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UBC Theses and Dissertations

The purification and properties of ribonucleases Roy, Kenneth Leo Joseph 1964

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THE PURIFICATION AND PROPERTIES OF RIBONUCLEASES by Kenneth Leo Joseph Roy B . S c , The U n i v e r s i t y of B r i t i s h Columbia, 1962 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science i n the Department of Biochemistry We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA May, 1964 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of • B r i t i s h Columbia,, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study, I f u r t h e r agree that per-m i s s i o n f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood that copying or p u b l i -c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission-Department of Biochemistry  The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada Date May 6. 1964  i ABSTRACT The p u r i f i c a t i o n of r i b o n u c l e a s e s and T£ from Ta-k a d i a s t a s e has been c a r r i e d out to provide enzymes of high pur-i t y and known s p e c i f i c i t y f o r s t r u c t u r a l s t u d i e s on s-RNA. These p u r i f i c a t i o n s i n v o l v e d a c i d treatment, acetone f r a c t i o n -a t i o n , g e l f i l t r a t i o n on Sephadex and ion-exchange chromatogra-phy on s u b s t i t u t e d c e l l u l o s e s . S a t i s f a c t o r i l y pure RNase T^ has been obtained and i t s s p e c i f i c i t y has been confirmed. Fur-t h e r p u r i f i c a t i o n of RNase 1^ might be d e s i r e a b l e but some st u d i e s using c h e m i c a l l y s y n t h e s i z e d s u b s t r a t e s have been c a r -r i e d out on the most h i g h l y p u r i f i e d f r a c t i o n y e t obtained. P a r t i a l p u r i f i c a t i o n of an RNase present i n a d i f f e r -ent d i a s t a s e p r e p a r a t i o n i n v o l v e d heat, a c i d , acetone f r a c t i o n -a t i o n and anion exchange chromatography. Information i s not y e t a v a i l a b l e on i t s s p e c i f i c i t y because of the small q u a n t i t i e s i s o l a t e d . i v ACKNOWLEDGEMENT The author wishes to express h i s most s i n c e r e thanks to Dr. Gr. M. Tener f o r h i s i n t e r e s t and guidance throughout t h i s work. The author a l s o would l i k e to acknowledge the under-standing and encouragement of h i s w i f e . The f i n a n c i a l a s s i s t a n c e of the N a t i o n a l Research C o u n c i l i s al s o a p p r e c i a t e d . i i TABLE OP CONTENTS Page I n t r o d u c t i o n . . 1 M a t e r i a l s and Methods 18 Experimental and R e s u l t s 22 Sy n t h e t i c Substrates 22 RNA P r e p a r a t i o n 24 Pa n c r e a t i c RNase 25 Mann Diastase 26 RNase T 1 P u r i f i c a t i o n 29 RNase T 2 P u r i f i c a t i o n 34 C h a r a c t e r i z a t i o n of RNase T^ . . . . . 40 C h a r a c t e r i z a t i o n of RNase T 2 42 D i s c u s s i o n . . . . . . . . . . . 44 Summary 56 B i b l i o g r a p h y 57 i i i ABBREVIATIONS AMP Adenosine monophosphate (3 or 2 ) CMP C y t i d i n e monophosphate (3 or 2 ) G-MP Guariosine monophosphate (3 or 2 ) UMP U r i d i n e monophosphate (3 or 2 ) CM- Carboxymethyl-DEAE- Di e t h y l a m i n o e t h y l SE- S u l f o e t h y l TEAE- T r i e t h y l a m i n o e t h y l DCC D i c y c l o h e x y l c a r b o d i i m i d e P o l y A Poly a d e n y l i c a c i d P o l y C Pol y c y t i d y l i c a c i d P o l y G Pol y g u a n y l i c a c i d P o l y I Poly i n o s i n i c a c i d P o l y U Poly u r i d y l i c a c i d RNase Ribonuclease UV U l t r a v i o l e t H i s 12 H i s t i d i n e i n the 12th p o s i t i o n i n p a n c r e a t i c RNase H i s 119 H i s t i d i n e i n the 119th p o s i t i o n i n p a n c r e a t i c RNase Lys 41 Lysine i n the 41st p o s i t i o n i n p a n c r e a t i c RNase 1 INTRODUCTION S t r u c t u r a l s t u d i e s on r i b o n u c l e i c a c i d s , i n p a r t i c -u l a r on the s o l u b l e or t r a n s f e r r i b o n u c l e i c a c i d s , r e q u i r e s e v e r a l h i g h l y p u r i f i e d and w e l l c h a r a c t e r i z e d r i b o n u c l e a s e s of known enzymatic s p e c i f i c i t y . T h i s t h e s i s d e s c r i b e s the p u r i f i c a t i o n and some of the p r o p e r t i e s of two such r i b o n u -c l e a s e s , the T^ and T 2 from Takadiastase. P a n c r e a t i c Ribonuclease The f i r s t d i s c o v e r e d and most thoroughly s t u d i e d r i b o n u c l e a s e (RNase) i s that of bovine pancreas which was c r y s t a l l i z e d by Moses Kunitz i n 1940 ( l ) . Over 20 years l a -t e r i t became the f i r s t enzyme i n which the primary sequence of amino acids was e s t a b l i s h e d . The sequence of r e a c t i o n s i n v o l v e d i n the h y d r o l y s i s of RNA by t h i s enzyme, and i t s s p e c i f i c i t y , were e l u c i d a t e d by Schmidt et a l . (2), Markham and Smith (3), Brown and Todd (4), and V o l k i n and Conn ( 5 ) . I t was shown that p a n c r e a t i c RNase c a t a l y z e d two separate r e -a c t i o n s , t r a n s e s t e r i f i c a t i o n and h y d r o l y s i s ,(3,4). Both r e -a c t i o n s were shown to be s p e c i f i c f o r phosphodiester linkages i i n v o l v i n g p y r i m i d i n e nucleoside 3 -phosphates (2,5). As i i shown below, t r a n s e s t e r i f i c a t i o n produces a 2 ,3 - c y c l i c phosphate which i s then opened i n a h y d r o l y t i c step to pro-duce the 3 -phosphate ( 4 ) . 2 R OCH; OH 0 OH 0—P=0 0 0 0 - P = 0 0" + R'OH R = H or Nu c l e o t i d e R = A l k y l group or Nucleoside Complete h y d r o l y s i s of RNA by RNase produces, as a r e s u l t of i t s pyri m i d i n e s p e c i f i c i t y , u r i d i n e 3 -phosphate, c y t i d i n e 3 -phosphate, and a s e r i e s of o l i g o n u c l e o t i d e s com-posed of purine n u c l e o t i d e r e s i d u e s terminated by a pyrimidine n u c l e o s i d e 3 -phosphate. Reports by Hakim (6) th a t p a n c r e a t i c RNase can hydrolyze the 2 ,3 - c y c l i c phosphates of adenosine and guanosine disagree with the r e s u l t s of Brown and Todd (4) and r e s u l t s obtained i n t h i s l a b o r a t o r y . Beers (7) has claimed than p a n c r e a t i c RNase can hydrolyze an a d e n y l i c a c i d polymer (pol y A) but no one has as y e t confirmed t h i s so i t would prob-a b l y be unwise to accept i t at the present time. Ribonucleases T^ and T^ Recently, c o n s i d e r a b l e i n t e r e s t has centered around the r i b o n u c l e a s e s of Takadiastase, an e x t r a c t of A s p e r g i l l u s oryzae. The presence of RNases i n Takadiastase w a s . f i r s t r e -ported by Kuninaka (8) i n 1954. In 1957 Egami and co-workers 3 showed the presence of at l e a s t two RNase a c t i v i t i e s and named them (major component) and T 2 (minor component). The T^ enzyme was p a r t i a l l y p u r i f i e d at that time and a few of i t s p r o p e r t i e s shown (9). The p r e l i m i n a r y r e s u l t s suggest-ed t h a t RNase T^ p r e f e r e n t i a l l y hydrolyzed those phosphodies-t e r bonds d i s t a l to the 3 -phosphate of gua n y l i c a c i d r e s i -dues i n RNA. They a l s o demonstrated the intermediate forma-i t t i o n of the 2 ,3 - c y c l i c phosphate of guanosine, and i t s sub-i sequent h y d r o l y s i s to guanosine 3 -phosphate. Takahashi (10, 11) and Rushizky and Sober (12) have p u r i f i e d the RNase T^ q u i t e h i g h l y and the l a t t e r have confirmed i t s absolute spe-c i f i c i t y . P a r t i a l p u r i f i c a t i o n of RNase was rep o r t e d by Naoi-Tada ert a l . (13) who observed p r e f e r e n t i a l h y d r o l y s i s of t RNA at phosphodiester bonds d i s t a l to the 3 -phosphates of i a d e n y l i c a c i d . The enzyme.was able to hydrolyze adenosine 2 , i i 3 - c y c l i c phosphate to the 3 -phosphate but the c y c l i c i n t e r -m e d i a t e s were not found i n t h e RNA d i g e s t s . The suggestion was made that RNase T 2 might have an absolute s p e c i f i c i t y f o r a d e n y l i c residues with contamination by another RNase. As y e t no s t u d i e s have been p u b l i s h e d on the r e l a t i v e r a t e s of hy-d r o l y s i s by t h i s enzyme of the d i f f e r e n t linkages i n RNA. Recent work by Rushizky and Sober has i n d i c a t e d t hat RNase T 2 i s a completely n o n - s p e c i f i c enzyme which merely cleaves those bonds i n v o l v i n g a d e n y l i c a c i d residues at a higher rate than others (14). 