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Crystallographic study of beef liver catalase
suturatrcl soditml eit rate at pII 8.2. I,:ugc dark brown crystals like those showrl in Fig. 1 were formed on the rralls of the dialysis ttrbitrg within 12 hr nft,er transfer to the sodiunt citrate. Only this crystal habit was ol~srrved, atrtl afler 8 mo of st nndittg. IIO t ransforniat ion to any cjt her form owtuwd. For S-ray examination, crystals new sealed in quartz capillaries by the conventional means, and dit’fraction photographs recorded on a Supper precession camera using nickel filtcrrd CuKcu radiation generated by an Elliot rotating anode sours. For rsamination with the electron micro scope, large crystals were crushed wit.h a glass rod in :I small volnmc of mother liqttor. The resulting fragnwnt,s Kere negativeI>- stained by placing a drop of the suspension on n carbon covered grid followrd by a drop of a 2’,‘, solnt~ion of uranyl acetate. Micrographs were taken on a JEOI~ 100-B operating at X0 kV and a magnifieat ion of .50,000. I:l!xUI‘TS
.\ precession photograph of the Okl zone (J;ig. 2) possesses m m symmetry with Ok0 and 001 reflections present only when k = 2n and 1 = 2n. The h0Z zone also showed m 712s\-mmetry with 1~00 present only when 11= 3~. Uppc’r lwels of both zones dcmonst,rated 1~2 111symmckr\- with no systematic absents rclativc to thus wro l~~~~ls. The crystal is, thtwfow, of orthorhombicO space group 1’211)1L?1 with (7 = S!J.l f 0..5 A, b = 140.0 f o.r, A, and (’ = 2:<1.2 rt 2.0 .I. Thc~ voluinc~ of ori(’ unit ccl1 is 2.38 X 10F K3, and the: dwrsity of thcl crystals has
EM. and eler. dill’. EM. and S-ray diff. EM. E.M. EM. Elec. diff. X-ray diff. S-ray diff. E&l X-ran- difl’. I’M. optical diff.
Thin
platelets
Ortho. I’R,21 Ortho. Ortho. Ortho. 12.2 1>2,2,;,
and
7 ,Si,‘;,
Ortho. dr) X-ray S-ray
dilf. diff. and EM.
E’IG.
75(,‘i. 57 ('
1. Rli~rtr~)hotc,gr:t~)~~ of orthclrhorrll)ic.
been mcasuwd on :L bromobc,rlxc~rlc:-x~lcrle gradknt to b(k p = 1.10 g/en?. Assumption of one molwulc per as\-mmctric unit yicllds a volume to mass ratio of 2.M AR/d&on and :I solvcwt volume cof 37 ‘,B which is in
beef liver
~:tlal:~sc~ tryst:&
good agrwment rvith compukd values for other protcin crystals (1s). It should bc pointed out that the volume of t,he trigonal unit, ccl1 occupied by solvent ~-as estimated to bc 70-75 % , hcncc, the orthorhombic
crystal form probably ariws from firmer intermolecular forws and a more stable lattice. The crystals giw $rong diffraction intensities to at least 2.S A resolution and show little widenw of radiation damage after 100 hr of caxposuro. E’igurcx 3 is an clrctron micrograph of the most, commonly obwrvcd view of the negstained orthorhombic crystals, atiwly she\\-ing the charactcbristic distribution of heavily st’ained areas \vhich have been interpretod as large chanwls running through the crystal in a dir&ion pc>rpc>ndicular t c) the planc~ of thr micrograph. Thaw channels form a hrtx$gonal agray with a wparation of about 125 A & 10 A. I ) Is~‘LM-51( )?J FIN:. 2. -X-ray diffraction photograph of t.he Ok! zone of an orthohombir beef liver catnl:~~ crystal. Tht= precession :tnglc is 8” and the crystal to film distance is 100 mm.
