standardization for the MnC’l:: solution was sought. Uiedermann and Schwarzenbach (13) described a modification of their EDTA titration method for lMg++ and Ca++, by which lead, manganese, and mercury could be determined. They prevented precipit,ation of the ammonia buffer simply by adding an excess of l:D’l’A before adding the buffer. Then they back-tit,rat,ed the excess EDTA with standard magnesium or zinc solut,ion, getting a reversed end point, i.e., blue to red. This reversed end point, dew not appear to I)e complctcly accur:ttje; expcricnce has shown that a slight but, significant amount, of EDTA is consumed in the normal t,itration from red to blue. Thus one overtitrates when using t,hc red end point. To keep the same end point, for the MnCL titration as for (‘a++ and Rig++ (II), a further modification was introduced. EDTA \v:ts added t,o the XInC12 in slight exccs~, as above; t,hen the cthanolaminr t)uffer--indicat,or solution was added. Nest,, however, a howl aliquot of a st andartl R~lgC12 solut,ion was introduced, and t,hc titrnt.ion of t,hc cxccss Mg(Zl~ xvas completed with EDTh to the usual cntl point,. Thus t,he tit,rat.ion value for the Mn(S is the difference between this total value and t,hc value for t,hc aliquot of MgClr It is import,ant that hlg+’ ('a* b bc t,hc ion added to complete the tit ration rather than (‘a++, because ~.hen weak, greenirh yellow color (not reversible 1)) was tried, the charactrristic, ranISDTA) of t,hc RIndye complex rcplacrd the strong red color of t,hc (‘adye pies. Since t,he (!:I l7’A gave posit ion begins at, TOO”), wcighcd, agrcemcnt, with this gravin&ric st,:lntl:LrtlizatiorI \v-it.hin 0.50/,. FeHO4 and (‘o(:12 jvcrc also found not to interact, wit,h tither I hc ct,l~:lllolnmille or the indicator when st:ttrtl:udizett by this method. It i H suggested, then, that salts of I)iv:tlcnt met,als having format,ion constants xvith J~~IITA llighcr t harr t.h:tt, of (‘a’ ‘ (14, 15) standardized by this simple procedure. Such metals can probubl~- bc directly include ;LIn’~+, FP+~, (‘o++, %n++, Cd’+, I%++, Ni++, (‘u++, ant1 llg++.

nh++

Determination of Free Al&d-Ion Concentration in the Presence of a Ligand In the presence of a ligand, varying amounts of free metal ions remain in solntion, depending uporI t.he t.otal amount of metal, total amount of ligand, and the dissociation constant of the metal-ligand complex. In the present studies it was necessary to km)\\- the jree metal-ion concentrations in t.he various enzyme solu tions, t,o compare lcgit,imately the enzyme data with the I)?IA-binding data (11) ; equally necessary to understanding the inhibition mechanism was knowledge of

.IC?IWATION

OF J)SASIS

311

I

the free metal-ion concentrations in the presence of the enzyme inllibitors, citrate and EL)TA. Rabin and Crook (16) have recent,ly emphasized the necessity for using jrce rnt,her than t&Z metal concentrations in considerations of the kinetics of metal-activated enzymes. Free metal-ion coucentrstions were estimated I)? these workers by a process of successive approximations; nut, \vhcn a large number of ditfcrent metal concent,ratjions is stIrdied, as here, this l~ccomcs a tediousprocess and lacks precision as well. Consequentl,v, an exact and relatively simple equation was derived. The dissociation equilibrium is rcpresentcd by:

wllrre I, - is t,hc ligand, AI’-+ is the metal , anti JAl the ~ornplr.~ fo~mcd. Whcrl sigrriticarrt amounts of other Imrrntl species arc formed, such as J&J- - or IAl2 #, this simple derivation cannot, be applied, of COIIT’SC.IIo\vever, all of the present of \vhich the onl? ligands appear to form (:a++, big-‘+, and .1tn++ complexes significant form is JAJ4 undrr t,he condit,ions of this stud)-. If the total ligan(l concentration is [LIT , the free lignnd concentration is [I,- -1, the total metal is [~I++], and t,he apparent concentrat~ion is [hljr , the free metal concentration equations hold, ate the ionic tlissocntion constant is lir, )r ,5 then the following strength for which KL %rwas tleterminetl:

[At++]= [M]y - [LA31

iI) (2)

(I,- -1 = [L],r -

(3)

Kr.1, = [I,- -][nr~+]/[LnrJ,

.%~l~l,rnc~ting J
01‘ [nr++] = z\r,y

[r,ar]

[rAr]/[rJ-

-I

Wt. (2) gives : [T,- -~I = ply+]

+ [I,]T -

[Al],,

13 )

~S~~l~siituling T5q. (3) into Ij:q. (1) gives:

Sn)wtitlrting

l’:(t. (4) into IQ.