4 B. s u b t i l i s Ribonuclease Another ribo n u c l e a s e f o r which a d i s t i n c t s p e c i f i -c i t y was claimed i s the e x t r a c e l l u l a r RNase of B a c i l l u s sub-t i l i s . Nishimura was the f i r s t to study t h i s enzyme and he concluded t h a t i t s s p e c i f i c i t y was complementary to t h a t of p a n c r e a t i c RNase. That i s , i t seemed to hydrolyze only phos-i phate e s t e r bonds d i s t a l to the 3 -phosphates of purine nuc-l e o t i d e s (15). R e i n v e s t i g a t i o n of t h i s enzyme by Rushizky et a l . (16), and by W h i t f e l d and W i t z e l (17) i n d i c a t e d a very complex s e r i e s of p r e f e r e n c e s , with only a very few linkages completely r e s i s t a n t to h y d r o l y s i s . Other Ribonucleases Although the four r i b o n u c l e a s e s d e s c r i b e d above are the most h i g h l y p u r i f i e d and w e l l c h a r a c t e r i z e d many others have been r e p o r t e d . Ribonucleases are so widely d i s t r i b u t e d t h a t probably a l l l i v i n g c e l l s produce one or more RNases. Many d i f f e r e n t p l a n t s have RNases, n e a r l y a l l capable of hy-i t i d r o l y z i n g RNA to 3 - n u c l e o t i d e s or the nucleoside 2 ,3 -cy-c l i c phosphates (18). An unusual p l a n t nuclease i s that from Mung Bean sprouts which was s t u d i e d by Sung and Laskowski (19). This enzyme hydrolyzes both RNA and DNA with the production of nu-t c l e o s i d e 5 -phosphates. I t shows no base s p e c i f i c i t y but seems to p r e f e r e n t i a l l y hydrolyze those bonds d i s t a l to the 5 -phosphates of adenosine and u r i d i n e (or thymidine i n DNA). A somewhat s i m i l a r RNase has been i s o l a t e d from l i v e r n u c l e i . 5 I t l i k e w i s e produces 5 -phosphates (20) but appears to be s p e c i f i c f o r RNA. Cunningham et ajL. (21) have d e s c r i b e d a nuclease from Staphylococcus pyogenes (M i c r o c o c c a l nuclease) i which degrades RNA and DNA to the 3 -phosphates of mono-and d i n u c l e o t i d e s . T h i s enzyme would probably not c a t a l y z e the t r a n s e s t e r i f i c a t i o n r e a c t i o n t y p i c a l of the four RNases des-c r i b e d p r e v i o u s l y . A great many other RNases have been r e -ported other than these few, but g e n e r a l l y have not been ex-t e n s i v e l y s t u d i e d . An i n t e r e s t i n g f e a t u r e of most rib o n u c l e a s e s i s t h e i r s t a b i l i t y . In general they can be subjected to strong a c i d s and high temperatures with very l i t t l e l o s s of a c t i v i t y . These two p r o p e r t i e s have f r e q u e n t l y been e x p l o i t e d i n the schemes devised f o r the p u r i f i c a t i o n of RNases. These s t a -b i l i t y p r o p e r t i e s can be a t r i b u t e d to t h e i r f a i r l y simple s t r u c t u r e s , at l e a s t i n those cases where the s t r u c t u r e s are known. Pa n c r e a t i c RNase, RNase T^, the B. s u b t i l i s RNase, and M i c r o c o c c a l nuclease have molecular weights under 14,000, and RNase i s thought to have a molecular weight no greater than 30,000. A f u r t h e r demonstration of t h e i r remarkable s t a b i l i t y i s the r e p o r t of Rushizky et_ a l . (22) who found t h a t p a n c r e a t i c RNase, B. s u b t i l i s RNase, M i c r o c o c c a l nuclease, and RNase T^ could a l l be e x t r a c t e d from aqueous s o l u t i o n i n t o phenol with f u l l r e t e n t i o n of a c t i v i t y on removal of the phe-n o l . When one considers the a b i l i t y of phenol to denature p r o t e i n s t h i s s t a b i l i t y i s remarkable. 6 Mechanisms of H y d r o l y s i s The mechanisms by which RNases hydrolyze RNA have not y e t been very e x t e n s i v e l y s t u d i e d , except i n the case of p a n c r e a t i c RNase. Even i n t h i s case the l i t e r a t u r e i s meager when compared to t h a t on the p r o t e o l y t i c enzymes. Mechanisms have been proposed f o r the chemical degradation of RNA and the simple phosphodiesters of n u c l e o s i d e s . I t has been shown t h a t i n those cases, as w e l l as i n the enzymatic h y d r o l y s i s , i i n u c l e o s i d e 2 ,3 - c y c l i c phosphates are i n t e r m e d i a t e s . A c i d C a t a l y s i s P r o t o n a t i o n of the primary phosphate d i s s o c i a t i o n of these phosphodiesters causes a l a r g e i n c rease i n the e l e c -t r o p h i l i c i t y of the phosphorus atom. This makes i t more sus-i c e p t i b l e to n u c l e o p h i l i c a t t a c k by the 2 -oxygen atom and i i leads to the formation of e i t h e r a 2 ,3 - c y c l i c d i e s t e r by « I I _ e l i m i n a t i o n of R , or a 2 ,3 - c y c l i c t r i e s t e r i f OH i s e l i m -i n a t e d . 7 A t t a c k by a water molecule would produce i n the • i f i r s t case a mixture of the n u c l e o s i d e 2 - and 3 -phosphates. In the second case a t t a c k by water would y i e l d a mixture of i t i t the 2 ,3 - c y c l i c d i e s t e r and the 2 - and 3 -phosphodiesters. t These three products would be e v e n t u a l l y hydrolyzed to 2 -t and 3 -phosphates. The c y c l i c t r i e s t e r shown above has not been demonstrated as an intermediate i n t h i s r e a c t i o n but i t s i e x i s t a n c e i s necessary to e x p l a i n the formation of the 2 -phosphodiesters. Szer and Shugar c l a i m to have syn t h e s i z e d compounds of t h i s s t r u c t u r e (23) but i n f o r m a t i o n on i t s s t a -b i l i t y i s not a v a i l a b l e . Brown e_t a l . (24) s y n t h e s i z e d d i -i methyl and d i b e n z y l e s t e r s of u r i d i n e 3 -phosphate and found i them to be unstable at a l l pHs, decomposing to u r i d i n e 2 -and 3 - phosphomonoesters. This extreme i n s t a b i l i t y i s a l -most c e r t a i n l y due to the presence of the v i c i n a l hydroxyl group. B a s i c C a t a l y s i s t In a l k a l i the 2 -hydroxyl group becomes i o n i z e d and the n e g a t i v e l y charged oxygen c a r r i e s out a n u c l e o p h i l i c a t -tack on the phosphorus. Due to the primary phosphate d i s s o -i t t c i a t i o n only -OR can act as a l e a v i n g group so only 2 ,3 -c y c l i c phosphate d i e s t e r s can be formed as products of t h i s t r a n s e s t e r i f i c a t i o n . 8 Two l i n e s of evidence tend to e l i m i n a t e the p o s s i -b i l i t y of an intermediate c y c l i c t r i e s t e r as p o s t u l a t e d f o r a c i d h y d r o l y s i s . F i r s t , no 2 .