Thr: fcat,urcs which impow the greatest constraints on any proposed packing arrangemrnt arc t,lw broad channels arrayed
in the hexagonal manner obsorvcd in the micrograph, the unit cell dimensions deduced from both thcl micrograph and from the X-ray data, and t)he space group sj.nlmetry derivc>d from th(b diffra&on pat’tcrn. Thew points are wconcilcd if one assumers a molecular packing arrangcmcwt similar to that shon-n schc~matically in I’ig. 4. .Assuming this unit ~11, the dimensions d;rivetl from the mic~ograph arc’ b,,,,. = 1’&5A and cE.Mb = ZO A which cor@atcs \vith bS-r:,5-= 140 A and c~-,,) = 232 A, allo\ving a reason able shrinkage upon drying. The length of the u axis corrwponds to a bit more than one molecular ~liicknws and from t8hclX-m\ results is S9 A in the \vet orthorhombic cyrstal. In t,liis model the molecules are packed with the required symmetry as spheres with a diumc%cr of roughly ‘73 K. The unit cell contains a large solvent cl~nmwl running through the center and coincident with a Y1axis
The packing arrangement shown in F’ig. 4 is virtually the same as that deduced b3 Rossman and Lnbaw from the X-ray data on the n-et P3121 crystals and Lab&w’s model for the dry crystal. As they pointed out, the ort,horhombic cell is derived from the t’rigonal cell by a different choice of (1 and h axes and by choosing 2 c axis length which is one-third of the 241 A trigonal axis. The orthorhombic cell which \ve propose is similarly related with an a axis equal to S9 A which is roughly one-third of the trigonal axis. It, seems possible that under some circumstances a s?,mmetry transition within tJhchcrystal could occur. Since the trigonal and orthorhombic forms are based on t)he same pwking arrangement, no t’ranslntion would be required. In the cast of a reduced state, (Jf hydration, the moleculrs in the trigonal crystals might8 worient themwlws thrOllgh
rot :ttiOn,
\i’ith(JUt
Chlging
pOSitiOll
FIG. 4. Orthogonal views of a proposed unit ~11 in orthorhornbic crystals of beef liver cat&se sho\ving the sgrnmctry relationships between the molecr~les. The hexagonal motif is apparent in the disposition of the spheres and t,he crystallographic trigonal unit cell is irldicated by the dashed lines. The levels of the asymmetric lmits refer to the orthorhombic system only and itr each view are with respect to the axis perpendicldar to the plane. The heavily stained channels are coincident with t.he 3, axes on the centers and cc,rners of the cell and parallel with u.
ill t11(~ Iattic~c~! to >%*ld th(b orthorhombic fornl. Pwcxd diff(wnt habits of wtalase crest :ds havt~ bwn obwrved to ccwxist in tlicb motlic~r licllwr and transform spontnrlc~c,usl\~ fl’~Illl Oll(’ morphologic~al form to another, implying sonw 1x1; of stabilit,y in tlw latt.icrs. \\ itli respect to hJ.drstion, the ortllorh~ )nlbic rr\-stals described hew ~)osses abuut 1.; “; Iws solvthnt than the trigonal cr>xt ds, aud thcl dry orthorhombic crptals studic,tl by Laban- contained none at, all. Iu adtiitiou, 11~0otliw types of catalaw cr>xtnls ltav(~ hccn cxunincd by S-ray diffraction, llorw livcsr cxtulasc (13) nud horw eythrucytc> catalaw (l-1). Both wew found to btr of oJ’t lrorh~ mbit spxcn groups and both contaill(td about ZOYC less solvent than the t rigolial (aatalaw cr\-stals. I,ouglo~~ (l(i) nllo\vcd crystals which he 11~1 iclwtific~d as belonging to space group P:l,?l to air dry. Optical diffraction pattcbrtls rw )rtl(J from micrographs of thcsc c~y.4 al.