(5) gives:

‘See the rcfrrcnccs rit,rd with the several dissociation constants quoted in this paper. Schubert (17) has shown that, a linear plot of [I,- J vs. I/f<,, is excellent. evidence for the formation of l,he IA1 complex; the data of ‘I‘al)lc I form such a linear plot, for RJn cit,ratc, :tntl an earlier study (II) sho~rd t.hc ,*:nnc for &Jr1 DN.1. 6 The applical~ility of F:q. (S) t,o DiYA binding depends, of course, on the v:didity of the respective met&DKA dissociation constants; these constants \Vere tletrrminctl and adcquatcly discurscd previously (1 I ).

IMomin~atio?~

of >I1II Citrate

Ihwciation

Constant

‘I‘hc s:lmr procedures WCI’C:uwtl here that were used in the I)revious study (I 1) for (‘a DX.4 and 1111 1)X.4, i.r., the ion~f~schangc rudioisotopc method of Schul)ert (17). The equilil,rntion ~wriotl \v:t’ 5 hr. ; rxh flask rant aincatl 30 mg. of resin. hSULTS

Jig-l-+ .Actimtion (4 DiVase I In Fig. 1 is show11 t~he vari:ltion in DS:IW 1 activity with Mg++ condata (1 lj. The cu~trntion. Also included :m: tho earlier Ng-binding cttuyme activity pxdlels fairly closely t,he snturat ion of DNA nith Mg++.’ On the :wtivntiou carve is Iwted tht: c,ollc,nllt,ratiol1~ti~l~ at, \vhic:h 1;The npplicahility of 1Cq. (11) t,o DNA assumes that the same binding sit,cs :~re involved for the three bivdent metals studied, IQ++, hln++, :md (!a++; this :~,~pears to be the case (11)) though conclusive proof has not been shown. It) must &o 1~ tssumcd that, the apparent dissociation consbnt, for one metal ia not altered by the presence of another metal, by virtue of some change, reversil)lc or not,, in t,he mncrostructure of DXA (we Disc~ssiora). 7 ‘J‘h:tt the two curves do not exactly coincide is not surprising; perhaps those sites first romplexetl by metId are the most, suscel)t,iblc t,o enzyme :rtt,ack. Or, if

P r^ n,~~---~~~------““-0 .= omymo eetivotion 6 so o= DNA binding ‘D 80 t

. /F-

1.0 2 -I0.9 g 40.8

$

0.7

j.

0.6

i$

0.5

-

0.4

H+

0.3

c

0.2

8 a

0.1

-O

360

300

10-6

10-S

IO‘4 [Metal

to-3 ++I

10-Z

IO”

f,ee

FIG. 2. DBase I activity as n function of Mrl++ concentrat,ion. For compnrison, the Mg++ activation from Fig. I is included. Ionic strength 0.15, pH 7.5-7.2. Shaded area denotes concentr:~f,ions of hin(‘lz which r:tuw DSA i,o gel.

opalescent, but no precipitate appeared wren n&r 4 days. On centrifugstion at 18,000 X g, a transparent gelatinous material (probably a manganese salt’ of DKA) came down. hiore appeared at higher &In++ concentrat,ions. Precipitation of D?;X by 0.15 31 NaCl below 5°C. (I 8) and gelation of DNA in concentrated MgSO, (19) have been reported. Ccc++Activation oj Ilh’ase I XXJwt~y (9), using a viscometric assay for DBase I, claimed that “calcium and iron have no activating effect.” In contrast, Greenstein and coworkers (z&22), measuring dialyznbility and acid-precipitability, claimed that Ca++ and many other salt,s did activate DKnse I. Sinw the all of these methods mny give nmbiguous rem& (see IXscussion), effect of Ca++ was studied with the direct, acid-liberation method. In Fig. 3 is shown the effect of Ca++ on DNase I activity. The maximal activity with Ca++ is approximately 6% of that with Mg++, at t,he pI1 and ionic strength used. Maximal ac:tivit,y was attained at a concentra-

;3f .E 2 :: = IB 340 10-5

, I o-4

I 10‘3 [co++]

I’Ic:. 3. 1)Sax

I

10-z

free

I activity 3s :t function of (‘a- ’ strength 0.15, $1 7.5-7.2.

coIlccrlt1.3t~ioIl.

IOlliC

O=Mg+++Ca++,observed X = Mg++ olone

l

=Mg+++Co++,expected

.Ol

0.1

IO

1.0

100

[Mg++@++]

Frc:. 4. l’:ffect of varying ratios of Mg++ to (:a++ on DNasc I activit.y. [(‘:~(!I?] + [hIgClr] = 0.050 M. Included for comparison is the effect of Mg++ alone (Fig. 1) at the same Mg++ concentrat,ions that exist in the Mg++ and Ca++ combinations. Effect of Ca++ alone is almost insignificant, on this rate scale. 0’s represent t)he sum of the activit,ics expected if the activity due to each metal were the product of its maximum activity (alone) times t)he fraction of the fot,al DXA which it saturates [Eq. (II)].

a ?