-phosphodiesters have been i s o -l a t e d from a l k a l i n e d i g e s t s although they have been found i n 18 a c i d h y d r o l y s a t e s . Second, a l k a l i n e h y d r o l y s i s i n 0 of 18 y e a s t RNA gave mononucleotides with only one atom of 0 per atom of phosphorus. Formation of an intermediate c y c l i c t r i -e s t e r would be expected to allow i n c o r p o r a t i o n of two atoms 18 of 0. H y d r o l y s i s of the c y c l i c d i e s t e r i s c a r r i e d out by _ t t d i r e c t a t t a c k by OH producing a mixture of the 2 - and 3 -phosphomonoesters. H y d r o l y s i s of RNA c a t a l y z e d by c e r t a i n d i v a l e n t c a -t i o n s has a l s o been repo r t e d (25,26). The mechanism i n t h i s case appears to be s i m i l a r to t h a t c a t a l y z e d by a c i d . Enzyme Mechanisms Mechanisms f o r the enzymatic h y d r o l y s i s of RNA by p a n c r e a t i c RNase have been proposed by W i t z e l (27) and F i n d -l a y et a l . (28). V i t z e l suggests t h a t the p y r i m i d i n e base i s not i n v o l v e d i n b i n d i n g to the enzyme but i s s p e c i f i c a l l y i n -v o l v e d i n c a t a l y s i s by hydrogen bonding to the 2 -hydroxyl group. He proposes that t h i s i n c r e a s e s the n u c l e o p h i l i c i t y of the 2 -oxygen i n the same way that a l k a l i does and thus causes t r a n s e s t e r i f i c a t i o n to occur forming the 2 ,3 - c y c l i c phosphate. In a d d i t i o n he suggests that the e l e c t r o p h i l i c i t y of the phosphorus i s in c r e a s e d by p r o t o n a t i o n by the enzyme. The a c t u a l sequence of r e a c t i o n s he proposes i s l ) Protona-t i o n of the phosphate by the enzyme 2) N u c l e o p h i l i c a t t a c k of the phosphorus by the 2 -oxygen. In t h i s second step the py r i m i d i n e base i s assumed to t r a n s f e r the proton from the 2 -oxygen to the R -oxygen causing the l o s s of that group. i* 0 A o A N N R R' E = ENZYME In the opening of the c y c l i c d i e s t e r W i t z e l suggests t h a t the enzyme again protonates the phosphate and that the pyr i m i d i n e a c t i v a t e s a water molecule by hydrogen bonding to 10 i t . The a c t i v a t e d water molecule i s then assumed to c a r r y out a n u c l e o p h i l i c a t t a c k on the phosphorus while the pyrimidine base t r a n s f e r s one of the protons from the water molecule to i the 2 -oxygen. The mechanism proposed by F i n d l a y e_t a J . c o n t r a s t s r a t h e r s h a r p l y with W i t z e l ' s i n that they consider the pyrim-i d i n e to be i n v o l v e d only i n the b i n d i n g of s u b s t r a t e to en-zyme, and not at a l l i n c a t a l y s i s . In t h e i r hypothesis two imidazole groups are i n v o l v e d , one protonated and the other not. In t h e i r f i r s t step the unprotonated h i s t i d i n e i s H-i bonded to the 2 -hydroxy! to i n c r e a s e the n u c l e o p h i l i c i t y of the oxygen while the protonated h i s t i d i n e t r a n s f e r s i t s pro-ton to the oxygen of the l e a v i n g group. In the second step they c o n s i d e r that the unproto-nated imidazole H-bonds to a water molecule to a c t i v a t e i t and t h a t the protonated imidazole t r a n s f e r s a proton to the 2 -oxygen to weaken the P-0 bond. In t h i s way the c y c l i c phos-i phate i s s p e c i f i c a l l y opened to the 3 -phosphate. 11 I f one c o n s i d e r s the p y r i m i d i n e to perform the func t i o n of the u n i o n i z e d imidazole one has a mechanism s i m i l a r but not i d e n t i c a l to W i t z e l ' s . T h i s i s due to V i t z e l ' s pro-p o s a l that the phosphate i s protonated by the enzyme and thus becomes very s u s c e p t i b l e to n u c l e o p h i l i c a t t a c k . In the me-chanism proposed by F i n d l a y et a J . the phosphate i s always i o n i z e d . V/ i t z e l bases h i s mechanism on s e v e r a l good argu-ments. F i r s t , the Km i s not p a r t i c u l a r l y dependant on the nature of the p y r i m i d i n e base while the ES ^3 E + P step i s . He assumes Km to be a measure of the enzyme's a b i l i t y to bind a g i v e n s u b s t r a t e while k^ i s assumed to be the r a t e of trans e s t e r i f i c a t i o n or h y d r o l y s i s . F u r t h e r , h i s s t u d i e s with d i -n u c l e o s i d e phosphates i n d i c a t e a very c o n s i d e r a b l e e f f e c t of the 5 - l i n k e d n u c l e o s i d e on k-j but very l i t t l e e f f e c t on Km. He i n t e r p r e t s t h i s as being due to tf-interaction between the p y r i m i d i n e base and the second base. The most e f f e c t i v e base f o r t h i s assumed '^'-interaction i s adenine, then guanine, cy-t o s i n e and f i n a l l y u r a c i l . T h i s -nr-interaction i s assumed to 12 i n c r e a s e the a b i l i t y of the 2-oxygen s u b s t i t u e n t on the p y r i -i midine to p o l a r i z e the 2 -OH bond and thus cause a f u r t h e r i i n c r e a s e i n the n u c l e o p h i l i c i t y of the 2 -oxygen. The methods t h a t W i t z e l has used were g e n e r a l l y k i n e t i c a n a l y s i s of r e a c -t i o n s using a wide v a r i e t y of s u b s t r a t e s . P i n d l a y et a l . based t h e i r mechanism on s t u d i e s of one r e a c t i o n — t h a t of hy-t i d r o l y s i s of c y t i d i n e 2 ,3 -phosphate. These i n c l u d e d s t u d i e s on pH dependence of the k i n e t i c parameters f o r t h i s r e a c t i o n , the e f f e c t s of i n e r t organic s o l v e n t s and a l c o h o l s , an attempt to determine the charge types of the groups at the a c t i v e cen-t e r , and the e f f e c t s and i n t e r a c t i o n s of i n h i b i t o r s with the enzyme (29, 30, 3.1, 32). T h e i r conclusions are t h a t the ac-t i v e s i t e contains two h i s t i d i n e r e s i d u e s , one protonated and the other not. The s t u d i e s of C r e s t f i e l d et a l . (33, 34) have shown t h a t a l k y l a t i o n of His-119 completely i n a c t i v a t e s pan-c r e a t i c RNase, while a l k y l a t i o n of His-12 does not completely i n a c t i v a t e i t . I t would seem then that F i n d l a y ejb a l . are probably c o r r e c t i n t h e i r f i n d i n g of the involvement of two h i s t i d i n e residues at the a c t i v e s i t e . W i t z e l has a d i f f e r e n t i n t e r p r e t a t i o n of how the two h i s t i d i n e s are i n v o l v e d (35). According to h i s hypothesis the p y r i m i d i n e base c a t a l y z e s both r e a c t i o n s when the phosphate i s protonated. He f i n d s t h a t imidazolium i o n does not c a t a l y z e the r e a c t i o n and t h e r e -f o r e the e l e c t r o p h i l i c i t y of the imidazolium proton must be i n c r e a s e d . This he f e e l s i s brought about by having a hydro-gen bond between an imidazole and an imidazolium i o n . S e v e r a l groups, i n p a r t i c u l a r C r e s t f i e l d et aJL. (33), have found that carboxymethylation of RNase has a pH optimum at 5.5. This i s about the same pH at which ¥itzel found the Km f o r s e v e r a l RNase subs t r a t e s to be lowest. Carboxymethylation of N - l of His-119 occurs i n 92% y i e l d , the other 8fo of carboxymethyla-t i o n o c c u r r i n g at N-3 of His-12. From t h i s i t has been pos-t u l a t e d t h at they are s u f f i c i e n t l y c l o s e together t h a t when one imidazole i s protonated i t a t t r a c t s an iodoacetate i o n and o r i e n t s i t i n such a way that the other imidazole i s a l -k y l a t e d . C r e s t f i e l d e_t a l . suggest from t h i s t h a t the two h i s t i d i n e residues are about 5A apart (34). This c o n c l u s i o n i s reached i n p a r t by the o b s e r v a t i o n that i f one residue i s a l k y l a t e d i t p r o t e c t s the other from a l k y l a t i o n . W i t z e l now suggests that the enzyme-substrate complex i n the ribonuclease r e a c t i o n i s an enzyme bound pentacovalent p h o s p h o t r i e s t e r . Since Lys-41 has been i m p l i c a t e d i n the a c t i v e s i t e of the enzyme by s e v e r a l groups W i t z e l proposes that i t s f u n c t i o n i s to s t a b i l i z e one of the negative charges on the p h o s p h o t r i -e s t e r . F u r t h e r he proposes that no d i e s t e r monoanion can act as an i n h i b i t o r of RNase unless i t can form the d i a n i o n i c i n -termediate. A l l d i a n i o n s should t h e r e f o r e be i n h i b i t o r s of RNase, and i n a number of cases t h i s has been shown. The f o l l o w i n g diagram i n d i c a t e s W i t z e l ' s r e v i s e d hy-p o t h e s i s . 