< ~hc~n-c~lthe presence of an apparent ?I axis \vhws tile S1 axis was expected to upI)c2r sugg(5tiiig a symmct’q- transition upon dc~hydrntiou. It. does not. seem unreasonable, t Ilc~rc~forc,that transformation bctwwn diff’(~r.c~~lt~ forms tloc~s occur which may bc dep(atltlcut, on thcb state of solvation. The cbstcwt 4)f the molecular reorkntat’ion that \\-orild b(> rquirod to effect, the symmetry 01:~1g~ ~YHM b(k considerab.ly reduced if nicA(4:ir s;!-mmrtry ~3s utilized. Indcc~l, I\-(’ s~qw(*t this to bc the cast. .\(‘h:NOWI,~i)C:~Il.:NTS I grnt~:f~~lly :lc,know-ledg? the patience ;tntl skill 01’ 111~. I,:l:~irlc T,enk for hrr techniral assistanw will, 1hr cl~c.tron n~irroswpe and I)r. Jon King
.\ precession photograph of the Okl zone (J;ig. 2) possesses m m symmetry with Ok0 and 001 reflections present only when k = 2n and 1 = 2n. The h0Z zone also showed m 712s\-mmetry with 1~00 present only when 11= 3~. Uppc’r lwels of both zones dcmonst,rated 1~2 111symmckr\- with no systematic absents rclativc to thus wro l~~~~ls. The crystal is, thtwfow, of orthorhombicO space group 1’211)1L?1 with (7 = S!J.l f 0..5 A, b = 140.0 f o.r, A, and (’ = 2:<1.2 rt 2.0 .I. Thc~ voluinc~ of ori(’ unit ccl1 is 2.38 X 10F K3, and the: dwrsity of thcl crystals has
EM. and eler. dill’. EM. and S-ray diff. EM. E.M. EM. Elec. diff. X-ray diff. S-ray diff. E&l X-ran- difl’. I’M. optical diff.
Thin
platelets
Ortho. I’R,21 Ortho. Ortho. Ortho. 12.2 1>2,2,;,
and
7 ,Si,‘;,
Ortho. dr) X-ray S-ray
dilf. diff. and EM.
E’IG.
75(,‘i. 57 ('
1. Rli~rtr~)hotc,gr:t~)~~ of orthclrhorrll)ic.
been mcasuwd on :L bromobc,rlxc~rlc:-x~lcrle gradknt to b(k p = 1.10 g/en?. Assumption of one molwulc per as\-mmctric unit yicllds a volume to mass ratio of 2.M AR/d&on and :I solvcwt volume cof 37 ‘,B which is in
beef liver
~:tlal:~sc~ tryst:&
good agrwment rvith compukd values for other protcin crystals (1s). It should bc pointed out that the volume of t,he trigonal unit, ccl1 occupied by solvent ~-as estimated to bc 70-75 % , hcncc, the orthorhombic
crystal form probably ariws from firmer intermolecular forws and a more stable lattice. The crystals giw $rong diffraction intensities to at least 2.S A resolution and show little widenw of radiation damage after 100 hr of caxposuro. E’igurcx 3 is an clrctron micrograph of the most, commonly obwrvcd view of the negstained orthorhombic crystals, atiwly she\\-ing the charactcbristic distribution of heavily st’ained areas \vhich have been interpretod as large chanwls running through the crystal in a dir&ion pc>rpc>ndicular t c) the planc~ of thr micrograph. Thaw channels form a hrtx$gonal agray with a wparation of about 125 A & 10 A. I ) Is~‘LM-51( )?J FIN:. 2. -X-ray diffraction photograph of t.he Ok! zone of an orthohombir beef liver catnl:~~ crystal. Tht= precession :tnglc is 8” and the crystal to film distance is 100 mm.