5 *

X = Mn++ alone x,3,-.

0s Mn+++ Ca++,expected

1 O.01

I .I

P

x 0

5

I

I

,

1.0

IO

100

[Mn++flCo++] FIG. 5. I%“ect of varying ratios of Mn++ to Ca++ on DNase I activity. [MnC’l?] + [CaCls] = 0.020 .W. Included for comparison is the effect of Mn++ alone (Fig. 2) at the same Mn++ concentrations that exist in the Mn++ and Cab+ combinations. Effect of Ca++ alone is insignificant on this rate scale. 0’s represent the sum of the activities expected if the activity due to each metal were the product of its maximum activity (alone) times the fraction of the total DNA which it saturates [RI. (ll)].

346

0 a Mn++

+ Mg++,obrerved

x = hall++

alone

+ = WI++

done

l

+Mg++.

* Mn++

+ htg++

done

expectQd

/‘

6 b”

I

0

I

I

IO

.I

.Ol

I 100

[M"++;Mg++]

Frc:. 6. I~Xrct of varying ratios of iLln’+ t,o bIgi+ on L)K:asc I activity. [R1n(‘12] + [JIg(‘lz] = 0.020 Al. Included is the sum of t,hc activities of Mn+-+ alone (Fig. 2) and RIg++ alone (Fig. 1) at tile same concentrations of each that exist in the I%++ and Mg++ combinations; +‘s reI)resent t,he Mn’+ contribution to this sum. 0’s wprcsent, the sum of the activities rxpected if the :tctivit.y due t,o each nwtal ucrc t hc I,roduct, of its maximum activity (alone) times the fraction of the total 1)NA \vhicxh iI sat,urntes [IQ. (ll)]. lnc~t:d wcrc t,he product of its masimal wtivity (\vhcll dolrc~) mult~ipliccl by t,hc fr:lAon of the total DNA which it’ saturates, at cwh ratio studied [Eq. (1 l)]. l!yd

f/f (‘n++ plus 1’1111++,Wld of iug++ p171.3.u?L++

‘I’his tlisco~cry of ;L synergistic* Ca++ cffcct 011 Mg++ :kctiwtioil glut.ur;~lly r:ktl t,hc question as to whether C:i++ and AIll++, and Al@+;\l~tl Aln+-I- ~vould act syrlcrgisticully ill combinntion. In t,hese stud& t IIC total cwllcentration of bivalent metal W:LS 0.02 31; t.hc iolk strclrgth \V:IS brought to 0.15 wit’h N&Y. Figure 5 sho\vs t,hat CA++ has wither :I st inlulutory nor inhibitory cffwt on Mn++ activ:Ltioii. Figure (i shop\-s that, Mu++ and Mg ++ summate in their activation when the ikIll to hlg ratio is higher than 1; below this ratio a partial summation is seen, which cal also be viewed as a partial mutual inhibition. AIn++ Einding 111 l!M hl&uty gaiirsc wn replace

(NJ Citrate

(9) obser\-cd that, in the T>S:wr I rc:wtioll “ma11magnesium and is equally cffect’ive at an equiv:tlcllt,

348

WIBERG

TABLE Determination

of Mn

Citrate

I 1Iissociation

Constant

Ionic strength of 0.15, pH 7.2, 5 hr. cquilihration [Saa citrate]

X 101

l/K11

x 103

0

1.16 I.-u

1.“5 I 2.50

2.Bi 2.45 3 -11

3.75

4.52

K!d,,

3 .!I5

1.96 1.80 1 .83 1.00 1.76 2.15

5.20

1.02

3 .:33

5.00 Av.

= l.Wl f

LiLrste

at 25°C. x

10”

c = 0.13 (u = ~t:~lid:trd tlevi:~i~ion).

molar concentration.” WC also found t,hat “citrate serws as a potelit inhihitor of the magnesium-ncti~at,ed enzyme” hut that “citrate has no such inhibitory effect when manganese is used ~1sthe metallic activator.” l’erhaps citrate hinds &In++ very poorly; however, :I search of t,he literature revealed no information on Mn++ binding by citrate. ConsequentSly the disso<’ion caonst):mt,for R4n &rate was determined under conditions approximating those used iu the enzyme-inhibition studies (helow). T:lble I gives the results of this esperimcnt. The disswiat~iol~ CWIstmt, Ir’ Mn citr:\f,,, was c&ulated to l)e I .90 X 10e4, with a standard deviation of 0.1:3 X lo-“. Values for KcZt citr;Ltr:rt, ionic skengths of 0.15 0.16 and pH 7.2-7.5 arc rcport,ed to he 7.1 X lo-‘% (37) and 6.03 & ‘1 v:~luc of 5.6 f 2.1 X IO-” is r(‘0.35 X IO-” (X3), \vliilc for K,,, citrL,t,,C ported (38). Thus vitr:Ltv appears to hind brn ++ three to four times more of citrate t,ight,ly t,h:tn it dots Cl++ or Jig++. ‘I’hcrefore, an ill:&ility to umples ,1111++ is not the cxpl:ulution of r\IcCarty’s findings. I~JuIbilior~