14 N H I ROCH V 1 ^ o w o . . / ^ o w o 7 I O-H H "0—P—0—R' h + I 3 (CH2)4 J + -0—Pv^-vH 0 -X NH-; R'OH (CH2)4 A l l other r i b o n u c l e a s e s which produce 3 -phosphates have been shown to f o l l o w the same sequence of r e a c t i o n s , t h a t i s , t r a n s e s t e r i f i c a t i o n f o l l o w e d by h y d r o l y s i s of the t i i intermediate 2 ,3 - c y c l i c phosphate. Some RNases open the 2 , i 3 - c y c l i c phosphates of some n u c l e o t i d e s very slowly and i n some cases not at a l l . W h i t f e l d and W i t z e l have p u b l i s h e d a note on the mechanism of RNase h y d r o l y s i s of RNA and f i n d some d i f f e r -ences between i t and p a n c r e a t i c RNase (36). For subs t r a t e s they used a number of d i n u c l e o s i d e phosphates and found the i e f f e c t i v e n e s s of the 5 - l i n k e d n u c l e o s i d e s i n promoting t r a n s e s t e r i f i c a t i o n decreased i n the order c y t i d i n e , adenosine, guanosine, u r a c i l . The r e l a t i v e e f f e c t i v e n e s s of the 3 - l i n k e d n u c l e o s i d e s decreased i n the order guanosine, i n o s i n e , xan-t h o s i n e , g l y o x a l guanosine. 15 Fur t h e r they found that the rates of t r a n s e s t e r i f i -c a t i o n of the d i n u c l e o s i d e phosphates was between 125 and 400 i t times the r a t e of h y d r o l y s i s of guanosine 2 ,3 - c y c l i c phos-phate. The c y c l i c phosphates of i n o s i n e , xanthosine, and g l y -o x a l guanosine were not hydrolyzed. The conclusions they reached were t h a t the T^ enzyme d i f f e r e d markedly from pan-c r e a t i c RNase i n i t s k i n e t i c behavior. Whereas p a n c r e a t i c i RNase i s very s t r o n g l y i n f l u e n c e d by the nature of the 5 -l i n k e d n u c l e o s i d e , T^ i s only i n f l u e n c e d to a minor extent. They i n t e r p r e t t h i s as meaning t h a t the guanine base cannot p l a y the same r o l e i n c a t a l y s i s d u r i n g r e a c t i o n with T^ as the p y r i m i d i n e bases p l a y during r e a c t i o n with p a n c r e a t i c RNase. Instead they suggest t h a t the guanine base i s i n v o l v e d i n b i n d i n g to the enzyme and that the base must be protonated at N - l or the oxygen on p o s i t i o n 6. McCully and Cantoni (37) have shown t h a t RNase T^ i s unable to hydrolyze those phosphodiester bonds d i s t a l to 1-methyl-guanosine and 6-hydroxy-2-dimethylaminopurine nucleo-s i d e . This i s i n accord with the proposed requirement f o r p r o t o n a t i o n of the base at N - l . However, t h e i r methods would i i not have shown the formation of a 2 ,3 - c y c l i c phosphate of e i t h e r of these n u c l e o s i d e s . Since the g l y o x a l d e r i v a t i v e of guanosine i s a s u b s t r a t e f o r the t r a n s e s t e r i f i c a t i o n r e a c t i o n but not the h y d r o l y s i s step the p o s s i b i l i t y remains that the i methylated guanosines 3 -phosphodiesters may a l s o be suscep-t i b l e to t h i s t r a n s e s t e r i f i c a t i o n . 16 W h i t f e l d and W i t z e l have a l s o s t u d i e d B. s u b t i l i s RNase using i n t h i s case homopolymers of IMP, AMP, CMP and UMP (38). In a d d i t i o n they used s e v e r a l smaller s u b s t r a t e s . They found t h a t the enzyme c a t a l y s e d t r a n s e s t e r i f i c a t i o n of purine p o l y n u c l e o t i d e s f a s t e r than pyrimidine and p o l y n u c l e o -t i d e s with 6-hydroxy s u b s t i t u e n t s f a s t e r than those with 6-amino s u b s t i t u e n t s . The r a t e s were P o l y I :> p o l y A >• p o l y U >. p o l y C and they c a l c u l a t e d t h a t p o l y G would have been broken down even f a s t e r than p o l y I . They p o s t u l a t e t h a t the d i s t a n c e between the 6-substituent and the c a t a l y t i c center f o r t r a n s e s t e r i f i c a t i o n i s c r i t i c a l . F u r t h e r they conclude t h a t the 6-substituent must act as a proton donor. They found i t h a t a d d i t i o n of a 3 -phosphate to a d i n u c l e o s i d e phosphate i n c r e a s e s the r a t e of t r a n s e s t e r i f i c a t i o n and that a f u r t h e r i n c r e a s e r e s u l t s from a d d i t i o n of another nuc l e o s i d e to t h i s i i phosphate. Another p o i n t i s t h a t guanosine 2 ,3 - c y c l i c phos-phate i s the only c y c l i c phosphate t h a t can be hydrolysed of the four a r i s i n g from RNA. As y e t no k i n e t i c s t u d i e s have been p u b l i s h e d d e a l -i n g with RNase T 2 and no mechanisms have been suggested. The most recent i n f o r m a t i o n on t h i s enzyme i n regard to i t s sub-s t r a t e preferences i s found i n the paper of Rushizky and So-ber (14). They showed that AMP (3 ) i s produced at a f a s t e r r a t e than the other mononucleotides. This i n i t s e l f i s un-u s u a l i n that a l l seem to be l i b e r a t e d at r a t e s u n l i k e those shown by any other enzyme. There have been no determinations 17 of r e l a t i v e r a t e s of h y d r o l y s i s of s u b s t r a t e s of known s t r u c -ture and no i n h i b i t o r s of t h i s enzyme have been r e p o r t e d . The purpose of the present i n v e s t i g a t i o n s was to p u r i f y RNases T^ and T£ from Takadiastase and to determine s p e c i f i c i t i e s u s i n g s y n t h e t i c s u b s t r a t e s . F u r t h e r , i n the event of n o n - s p e c i f i c nuclease a c t i v i t y i t was intended t h a t r e l a t i v e r a t e s of h y d r o l y s i s and t r a n s e s t e r i f i c a t i o n be de-termined. An i n v e s t i g a t i o n of the e f f e c t s of v a r i o u s i n h i b -i t o r s was a l s o considered necessary to f u l l y c h a r a c t e r i z e i i the a c t i v i t i e s . The use of n u c l e o s i d e 2 ,3 - c y c l i c phosphates i and n u c l e o s i d e 3 -phosphate p r o p y l e s t e r s made p o s s i b l e the study of both the t r a n s e s t e r i f i c a t i o n step and the h y d r o l y t i c step, without the complications introduced by the presence of a second base as found i n s t u d i e s u s i n g d i n u c l e o s i d e phos-phates and d i n u c l e o t i d e s . 