Thr: fcat,urcs which impow the greatest constraints on any proposed packing arrangemrnt arc t,lw broad channels arrayed
in the hexagonal manner obsorvcd in the micrograph, the unit cell dimensions deduced from both thcl micrograph and from the X-ray data, and t)he space group sj.nlmetry derivc>d from th(b diffra&on pat’tcrn. Thew points are wconcilcd if one assumers a molecular packing arrangcmcwt similar to that shon-n schc~matically in I’ig. 4. .Assuming this unit ~11, the dimensions d;rivetl from the mic~ograph arc’ b,,,,. = 1’&5A and cE.Mb = ZO A which cor@atcs \vith bS-r:,5-= 140 A and c~-,,) = 232 A, allo\ving a reason able shrinkage upon drying. The length of the u axis corrwponds to a bit more than one molecular ~liicknws and from t8hclX-m\ results is S9 A in the \vet orthorhombic cyrstal. In t,liis model the molecules are packed with the required symmetry as spheres with a diumc%cr of roughly ‘73 K. The unit cell contains a large solvent cl~nmwl running through the center and coincident with a Y1axis
The packing arrangement shown in F’ig. 4 is virtually the same as that deduced b3 Rossman and Lnbaw from the X-ray data on the n-et P3121 crystals and Lab&w’s model for the dry crystal. As they pointed out, the ort,horhombic cell is derived from the t’rigonal cell by a different choice of (1 and h axes and by choosing 2 c axis length which is one-third of the 241 A trigonal axis. The orthorhombic cell which \ve propose is similarly related with an a axis equal to S9 A which is roughly one-third of the trigonal axis. It, seems possible that under some circumstances a s?,mmetry transition within tJhchcrystal could occur. Since the trigonal and orthorhombic forms are based on t)he same pwking arrangement, no t’ranslntion would be required. In the cast of a reduced state, (Jf hydration, the moleculrs in the trigonal crystals might8 worient themwlws thrOllgh
rot :ttiOn,
\i’ith(JUt
Chlging
pOSitiOll
FIG. 4. Orthogonal views of a proposed unit ~11 in orthorhornbic crystals of beef liver cat&se sho\ving the sgrnmctry relationships between the molecr~les. The hexagonal motif is apparent in the disposition of the spheres and t,he crystallographic trigonal unit cell is irldicated by the dashed lines. The levels of the asymmetric lmits refer to the orthorhombic system only and itr each view are with respect to the axis perpendicldar to the plane. The heavily stained channels are coincident with t.he 3, axes on the centers and cc,rners of the cell and parallel with u.
ill t11(~ Iattic~c~! to >%*ld th(b orthorhombic fornl. Pwcxd diff(wnt habits of wtalase crest :ds havt~ bwn obwrved to ccwxist in tlicb motlic~r licllwr and transform spontnrlc~c,usl\~ fl’~Illl Oll(’ morphologic~al form to another, implying sonw 1x1; of stabilit,y in tlw latt.icrs. \\ itli respect to hJ.drstion, the ortllorh~ )nlbic rr\-stals described hew ~)osses abuut 1.; “; Iws solvthnt than the trigonal cr>xt ds, aud thcl dry orthorhombic crptals studic,tl by Laban- contained none at, all. Iu adtiitiou, 11~0otliw types of catalaw cr>xtnls ltav(~ hccn cxunincd by S-ray diffraction, llorw livcsr cxtulasc (13) nud horw eythrucytc> catalaw (l-1). Both wew found to btr of oJ’t lrorh~ mbit spxcn groups and both contaill(td about ZOYC less solvent than the t rigolial (aatalaw cr\-stals. I,ouglo~~ (l(i) nllo\vcd crystals which he 11~1 iclwtific~d as belonging to space group P:l,?l to air dry. Optical diffraction pattcbrtls rw )rtl(J from micrographs of thcsc c~y.4 al.< ~hc~n-c~lthe presence of an apparent ?I axis \vhws tile S1 axis was expected to upI)c2r sugg(5tiiig a symmct’q- transition upon dc~hydrntiou. It. does not. seem unreasonable, t Ilc~rc~forc,that transformation bctwwn diff’(~r.c~~lt~ forms tloc~s occur which may bc dep(atltlcut, on thcb state of solvation. The cbstcwt 4)f the molecular reorkntat’ion that \\-orild b(> rquirod to effect, the symmetry 01:~1g~ ~YHM b(k considerab.ly reduced if nicA(4:ir s;!-mmrtry ~3s utilized. Indcc~l, I\-(’ s~qw(*t this to bc the cast. .\(‘h:NOWI,~i)C:~Il.:NTS I grnt~:f~~lly :lc,know-ledg? the patience ;tntl skill 01’ 111~. I,:l:~irlc T,enk for hrr techniral assistanw will, 1hr cl~c.tron n~irroswpe and I)r. Jon King