0s DNase

I by C’itrfrfc

Siiicae c:itr:rt,e complexes ml++ evc’n better than it does Rig++, :L winITestigation of McCarty’s apparent.ly anomalous findings nppearcd warranted. One discrepancy already was apparent, between his data and those report,ed here, viz., McCarty found Mn++ and Mg++ equally &i(:ient, a&\rat,ors, \vhere:w here (Icig. 2) M~I++ \V:IS found to he far t>ett,er. This discrepawy may, however, he :ltt,rihut:lblc to :I number of factors : McCart,y used a noncrystalline enzyme, :L differcllt prep:wat,ion of DNA, gelut,in as an enzyme st,abilizer, and a viscometric assay-

Calculated

f a ;‘ s -=_ 9 2 (h) 0.001 dl hlnCl, + 0.0012 111N:t:$ citrate (rii r:~tc added to Mn++ I)efore I)NA) (c) 0.001 111MnClz + 0.0012 M Naa citrate (citrate added to &In++ after DNS) (,I) 0.0022 M hZnC12 + 0.0012 M ?rTaa citrate

0.001 M MgCL + 0.0012 111Nns cit,r:lie (citrate z~dtletl to Rig++ before I1N.4) i!/) 0.001 M MgClz + 0.0012 M X:1?. citrate (citrate added to Mg++ after DSA) (16) 0.0022 ?V MgClr + 0.0012 31 Naa cit,rate

1

700 2x i

100 1 5,l * 5

28 It

1 54 z!z 5

53 zt 1 I 10 *

IO

(I)

‘I

which assay mny not reflect the differences in rate given by the two metals, since t,he viscosity c*hangcs are essentially complete txforc t,he enzyme has released much acid (1). In the experiment outlined in Table 11, relat,iwly low metal c’ollcentwtions had t,o lx used because of cikate’s Ml’ering capwity. The data are correct,ed for this additional huffering.g Table II shows that 9 The tlelnsity

correction of

t,hr

factor

p-nitrophcnol

was determined by following the change in optical on :ttltlilioll of IIC’l in t,lw prw:pnw of ((I) only hIg+’

Efect

of EDTA

on the Mu++-Activated

TABLE III DNase I Reaction

at p = 0.15 and pH 7.5-7.2 Rate,

(a) 0.0002 M MnCl~ (6) 0.0002 M MnC12 + 0.00022 111l!DTA (c) 0.0002 M MnCl2 + 0.00022 iVf El)TA (using 10X the concn. of enzyme) (d) 0.00032 M MnClg + 0.00022 111 I
as % of (a)

100 O&5 Of5 100 *

20

the observed rates with Mn++ in t,he presrncae of caitrat,e arc higher than those which would be expected purely 011 t,hc basis of t,he (~alculatt~d concentrations of free Mn ++; in fact, no inhibition at all is seen, witJhin the precision of the da& In this respect the results of McCarty are confirmed, inferring that the Mn citrate complex can still wtivate DNase I. Mg++, on the other hand, behaves as expected in the presence of citrate (Table II), in that t,he observed rates compare closely with those expected from the c*alculated free Mg++ concentrations. Perhaps the most important fact, emerging from these st)udies is that citrat,e is :L rather poor inhibitor of even the Mg ++-activated DSasc I rewt ioll, contrary to the caommonly held belief.“’ Znhibition

of DNnse Z by EDT/l

In the MgPcitrnte system above, the enzyme wtivit,y n-as &rict,ly :L function of the free Mg++ concentration. Consequently a stronger Mg++complexing agent, e.g., EDTA, would be expecked to provide better inhibition. The dissociation constant of t)he EDTA complex with Mg++ is 1.2 X 10Pg; with Mn++ it is 1.6 X IO-l4 (14). Since citrate failed to inhibit the Mn++-activated enzyme, EDTA was tested against Mn++. The data, in Table III, are corrected for the EDTA buffering, as NXS done with citrate. The final enzyme ooncelltration \vas 0.53 Fg./ml. in all but (c), where ten times this amount was used. It is apparent that, EDTA completely inhibits the Mn ++-activated reaction; Expt. (d) indicates

that

EDTA

is not

inhibiting

in any

\vay

other

t,han

through

its

or >\Zn++, (6) only citrate, and (c) citrate plus an equivalent amount. of &lg++ 01 bin++. In (c) very little buffering was seen, presumably t)ec:tuse of a sharp drop in the pk’, of citrate due to chelation. 10 In McCarty’s original inhibition studies (9), 0.003 112magnesium in 0.01 111 32~~ citrate gave no observable enzyme rate. h‘onetheless, assuming nn ionic strength of 0.15 (it was at least 0.07), calculations [Sq. is)] indicxte that nrnrly 10% of the added Mg++ still remained as jree M&+.