18 MATERIALS AND METHODS Takadiastase powder (Sanzyme R) was purchased from the Sankyo Co. L t d . , Tokyo, Japan. Another A. oryzae d i a s t a s e p r e p a r a t i o n was purchased from Mann L a b o r a t o r i e s , New York. Fresh pressed yeast (Flieschmann) was purchased from Standard Brands Inc., Richmond, B. C. The mixed 2 - and 3 -phosphates of adenosine, u r i -dine and c y t i d i n e were purchased from Schwartz B i o r e s e a r c h , Inc., Orangeburg, New York. Guanosine 2 (3 )-phosphate (mixed isomers) was pur-chased from C. F. Boehringer, Mannheim, Germany. Adsorbents DEAE-, CM- and S E - c e l l u l o s e were purchased from Brown Company, B e r l i n , N. H. T E A E - c e l l u l o s e was obtained from Calbiochem, Los Angeles. Before use the adsorbents were suspended i n tap wa-t e r and f i n e p a r t i c l e s removed by repeated d e c a n t a t i o n . C o l -umns were packed i n 2M ammonium carbonate under 2-4 l b . a i r pr e s s u r e . A l l columns were washed with 2M ammonium carbonate u n t i l the o p t i c a l d e n s i t y (260 m\i) of the e f f l u e n t was as low as that of the i n f l u e n t s o l u t i o n . The columns were then washed with at l e a s t 3 column volumes of d i s t i l l e d water to remove ammonium carbonate. Columns were converted to other i o n forms ( i f necessary) by washing with the appropriate so-l u t i o n . P r i o r to l o a d i n g , a l l columns were e q u i l i b r a t e d with the s t a r t i n g s o l u t i o n , which was a l s o used to wash i n the ap-19 p l i e d s o l u t i o n . A l l e l u t i o n s were c a r r i e d out u s i n g l i n e a r g r a d i e n t s of s a l t s , and, i n some cases pH. Sephadex Sephadex G-25 and G-75 were purchased from Pharma-c i a A. B.,. Uppsala, Sweden. Columns of Sephadex were prepared from g e l p a r t i c l e s which had been allowed to swell f o r at l e a s t 48 hours. The f i n e p a r t i c l e s were removed by repeated decantation and the coarse p a r t i c l e s r i n s e d s e v e r a l times with the s o l u t i o n to be used. Columns were packed slowly under g r a v i t y to ensure even packing. When completed a piece of f i l t e r paper was put on top of the g e l to prevent disturbances to the top. The c o l -umns were loaded i n the usual manner f o r g e l f i l t r a t i o n on Sephadex. Ribonuclease Assays A. The standard assay procedure used i n v o l v e d the a d d i t i o n of 0.1 ml of p r o p e r l y d i l u t e d enzyme (up to 7 units/ml) to 0.4 ml of 0.2M ammonium acetate b u f f e r (pH 4.5 f o r > pH 7.5 f o r T ^ ) . To t h i s was added 0.5 ml of RNA s o l u t i o n (140 O.D. 9 6 0/ml) and the mixture was incubated f o r 15 min at 37°C. At t h i s time 0.25 ml of c o l d 25% p e r c h l o r i c a c i d plus 0.75% u r a n y l acetate was added. The p r e c i p i t a t e was c e n t r i f u g e d out at 0°C and 0.5 ml of the supernatant was d i l u t e d to 5 ml. The O.D^^Q of t h i s s o l u t i o n was then determined u s i n g .the proper blanks. A u n i t of enzyme a c t i v i t y was d e f i n e d as that q u a n t i t y of enzyme which would produce an increase of one O.D. u n i t . (An O.D. 20 u n i t i s that amount of m a t e r i a l per ml which, i n a 1 cm l i g h t path gives a spectrophotometric reading of one.) This assay gave a l i n e a r response up to an O.D.^q of about 0.7. A l l UV ab s o r p t i o n measurements were c a r r i e d out on e i t h e r a Cary 11 Recording Spectrophotometer or a Z e i s s PMQ I I spectrophotometer. The Cary 11 was used f o r a l l s p e c t r a . t t B. Assays using n u c l e o s i d e 2 ,3 - c y c l i c phosphates and nucl e o s i d e t i i 3 -phosphate p r o p y l e s t e r s (or mixed 2 - and 3 -isomers) were c a r r i e d out i n 1 ml tubes with greased ground gl a s s stoppers at 37°C. At v a r i o u s times a l i q u o t s were removed and spotted on paper (Whatman 1 or 40). The chromatograms were developed i n a solvent system c o n t a i n i n g i s o p r o p a n o l , ammonium hydroxide, and water i n a r a t i o 7:1:2. A f t e r development, the papers were d r i e d and UV absorbing areas cut out. These were e l u t e d with d i s t i l l e d water and the o p t i c a l d e n s i t i e s determined against the appropriate blanks from c l e a r areas of the same R^ on the chromatograms. TABLE 1 R^ Values of Substrates and Products i n the Isopropyl A l c o h o l : Ammonium Hydroxide:Water (7:1:2) System (40) Compound R^ Adenosine 2 1(3')-phosphate .08 Adenosine 2 ' , 3 ' - c y c l i c phosphate .42 Adenosine 2 1(3')-phosphate pr o p y l e s t e r .56 C y t i d i n e 2'(3' )-phosphate .07 C y t i d i n e 2 ' , 3 ' - c y c l i c phosphate .31 C y t i d i n e 2'(3')-phosphate pr o p y l e s t e r ,54 Guanosine 2 1(3 1)-phosphate .05 Guanosine 2 ' , 3 ' - c y c l i c phosphate .28 Guanosine 2 1(3 1)-phosphate p r o p y l e s t e r .47 U r i d i n e 2'(3 1)-phosphate .06 U r i d i n e 2 ' , 3 ' - c y c l i c phosphate ^30 U r i d i n e 2'(3')-phosphate pr o p y l e s t e r .45 21 These R^ values tend to increase with the age of the sol v e n t system and are accurate only f o r the f r e s h l y prepared system. C. A t h i r d assay was used to f o l l o w r a t e s of h y d r o l y s i s of the • t p y r i m i d i n e " n u c l e o s i d e 2 ,3 - c y c l i c phosphates. H y d r o l y s i s of t i u r i d i n e and c y t i d i n e 2 ,3 - c y c l i c phosphates was followed d i -r e c t l y by measuring the increase i n O.D.^y^ and O.D^g^ r e -i s p e c t i v e l y produced on formation of the corresponding 3 -phos-phates (39). For measurement of RNase 1^ a c t i v i t y i ncubations were c a r r i e d out i n the c e l l compartment of the spectrophoto-meter. The temperature (37°C) was maintained by passing o i l from a t h e r m o s t a t - c o n t r o l l e d c i r c u l a t i n g pump through the ja c k e t e d c e l l compartment. The c e l l s were warmed to 37° i n a water bath before the enzyme was added. Complete d i g e s t i o n of u r i d i n e 2 ,3 - c y c l i c phosphate produced a 29.6?fc increase i n O.D.27C5 and c y t i d i n e 2 ,3 - c y c l i c phosphate a 33% increase i n O.D. 2gtj. 22 EXPERIMENTAL AND RESULTS  S y n t h e t i c Substrates A. C y c l i c Phosphates i t P r e p a r a t i o n of Nucleoside 2 ,3 - c y c l i c phosphates was by the method of Smith, Moffat and Khorana (41) or by the m o d i f i e d procedure of Smith and Khorana (42). The f i r s t of . i i these i n v o l v e d d i s s o l v i n g the n u c l e o t i d e (mixed 2 - and 3 -isomers) i n 2M ammonium hydroxide and formamide. To t h i s was added a f i v e - f o l d excess of d i c y c l o h e x y l c a r b o d i i m i d e (DCC) i n t - b u t y l a l c o h o l . The r e s u l t i n g s o l u t i o n was then r e f l u x e d f o r 2 l/2 hours, cooled, and ammonium hydroxide and t - b u t y l a l c o h o l were removed by evaporation under reduced pressure. T h i s was f o l l o w e d by a d d i t i o n of water, f i l t r a t i o n , e x t r a c t i o n w i t h ether, and f i n a l l y p r e c i p i t a t i o n of the product from ace-tone s o l u t i o n as the barium s a l t by the a d d i t i o n of barium i o d i d e . The q u a n t i t a t i v e y i e l d s claimed have not been a c h i e -ved, but y i e l d s of 80$ of b e t t e r have been obtained. The m o d i f i c a t i o n r e f e r r e d to used an aqueous s o l u t i o n of t r i e t h y l -amine to d i s s o l v e the n u c l e o t i d e s f o l l o w e d by a d d i t i o n of a f i v e - f o l d excess of DCC i n methanol. The r e s u l t i n g c l e a r so-l u t i o n was allowed to stand at room temperature f o r 8 hours (or more) and worked up i n the same manner as b e f o r e . In order to avoid d i v a l e n t c a t i o n s i n the prepara-t i o n s , a number of preparations have been p u r i f i e d d i r e c t l y , a f t e r e x t r a c t i o n with ether, by chromatography on columns of D E A E - c e l l u l o s e . The adsorbed n u c l e o t i d e s , c y c l i c and open, 23 were w e l l separated by e l u t i o n with a l i n e a r g r a d i e n t of am-monium carbonate. Repeated evaporation to .dryness under r e -duced pressure was s u f f i c i e n t to remove a l l of the s a l t . Nucleoside Phosphate Propyl E s t e r s i i Nucleoside 2 - and 3 -phosphate p r o p y l e s t e r s were prepared by the method of Tener and Khorana (40). As s t a r t i n g m a t e r i a l n u c l e o s i d e 2 ,3 - c y c l i c phosphates were used. They were subjected to a c i d c a t a l y z e d t r a n s e s t e r i f i c a t i o n i n an anhydrous s o l u t i o n of dioxane and n-propanol. The r e a c t i o n was stopped by evacuating to remove the h y d r o c h l o r i c a c i d . P u r i f i c a t i o n was then c a r r i e d out chromatographically on paper i n the isopropanol-ammonium hydroxide-water (7:1:2) system. i i S e p a r a t i o n of the 2 - and 3 -isomers of the adenosine and guan-osine phosphate p r o p y l e s t e r s was c a r r i e d out by chromatography on DEAE-and T E A E - c e l l u l o s e s , r e s p e c t i v e l y . i Synthesis of the pure 3 -isomers of u r i d i n e and cy-t i d i n e p r o p y l phosphates was achieved e n z y m a t i c a l l y using pan-c r e a t i c RNase. Incubation at room temperature of the enzyme i t and 2 ,3 - c y c l i c phosphate i n the presence of 80% n-propanol produced the d e s i r e d compounds (30). RNase was removed by f o r c i n g the s o l u t i o n very r a p i d l y through a column of S E - c e l l -u l o s e which adsorbs RNase but not the n u c l e o t i d e s . Chromato-graphy of the RNase-free s o l u t i o n on DEAE-cellulose gave the 3 -phosphate p r o p y l e s t e r s . In a l l of these syntheses i d e n t i f i c a t i o n of the UV absorbing m a t e r i a l e l u t e d from the ion-exchange columns was 24 i by paper chromatography. F u r t h e r , i n s y n t h e s i s of mixed 2 -i and 3 - p r o p y l e s t e r s the isomer e l u t e d from anion exchange i columns at lower i o n i c s t r e n g t h was the 2 - e s t e r . T h i s was proved by comparing t h e i r s u s c e p t i b i l i t y to enzymatic h y d r o l -y s i s . RNA P r e p a r a t i o n High molecular weight y e a s t RNA was prepared by the method of C r e s t f i e l d , Smith and A l l a n (43). T h i s procedure i n v o l v e d the breaking of the yeast c e l l w a l l by e x t r a c t i o n w i t h hot sodium dodecyl s u l f a t e . The s o l u t i o n was cooled r a -p i d l y to 0 C and c e l l d e b r i s was c e n t r i fuged o f f at CTC. The RNA was p r e c i p i t a t e d from the supernatant by a d d i t i o n to two volumes of c o l d 95% e t h a n o l . The p r e c i p i t a t e was washed twice w i t h 67fo ethanol and l e f t overnight i n 80$ ethanol. The pre-c i p i t a t e was then c o l l e c t e d by c e n t r i f u g a t i o n , d i s s o l v e d i n d i s t i l l e d water and c e n t r i f u g e d at 25,000 RPM f o r one hour. The supernatant was made 1M i n sodium c h l o r i d e and the r e s u l t -i n g p r e c i p i t a t e c o l l e c t e d . The y i e l d reported by C r e s t f i e l d et a l . (43) was about 0.9 g per 100 g of f r e s h pressed y e a s t . However, i n our hands the y i e l d s v a r i e d from .2 to .65 g per 100 g of pressed yeast, depending on i t s age. Much lower y i e l d s were obtained when a c t i v e dry yeast was used i n s t e a d of f r e s h pressed y e a s t . M o d i f i c a t i o n s of t h i s procedure were introduced to reduce contamination and to same time. These i n c l u d e d e x -t r a c t i o n of the aqueous RNA s o l u t i o n with phenol, followed by 25 e x t r a c t i o n of the aqueous phase with ether. In t h i s step a l l RNases and most other p r o t e i n was e l i m i n a t e d . High speed cen-t i f u g a t i o n was r e p l a c e d by f i l t r a t i o n through C e l i t e to c l a r -i f y the f i n a l s o l u t i o n . RNA was s t o r e d f r o z e n i n small tubes at a concen-t r a t i o n of 140 0-D.26Q u n ^ " t s P e r m^-' P a n c r e a t i c RNase P r e l i m i n a r y experiments were c a r r i e d out with pan-c r e a t i c RNase to determine the f e a s i b i l i t y of using nucleoside • i i « 2 (3 )-phosphate p r o p y l e s t e r s and n u c l e o s i d e 2 ,3 - c y c l i c phosphates as s u b s t r a t e s f o r the determination of the s p e c i -f i c i t y of the RNases and of the r a t e s of h y d r o l y s i s of the s u s c e p t i b l e bonds. A l l of these e a r l y experiments were c a r r i e d out at 37° with i n c u b a t i o n mixtures composed of equal p a r t s of nucleo-s i d e 2 (3 )-phosphate p r o p y l e s t e r s o l u t i o n (200 O.D./ml, 36% of which was the 2 -isomer) and b u f f e r e d RNase s o l u t i o n s of v a r y i n g c o n c e n t r a t i o n . K i n e t i c s t u d i e s on p a n c r e a t i c RNase h y d r o l y s i s of these s y n t h e t i c s u b s t r a t e s are complicated by two f a c t o r s . i F i r s t i s the presence of the 2 -isomer of the p r o p y l phosphates. These e s t e r s are known to be strong competitive i n h i b i t o r s of t h i s enzyme (32). Second i s the production by the r e a c t i o n i of nucleoside 3 -phosphates which are a l s o potent competitive i n h i b i t o r s . As a r e s u l t i t was extremely d i f f i c u l t to obtain data which f o l l o w e d zero or f i r s t order k i n e t i c s . 26 Since t h i s work was c a r r i e d out p r i o r to the prepar-t a t i o n of pure 3 -isomers of the nucl e o s i d e phosphate pro p y l e s t e r s no q u a n t i t a t i v e i n f o r m a t i o n was obtained on the course of the r e a c t i o n with these s u b s t r a t e s . Incubation of p a n c r e a t i c RNase A and B f r a c t i o n s w i t h a l l four p r o p y l e s t e r s showed that the pr o p y l e s t e r s of gu a n y l i c and a d e n y l i c acids were completely r e s i s t a n t to both the A and B f r a c t i o n s under c o n d i t i o n s i n which very r a p i d h y d r o l y s i s of c y t i d y l i c and u r i d y l i c a c i d propyl e s t e r s oc-c u r r e d . Mann Diastase An A. oryzae d i a s t a s e e x t r a c t from Mann L a b o r a t o r i e s , New York, was s t u d i e d to determine the presence or absence of RNases (T^ and T 2 ) . Incubation of an aqueous e x t r a c t of t h i s powder wi t h the nucl e o s i d e 2 (3 )-phosphate pro p y l e s t e r s l e d to the formation of n u c l e o s i d e s , thus i n d i c a t i n g the probable presence of both an RNase a c t i v i t y and a phosphatase a c t i v i t y . Heating the aqueous e x t r a c t at pH 1.5 for.two minu-te s at 80°C, f o l l o w e d by c o o l i n g and n e u t r a l i z a t i o n completely e l i m i n a t e d the phosphatase a c t i v i t y . Incubation of a sample of t h i s a c i d and heat t r e a t e d i i s o l u t i o n with each of the four mixed 2 - and 3 -nucle o s i d e phosphate p r o p y l e s t e r s o l u t i o n s showed the pro d u c t i o n of nu-c l e o s i d e 3 -phosphates i n a l l cases. A c t i v i t y a gainst the p r o p y l e s t e r of ad e n y l i c a c i d 27 was the g r e a t e s t , with that a g a i n s t the es t e r s of u r i d y l i c and c y t i d y l i c a cids much l e s s . H y d r o l y s i s of the p r o p y l e s t e r of gu a n y l i c a c i d was not de t e c t a b l e u n t i l 24 hours had elapsed. Under the c o n d i t i o n s used the time r e q u i r e d f o r 25$ h y d r o l y s i s i of the s u s c e p t i b l e 3 - s u b s t r a t e s was two hours f o r adenosine i i 3 -phosphate p r o p y l e s t e r , ten hours f o r c y t i d i n e 3 -phosphate t p r o p y l e s t e r and seventeen hours f o r u r i d i n e 3 -phosphate pro-p y l e s t e r . When assayed using RNA as substrate p r a c t i c a l l y no a c t i v i t y was observed. However, when 0.01M EDTA was added to the b u f f e r s o l u t i o n a c t i v i t y was observed. P u r i f i c a t i o n of the enzyme was attempted by DEAE-c e l l u l o s e column chromatography u s i n g a 2.2 cm column packed to a height of 22 cm. To t h i s was a p p l i e d 12 ml of the d i a s -tase e x t r a c t (about 2 g o f . p r o t e i n ) which had been a c i d and heat t r e a t e d as be f o r e . E l u t i o n was with 0.001M phosphate b u f f e r (pH 7) and a 2 1 l i n e a r g r a d i e n t of sodium c h l o r i d e to 0.5M. E i g h t y - f i v e p e r c e n t of the RNase a c t i v i t y a p p l i e d was recovered i n a 56 ml f r a c t i o n which emerged from the column a f t e r 1100 ml of eluant had passed