IY~lnoLd

of tht

, since the rat,c here is within of fret Mn++ given by 0.0001 111Mn++ (Fig. 2).

esperimcnlal

crro~

I~IsclxsIo~ LLnl ctnlloszrbstrates” full l>Nasc I xt.ivity has now b.xn shwx to rcquirc sat~uration l)TA-binding sites when either l\ilg++ or &In++ is the :rctiv:lt,iIrg metal, this rnzymc c’an he added to a rapidly growing list, of cnzymrs which fit into Najjar’s theory of met~allosubstrates (X9), in which thv ~,rue substrate is t,he mct’al complex. Lardy (40) pointed out “the app:trcut,ly universal requirement for bivalent vations” in cnzpmic phosphoryl:tt~ious involving adeuoainc triphosphatc (L21’1’), and more rewnt c~\~idcnw \vit,h many swh enzymes indicates t,hat .YlT’ participates ;IS (‘on:L metal complex (33, 41-M). Similarly, a mcta-pyrophosphate plcs appwrs to be the true substrate of wrt~ain pyrophosphatuscs (,::I ) 45, 46). Sillw

Ot’

ilctiunlion

by l’airs

of

Xclals

‘l’hc xtivat ion behavior of the pairs of metals indicates that n simple “met ~rllosuhst,late” theory af’fords only a partial description of the nwvhultism of metal act~ivatiou. Several explanations can he offered foI the data of Figs. Hi, all of ivhich are equally plansihle, eclually un~JI’OVCI~, and appear equally amcnahlc to esperimtwtnl test : (0,) l’crhaps the presenw of one metal (e.g., Cn++) alters the binding or spatial arrangement of the other metal (e.g., Mg++) in a cmxgy” Irc~ighl)orillg DNA site so as t,o ilwreasc the suswptibility of the neighI)orilrg site to cwzyme :ltt:wk. (b) 1t is pol~uhlr that there :II’CLsever:~l molccwlar spwiw, or frwtiwls ( 18, -!7) ii1 the 1)X:1 prcp:wtioii st,udicd here; tlicsc Inay dift’er ill tlicir l,c,clliirr:nlciits for mct:d a&\-:rt,ors. T-Towc\w, this \vould vsplaill 110 II~OIY~ th:u~ a simple summat~ion of rates with metal wrnhinations, unless t 11~ follo\viiig wxc also t,ruc: (c) 1111undcgrnded fraction of I>?uTAnot susceptible to Jig-activated hreakdowll (hut st,ill susceptible to slow Ca-activated hreakdon-n) m:IJ rapidly hecomc Mg-susceptible upon hydrolysis of a very few bonds under C,~-z~tirat,ion.” ‘ ‘ (d) It is vonceivnhle that t1v-o enzyme sites part,icipute in the metal I’ .\ ht utly of t hr simult :mwws hintling IL .Z 111rl:1l~:t(:tiV:ttioll stud\, \vith c:rctI

of t \vo tnct:tls fraction n odd

by DS.4 is nertl(~(l tw c~nlightrrliug.

activation. Then t)he wtivation dat,a could be satisfactorily explained by assigning to eu& metal a rank for each sit,e, according to its activating efficiency. At site I this rank \vould be &In++ = Ca++ > Rig++ (t,he ionic radii lit in t,he same order: l\ln++ > Ca++ > Rig++). At site II the rank would be Mu++ > Rig++ >> Ca++, the same rank as theil tendency to form coordination complexes (15, 18) .l:j The data raise several more quest,ions n-hich must be anwertd I)cfore WChave a clear picture of the metal activat,ion: (a) Does each a&\-ating metal illflucwe t,hr ellxyme t,o hydrolyze the same, or different bonds? (b) Is t,he same tot,al number of bonds hydrolyzed, from metal to metal? Only iUg++ has been studied in this rcspecst (1, 6). Kcwrsibility

0s L~NAlll&xl

Binding

I1 is conceivnblc that the binding of metals, particularly at saturating c~onccnt~rtltions, may result in some irreversible change in the DNA macrost,ruc,t,ure. Such cJj,T&s wo~dd then have to hc incoked in cxplaininy the metal-activation data. However, the only swh irreversible cahangts (ot,her than hydrolytic) reported t
ACTIVATION