through. This f r a c t i o n contained 36 C D ^ g Q u n i t s of p r o t e i n . A c t i v i t y of t h i s s o l u t i o n against the p r o p y l e s t e r s was.followed u s i n g a b u f f e r which i n c l u d e d EDTA (0.01M). In t h i s case the r a t e s of h y d r o l y s i s of a l l four p r o p y l e s t e r s were of the same order of magnitude. E i t h e r the i n c l u s i o n of EDTA or the column chromatography seemed to i n a c t i v a t e or r e -28 move some i n h i b i t o r present i n the enzyme. Acetone F r a c t i o n a t i o n An attempt to f r a c t i o n a t e the RNase a c t i v i t y of a crude, heat and a c i d t r e a t e d e x t r a c t with ammonium s u l f a t e was u n s u c c e s s f u l , but acetone f r a c t i o n a t i o n proved u s e f u l . I t was found t h a t the a d d i t i o n of i n c r e a s i n g amounts of acetone to crude a c i d and heat t r e a t e d enzyme s o l u t i o n s produced a pre-c i p i t a t e . The p r e c i p i t a t e which formed between one and three volumes of acetone at room temperature contained g r e a t e r than 85% of the RNase a c t i v i t y . Chromatographic F r a c t i o n a t i o n DEAE-cellulose column chromatography (column s i z e = 3.7 x 37 cm) was c a r r i e d out on a s o l u t i o n which had been sub-j e c t e d to heat and a c i d treatment f o l l o w e d by acetone f r a c t i o n -a t i o n . A t o t a l of 280 u n i t s were loaded on and a peak of ac-t i v i t y was recovered which contained about 190 u n i t s . This peak contained about 16.8 O.D^gQ u n i t s of p r o t e i n from ap-proximately 5 g of m a t e r i a l i n the a c e t o n e f r a c t i o n a t e d e x t r a c t . Samples of t h i s p r e p a r a t i o n were incubated with each of the four n u c l e o s i d e phosphate p r o p y l e s t e r s and each of the f o u r nucleoside 2 ,3 - c y c l i c phosphates. No h y d r o l y s i s of any of these was observed, even a f t e r 48 hours. One e x p l a n a t i o n f o r t h i s anomalous r e s u l t i s that too low a c o n c e n t r a t i o n of enzyme was used. On the other hand i t might represent the i s o l a t i o n of an RNase devoid of simple d i e s t e r a s e a c t i v i t y . Therefore, f u r t h e r s t u d i e s on t h i s f r a c -29 t i o n are needed before a f i n a l c o n c l u s i o n can be reached. Ribonuclease P u r i f i c a t i o n Two procedures have been reported f o r the prepara-t i o n of RNase T^ from Takadiastase, t h a t of Takahashi (10,11) and that of Rushizky and Sober (12). In the present work the f i r s t p r e p a r a t i o n of the T^ enzyme followed a modified proced-ure, d e r i v e d from t h a t of Takahashi. A. Method of Takahashi (Modified) 1) A c i d and Heat Treatment 80 g of Takadiastase powder was e x t r a c t e d with 150 ml of water f o r an hour and then c e n t r i f u g e d f o r an hour at 2000 RPM. The supernatant was removed and the p r e c i p i t a t e again e x t r a c t e d with 150 ml of water as before. The process was repeated a t h i r d time. The combined aqueous e x t r a c t was a c i d i f i e d slowly with h y d r o c h l o r i c a c i d to pH 1.5. I t was then heated to 80°C f o r 2 minutes i n 200 ml l o t s . The heated s o l u -t i o n s were cooled as r a p i d l y as p o s s i b l e and c e n t r i f u g e d at 2000 RPM and the p r e c i p i t a t e d i s c a r d e d . The s u p e r n a t a n t was brought to pH 5.8 with ammonium hydroxide and c e n t r i f u g e d again at 2000 RPM. 2) Acetone P r e c i p i t a t i o n The supernatant was added to three volumes of ace-tone at 0% and the p r e c i p i t a t e which formed was c o l l e c t e d by cen-t r i f u g a t i o n at 2000 RPM. The supernatant acetone s o l u t i o n was d i s c a r d e d . The p r e c i p i t a t e was e x t r a c t e d very thoroughly with 100 ml of 0.005M phosphate b u f f e r (pH 7) and c e n t r i f u g e d at 30 9000 RPM f o r 30 minutes. 3) DEAE-cellulose chromatography - Column 1 The 9000 RPM supernatant was loaded on a DEAE-cel-l u l o s e column (4 x 45 cm) which had p r e v i o u s l y been e q u i l i b r a -t e d with .005M phosphate (pH 7.0). E l u t i o n was c a r r i e d out w i t h a l i n e a r g r a d i e n t from 0 to 0.5M sodium c h l o r i d e , and with both chambers of the mixing apparatus 0.005M i n pH 7.0 phosphate b u f f e r . About 23,500 0.D. 2g 0 u n i t s were e l u t e d from t h i s column of which 1385 were i n the region of maximum RNase a c t i v i t y . The t o t a l a c t i v i t y of t h i s peak was about 520,000 u n i t s . 4) Phenol E x t r a c t i o n RNase was e x t r a c t e d from the above s o l u t i o n with phenol by the method of Rushizky et. a_l. (22). The enzyme was f o r c e d out of the phenol by the a d d i t i o n of ten volumes of d i e t h y l ether which had been f r e e d of peroxides by the a d d i -t i o n of water and calcium hydride followed by f i l t r a t i o n (44). The r e s u l t i n g s o l u t i o n was e x t r a c t e d three times with 0.1M am-monium bicarbonate (pH 7.8), using i n each case a volume of the bicarbonate s o l u t i o n equal to the o r i g i n a l volume of phe-n o l . The combined aqueous phase was e x t r a c t e d three times w i t h ether to remove phenol and f r e e d of ether by p a r t i a l evacuation. 5) DEAE-Cellulose Chromatography - Column 2 The r e s u l t i n g RNase s o l u t i o n was loaded d i r e c t l y onto another DEAE-cellulose column (2.2 x 105 cm) and e l u t e d with 31 a l i n e a r g r a d i e n t from 0.05M sodium phosphate (pH 7.0") to 0.15 M sodium phosphate plus ,2M sodium c h l o r i d e . Of the 300 O.D^g e l u t e d from t h i s second column about 60 O.D. u n i t s were i n the peak of maximum a c t i v i t y . This contained about 255,000 u n i t s of RNase which was phenol e x t r a c t e d as before and f o r c e d out of phenol as d e s c r i b e d p r e v i o u s l y . 6) DEAE-Cellulose Chromatography - Column 3 The aqueous s o l u t i o n recovered was loaded onto a t h i r d DEAE c e l l u l o s e column ( l x 105 cm). This was e l u t e d with a g r a d i e n t of 1 1 of 0.05M sodium phosphate (adjusted to pH 7.0) to 1 1 of 0.1M sodium phosphate (pH 7.0) plus 0.2M sodium c h l o r i d e . A sharp peak of a c t i v i t y was e l u t e d which c o i n c i d e d w i t h a peak of u l t r a v i o l e t absorbing m a t e r i a l ( F i g . l ) . This peak contained 220,000 of the 255,000 u n i t s of RNase a c t i v i t y a p p l i e d and a t o t a l of 26.5 O.D^gQ.* Using, the e x t i n c t i o n value of Rushizky and Sober (12) f o r RNase T^ t h i s corresponds to 15.5 mg of p r o t e i n . B. Method of Rushizky and Sober (Modified) 1) A c i d E x t r a c t i o n A p r e p a r a t i o n of RNase T^ was c a r r i e d out us i n g m o d i f i c a t i o n s of the procedure d e s c r i b e d by Rushizky and Sober (12). 200 g of Takadiastase was mixed with 1 1 of d i s t i l l e d water and s t i r r e d at room temperature f o r 2 l/2 hours. Con-c e n t r a t e d s u l f u r i c a c i d was added to b r i n g the pH of a 1 i n 100 d i l u t i o n to 2.6 and the s o l u t i o n was allowed to stand i n the c o l d room f o r 48 hours. The pH was r a i s e d to 5.3 with 51 33 ml of concentrated NH^OH. A c l e a r dark s o l u t i o n was obtained by c e n t r i f u g a t i o n at 8000 x g f o r one hour. This s o l u t i o n was e x h a u s t i v e l y d i a l y z e d a g a i n s t s e v e r a l changes of d i s t i l l e d wa-t e r . 