OF DNASE

I

353

furnished a high enough ionic strength to maintain the decreased optical density in spite of removing free Mg++ ions. (b) The inhibition studies in this paper (q.v.) point out that citrate does not completely complex Mg++ and thus did not necessarily reverse the DNA-Mg complex presumably present in Cavalieri’s study. (c) A previous paper from this laboratory (11) reported the observation of complexes of DNA with Mg ++, Ca++, and Mn++, and gave the respective dissociation constants. Thus it is apparent that the reversibility or irreversibility of the effects of bivalent metals on DNA is still an open question. Inhibition

of DNase I

It is strongly suspected that there has been a significant number of anomalous results reported in the literature, caused by a misplaced trust in citrate as a DNase I inhibitor. Such studies surely should be re-evaluated, substituting EDTA, particularly in those methods of preparation of DNA now utilizing citrate to inhibit DNase I in the tissues (57-62). Even though there is ample evidence that another DNA-degrading enzyme, DNase II, is quite widely distributed in the mammalian organism (3, 63-66) and requires no metal activator, it is only reasonable to inhibit maximally the DNase I that may be present. McCarty’s conclusion that Mg++ is probably the natural activator of DNase I (9) now appears unwarranted. His reasoning follows: (a) Citrate inhibited the DNase I of fresh tissue; (b) citrate also inhibited the Mg-activated reaction but not the Mn-activated reaction; (c) therefore Mg++ must be the natural activator. Perhaps the natural activator is a combination of Ca++ and Mg++. This would offer the cells a balanced regulator of DNase I activity, in that considerable changes in Ca++ to Mg++ ratios might occur without any change in the total metal concentration. On the Measurement of DNase I Activity In 1946, Carter and Greenstein (20) and in 1947, Greenstein, Carter, and Chalkley (21) reported that salts of a large number of univalent and bivalent cations “activated” purified preparations of DNase. Their main assay method involved dialyzing a solution of DNA and enzyme against the various salt solutions and measuring the rate of appearance in the dialyzate of material absorbing in the ultraviolet. Thus enzyme activity and dialyzability were measured simultaneously; that the great-

354

WIBERG

est effect of the salts was on dialyzability and not on the enzyme is indicated by the following evidence: These same workers (20) found that “the appearance of dialyaable components from (ribonuclease) digests of ribosenucleic acid is not per se a measurement of enzymic activity, for the same effect is accomplished nonenzymically by electrolytes,” and “no acid groups are liberated in the process, suggesting that the process is one of desaggregation. . .” Because salts did not render DNA dialyzable,14 these workers apparently concluded that DNA dialyzability was a function of enzymic activity alone. However, in 1952, Tamm, Shapiro, and Chargaff (67) observed that high concentrations of Mg++ rendered dialyzable a number of partially depolymerized or degraded products of DNA, but not the original DNA. The indications were, however, that few or none of the phosphate bonds were broken in the process. They concluded that Mg++ “brought about a nonenzymic disintegration of the degradation product’s of DNA.” The similarity between the DNA degradation products and RNA is obvious, the point being that very little enzymic activity need occur before DNA is rendered dialyzable by salts alone. Similar arguments can be applied to render questionable the conclusions in 1951 of Miyaji and Greenstein (22). They reported that Mg++, Mn++, and Co++ activated DNase I to the same extent, while Ca++, Fe++, Ba++, Sr++, Ni++, Cd++, Zn++, and an acetate buffer activated to a lesser but definite extent. The enzyme assay was based on the assumption that the extent of enzymic degradation was proportional to the amount of ultraviolet-absorbing material remaining in the incubation mixture after the addition of acid and centrifugation of any precipitate. That this assumption may be frequently in error is indicated by Kunitz’ finding (1) that “gelatin (and also neopeptone), even in very low concentrations, was found to interfere greatly with the precipitation of NaTNA (DNA) by strong acids.” Even though Miyaji and Greenstein do not mention using gelatin, it is quite probable that with the variety of salts, and with the wide range of salt concentrations and pH which they studied, much interference with acid precipitability occurred, at least for partially degraded DNA if not for the original DNA-as is the case with dialyzability (above). Such interference would give false positive reactions. These workers gave no indication that control “reactions” were run in which no enzyme was added, to test the effect of salts alone on the “rates” so assayed. False negative reactions could be I4 For a recent

study

of DNA

and RNA

dialyeability

Bee Ref.

(11).

ol~~~vetl if an effect owurrcd similar to that reported in the present st,udy, 11:m~rly that MnC12 :~t)ovc 0.03 ill mists grlation of L)KiZ and a scttliilg out> of :L fiiw prwipitatc :ift,er the cwzyme is added. IIob~~vcr, 110suc*h cffwt \V:LSrrportcd l)y these xorkers, ill spite of their having used even higher concentrations of MnCl2 and other salt)s. ‘I’hrse complic:~ting factors point up the desirability of using a method which I’ollo\~s the libcrntion of acid by DBase, as t,his is the most fundamcllt:~l wflwtiolr of its :wti\:ity in hydrolyzing phosphodiester bonds. ‘I’hc particwlar acid-production method (12) used in the present stud) is ilot entirc~ly imnirukc t,o c7iticisni; it. seems advis:~t~le iii future studies to 11X' :I “pi-I-stat” apparxtus such as t,h:lt described 1)~ ?ii&nds :wd C1:~1rlw~l(68) and already used ill studyilig DN:Lw I by Thomas (C;!,) ulcl 1)~ S~liumakrr rt al. (70). 13~ :iutuniatiwlly :tddiug titrailt, to maiilt;liii :I cak3taant81111,such :tii :tppar:Ltus c~lirnin:ltcs lxkh thr: small pI I ixngc (0.3 unit,) and the last noil-etisrnti:il ingrrdielltj (p-llitropheilol t)ufi’c~) required by C:Lv:Llieri’s mrt~hod ( 12). In routine assays for DSuse I activity, e.g., in tissues, it appears ;tdvisable to use a combination of C:l++ and AIg++ in :L ratio of approxim:ltely 1 : 10, respectively, at n total conc~entr:tt,ion of about 0.05 111. ‘L’his comtklation gives over t)hree t,imes the rate seen with the commonly 1lsrt1 Mg++ alo~le. The combinntion is prrfcrablr to Mn++ :done hecause (Fig. 2) ant1 110activity plateau w:w seen \wsus Nil++ caicciitrat,ion t bus the rate would lx somewhnt~ sensitive to sm:~ll changes in the cwlchclitr:rtion of Mn++ \vhich might] occur from many factors in tissue c>xtr:tct,s. Also, t,hc gelation of L>KA seen at high Mn++ concentrations ni:iktrs it advisable to :L\‘oid Rlii++ concentrations c\-en approaching ihcsc. With Mg++ and Ca++ in conihiii:~tion, :L constnnt rate obtains O\YT n mow than fivefold change in the ratio; this would provide an c#wtive *‘l)uffcr” against small ch:indL:, (11sl.i in the c~ollc~c~l~tr:~tio~~ of either ioll cxuwd by introducing tissue: extwcts. 1: ‘~II:L~, such ch:~ugw in lIN:~sc I activity do occur upon introduction of tissue c,st r:wt,s :~ppc:~rs ~~rol~al~lc. A rcccnt report by Feiustcin and Green (71) indieakd the prrscncr of a DNase 1 :tcfiv:Ltor in rat plasma. when 0.0-K--0.09 .I1 A2g++ was pwwut, in the iricuh:Ltion medium. ‘I’h:lt this “activator” may haw been C3++ is sho~vn I)y the following: (a) hcntjing the pl:wmn for 5 min. at) 100°C’. caused no loss of :Lctivitjy; (b) the activity reached a plateau as more and more I)l:Lsm:t w:w :~tltlrtl; (c,) tlw m:rsimum pcrrcritage incrensc in rate given I)y thr plasma was of the same uxrgnitude as that given by (‘a’+ (Fig. 4). Alt,hough the ?vIg’+ to (‘a++ I :tt.io prwcnt, at m:kn:~l :wtivit,v wv:w approximately 250 (assuming a plasma (:a++ cotrccnt,lntion of IO mg.%o), as compxrrd n-ith 25 observed in the present stlltly, thcb tlilf’c~rrncw Iwt \VWII 1hc, mcl hods m:ry :tccourlt~ for t.his diff’erf~nw.

:~C!liSOWLEDGMEKT

The author is indebl,rd to Dr. William valuable crit,icisms during this study.

14‘.IX~~um:ur for his encouragement

and

J~T)I,ENDUM

Aft,er these studies were completed it became npl)srent, from bhe work of Thomas (72) and of Cavalicri et ul. (51) that, the D?;A preparations used in both the previous (11) and the present studies had been “dilution dermturcd,” as evidenced by their relatively high ultraviolet :tbsorlx~nce (11). Because the same preparations were used for both studies, all the results rel)ortcd are mutually consistent and valid J’or these prepatxtiorts. Nonetheless, iowbinding :tnd mc~t:llwtivation studies should he performed on DNA prepred I)y the wmc method (62) t:king prolxr l)rrc:tut iow (51) t 0 avoid (l(~Il:lt ur:Lt,ion, lo wc I\ 11x1 clill’cwnws, if ally, might, :q)p~~:~r. SuhlhlnI~Y

1. The activation of deoxyribonu~le:Isc I (DBase I) by Mg++, lLln++, and Ca++ has been studied at 27”C., at :~II ionic strength of 0.15, and at, a pI3 of 7.5-7.2. Alone, the metals rank as nctivat’ors in the order Mll++

>

3rg++

>>

c:t++.

2. ,Wivity in the presence of Mg++ or Mn++ has been show1 to require the formation of a “met,allosubstrate.” 3. In a study of sctivstjion by pairs of these metals, CL++ was found to be a potent synergist in the Mg-wtivated reaction, increasing the maximum rut,e more than threefold, to give a rate compnrablc to that, given by J/Iii++ alone. Several possible explnnut~ions are suggested. 4. ,4 rapid method is described for standardizing solut’ions of a large number of bivalcnt metal salts. 5. Several pit,falls in DNase I assay methods arc discussed and modifications suggested. 6. The inhibition of DBase I by citr:ke has been studied and is t,o acid is suggested to replacze be deprecated; ethylelledinminetetrancet,i(~ citrate. 7. The dissociation constant of Mn cit,rate was determined to be 1.90 & 0.13 X 10m4,using an ion-exchange rndioisotopc m&hod. REFERENCES

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G~SSS, eds.),

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et Hiophp. ,I& 8, 424 (1052). s. A., NODA, I,., ASI) T,.~RDY, II. A., J. &ol. (,%m. 210, 65 (1954). 4.3. KREBS, 13. Cr., ANI) FIXHER, E. H., Riochim. cl l?ioph.tJs. Ack 20, 150 (1956). 11. ~~ARSH, (:., END RILITZER, W., Arch. Hiochetn. Hiophys. 60, -1% (1956). 45. ROBBINS, E:. A., STI.LHERG, M. l'., .4x1) DOYER, 2'. D., AWN. B~o&wL. nip-

42. I~UBY,

phys. 64, 215 (1955). 46. IIARSII, (:., .4ND ~~ILITZER, w., ,,i,ch. ~ioche,,~. ~~io~h~/s. 60, 439 (1956). 47. USXDICH, A., FREXO, J. It., ROSENKRANZ, H. S., .~NI) BEISER, 8. >I,, .J. .4~. Chem. Sot. 77, 3671 (1955). -18. I~JERRTX, J., “hfetal Amminc Formation in Aqueous Solution.” 1’. H:~asc and Son, Copenhagen, 1941. a!). WATSON, J. l)., AND (JRICK, F. H. C., Colt! Spring /Iarbor S1p71osirr. Qua&. BioZ. 18, 123 (1953). 50. DI~KKER, (‘. A., .4iw S~HACII~X~N, II. Ii., Ptw. N&l. Acrcd. &A. li. S. 40, 894 (1951). 51. (!A~AI,IERI, 1,. I?., Resow, lkl., .~NI) ~~OSESBERC, 8. II., J. ilvr. Chew. SOC. 78, 5239 (1956). 52. GULLAND, J. M., Jowar;, 11. O., .~NI) TAYLOR, 1%. F. W., .I. L'~LWL Sot. 1947, 1131. 53. LEE, W. A., ASI) PEACOCKE, A. It., J. Chem. Sot. 1951, 3361. 54. SHACK, J., AXD THORIPSETT, J. M., 1. Hiol. (Ihem. 197, 17 (11152). 55. SHACK, J., JENKINS, I<. J., .~NI) ‘~OMPSET’L’, J. II.. J. Viol. (“herr~. 203, 373 (1953). %. ('AVALIERI, I>., v/. LEN/. Chwr. 80~. 74, 1242 (1!)52). l,l. l,., AXI) l,Anln, (:. 31.. -7. &o/. (‘hem. 176, 685 (1!)48). 57. PETERMhNS, 58. C:HARGBFF, E., .4NI) ZAMENJIOF, S., J. NiO/. ('hf%Z. 173, 327 (1948,. 50. %MENJIOF, S., AXD (:HARGAFF, l<., J. Viol. Chetn. 187, 1 (1950). 60. ZAMENHOF, S., SHETTLES, 1,. R., AND ('HARGAFF, II;., Satire 165, 7.56 (19,5(I). 61. CHARGAFF, IX., LIPSHITZ, It., GREES, (1., 4x1) Honss, Ll. II:., J. IZiol. Chem. 192, 223 (1951 j 62. KAY, I<. R. M., SIMMONS, ?;. S., ASI) l)OIJn-CE, A. I,., J. .lv/. (‘hem. Sot. 74, li2f (1952). 63. .LII,LFREY, V., ANI) ~IIRSKY, A. I<;., J. Gcn. Phqsiol. 36, 227 (195’L). l,., STARIC, C;., ~(OHS 64. SIEBERT, c;., Tizai(;, K., ~,rX!~us-~,.%Xc:, 8.. HERKERT, XCJAER, G., ANJ) JWKEJ,, H., %. phpiol. Chew. 295, 229 (1953). 65, Iiosz.4~1~4, T. It., SCHREIXR, K., AND Z%I,TX~X, K. l., Riochim. et fziioph!/,s. ilcta 16, 194 (1954). 66. KOWLESSAR, 0. D., ALT&I.QN, K. I., ASU HEMPELLIS, 1,. H., Arch. Biochevb. Biophys. 64, 355 (1955). 67. Ta~nr, C., SHAPIRO, H. S., AND C'HARGAFF, li:., .I. Biol. Cheer. 199, 313 (1952). 68. NIELANDS, J. B.! APED Ca~xos, hr. D., Anal. C’henl. 27, 29 (1955). 69. THOMAS, C. A., JR., J. Anz. Chem. Sot. 78, 1861 (1956). 70. SCHUMAKER, Ii. I9‘., RICH.~RI)S, 15. G., ,4sn &AACIIR~.~N, H. K., .J. dl?r~. Chem. Sot. 78, 4230 (1956). il. FEINSTEIN, R. N., ANU GREEN, 1”. O., ,jlrr.h. Hiochcv!. Uioph!/s. 60, 502 (1956). 72. 1’HOMAS, It., Riochim. et Biophys. ilcta 14, 231 (1954).