2) Acetone P r e c i p i t a t i o n Ammonium acetate (pH 5.3) was added to b r i n g the so-l u t i o n to 0.1M and i t was added to 2.5 volumes of -15° acetone. The r e s u l t i n g p r e c i p i t a t e was d i s s o l v e d i n d i s t i l l e d water and c l a r i f i e d by c e n t r i f u g i n g f o r 45 min at 10,000 x g. 3) Gel F i l t r a t i o n The volume of the 10,000 x g supernatant was reduced to 110 ml on a r o t a r y evaporator at 30°C and loaded on a c o l -umn of Sephadex G-75 (7.5 x 42 cm) f o r g e l f i l t r a t i o n chromat-ography. The T^ and a c t i v i t i e s were separated to a c e r t a i n extent on t h i s column. The T^ f r a c t i o n was f u r t h e r p u r i f i e d by DEAE-cellulose column chromatography, e x t r a c t i o n i n t o phe-n o l , f u r t h e r DEAE-cellulose chromatography, g e l f i l t r a t i o n on Sephadex G-27, and a t h i r d chromatography on DEA E - c e l l u l o s e . TABLE I I RNase T^ P u r i f i c a t i o n Procedure O.D. 280 A c i d E x t r a c t i o n Acetone P r e c i p i t a t i o n Sephadex G-75 DEAE Column I Phenol E x t r a c t i o n DEAE Column I I DEAE Column I I I 103,820 14,080 8,232 1,567 656 148 84 Volume 1160 1110 . 980 1045 650 290 240 TT . , S p e c i f i c U n i t s / , . .. A c t i v i t y 2,500,000 1,727,000 1,234,800 1,200,000 540,000 24 123 150 760 820 485,750 3280 380,000 4530 Using the e x t i n c t i o n value of Rushizky and Sober of 34 1.7/mg f o r RNase T^, the q u a n t i t y recovered i s about 50 mg. Since 200 g of Takadiastase c o n t a i n about 2.5 x 10^ u n i t s of RNase a c t i v i t y , t h i s may then be considered a 630 f o l d p u r i -f i c a t i o n with 15.8$ recovery. Ribonuclease T_2 P u r i f i c a t i o n This p r e p a r a t i o n was c a r r i e d out by m o d i f i c a t i o n s to the procedure d e s c r i b e d by Rushizky and Sober (14). A. F i r s t P r e p a r a t i o n 1) A c i d E x t r a c t i o n 80 g of Takadiastase powder were mixed with 400 ml of d i s t i l l e d water and s t i r r e d at room temperature f o r two hours. The mixture was then cooled to 5° and 36N s u l f u r i c a c i d added to lower the pH to 0.6. A f t e r 48 hr the pH was r a i s e d to 5.3 with ammonium hydroxide and the whole s o l u t i o n was c e n t r i f u g e d f o r one hour at 1000 x g. 2) Acetone P r e c i p i t a t i o n The dark supernatant was added to 2.5 volumes of -16 acetone. The p r e c i p i t a t e was c o l l e c t e d and d i s s o l v e d i n 0.05M phosphate b u f f e r (pH 7.0). This was c e n t r i f u g e d at 8000 x g and the p r e c i p i t a t e d i s c a r d e d . 3) Sephadex G-25 Gel F i l t r a t i o n The 8000 x g supernatant was concentrated and passed through a 600 ml bed volume column of Sephadex G-25. The Se-phadex had been p r e v i o u s l y e q u i l i b r a t e d with 0.05M phosphate (pH 7.0) and was used i n s t e a d of the d i a l y s i s step recommended by Rushizky and Sober (14). 35 4) DEAE-Cellulose Chromatography - Column 1 The enzyme recovered from the Sephadex column was loaded d i r e c t l y onto a DEAE-cellulose (phosphate) column (3.0 x 80 cm). This column was e l u t e d using a t o t a l of 4 1 of e l u a n t i n a l i n e a r g r a d i e n t from 0.05M phosphate (pH 7.0) to 0.15M phosphate plus 0.2M sodium c h l o r i d e (pH 7.0). The a c t i v i t y was e l u t e d before the T^ but was not completely se-parated from i t . The volume of the T 2 f r a c t i o n was reduced to about 70 ml by using a r o t a r y evaporator at low temperature (30°C). 5) Sephadex G-75 Gel F i l t r a t i o n A column of Sephadex G-75 (4 x 40 cm) was p r e e q u i l i -b r a t e d with 0.05M phosphate (pH 7.0) and loaded with 75 ml of concentrated RNase T,,. The O.D.2gQ of the e f f l u e n t showed a small peak of a b s o r p t i o n followed by a much l a r g e r peak. RNase T 2 a c t i v i t y was a s s o c i a t e d only with the e a r l i e r peak ( F i g . 2). 6) DEAE-cellulose Chromatography - Column 2 V The a c t i v i t y e l u t e d o f f the Sephadex column was l o a d -ed d i r e c t l y on the second DEAE-cellulose column (3 x 80 cm). E l u t i o n was c a r r i e d out e x a c t l y as with the f i r s t DEAE column. In t h i s case the a c t i v i t y was e l u t e d i n a s p l i t peak, both por-t i o n s of which had i d e n t i c a l pH optima f o r a c t i v i t y . The whole r e g i o n of RNase a c t i v i t y was again reduced i n volume and sub-j e c t e d to d i a l y s i s a g a i n s t d i s t i l l e d water. 7) Attempted CM-cellulose Column Chromatography The d i a l y z e d T„ s o l u t i o n was loaded onto a CM-cellu-F i g u r e 2. Sephadex G-75 chromatography of RNase T 37 l o s e column (2.2 x 90 cm) which had p r e v i o u s l y e q u i l i b r a t e d w i t h 0.005M ammonium acetate (pH 4.35). I t was washed i n with one l i t r e of the above s o l u t i o n and g r a d i e n t e l u t i o n began to a l i m i t of 0.125M ( N H 4 ) 2 C 0 3 (pH 7.6). At t h i s p o i n t the ac-t i v i t y was l o s t and i t i s now f e l t t h a t i t was probably not adsorbed and washed through the column at the f r o n t . Second P r e p a r a t i o n 1) DEAE-Cellulose Column Chromatography A second p r e p a r a t i o n of RNase T 2 was c a r r i e d out i n c o n j u n c t i o n with the second RNase T^ p r e p a r a t i o n d e s c r i b e d p r e v i o u s l y . The RNase T 2 f r a c t i o n separated from T^ by g e l f i l t r a t i o n on Sephadex G-75 was chromatographed on DEAE-cellu-l o s e as d e s c r i b e d p r e v i o u s l y . The s p l i t peak of a c t i v i t y was again observed. 2) CM-Cellulose Column Chromatography - Column 1 A f t e r c o n c e n t r a t i o n and d i a l y s i s the enzyme was l o a d -ed onto a CM-cellulose column (3 x 90 cm) and washed i n as d e s c r i b e d p r e v i o u s l y . In t h i s c a s e the f r o n t was c o l l e c t e d and was found to c o n t a i n n e a r l y a l l of the p r o t e i n a p p l i e d to the column. A small peak of O.D.2gQ was e l u t e d much l a t e r and found to have a high s p e c i f i c a c t i v i t y . However, the m a j o r i t y (>»90%) of the a c t i v i t y was e l u t e d with the f r o n t . 3) CM-Cellulose Column Chromatography - Column 2 This m a t e r i a l which was not adsorbed was taken to a small volume on the r o t a r y evaporator and d i a l y z e d e x h a u s t i v e l y a g a i n s t running tap water, then a g a i n s t d i s t i l l e d water and 38 f i n a l l y a g a i n s t the s t a r t i n g b u f f e r (ammonium acetate - .005 M, pH 4.35). The pH of the enzyme s o l u t i o n was then checked (4.35) and i t was d i l u t e d with two volumes of the s t a r t i n g b u f f e r . I t was then loaded on the same, r e - e q u i l i b r a t e d CM-c e l l u l o s e column. This time a g r e a t e r p r o p o r t i o n of the ma-t e r i a l was adsorbed, but again, the m a j o r i t y of the RNase ac-t i v i t y passed through the column at the f r o n t ( F i g . 3). 4) S E - C e l l u l o s e Column Chromatography - Column 1 Since t h i s m a t e r i a l was not adsorbed i t was thought t h a t S E - c e l l u l o s e might adsorb the enzyme more f i r m l y . There-f o r e a small column (2 x 20 cm) was packed with S E - c e l l u l o s e and e q u i l i b r a t e d with 0.1M.acetic a c i d , adjusted to pH 3.5 with ammonium hydroxide. The enzyme s o l u t i o n was concentrated, d i -a l y z e d and made 0.1M i n a c e t i c a c i d , then adjusted to pH 3.5 w i t h ammonium hydroxide. I t was then loaded on the S E - c e l l u -l o s e column and washed thoroughly with the e q u i l i b r a t i n g s o l u -t i o n . A l i n e a r g r a d i e n t ( t o t a l v o l 1 l ) was a p p l i e d from the e q u i l i b r a t i n g s o l u t i o n to 0.6M ammonium carbonate (pH 7.8). The main peak of O'D^gQ an<^- RNase a c t i v i t y was e l u t e d very e a r l y i n the g r a d i e n t . However, the m a t e r i a l which washed through before the g r a d i e n t was s t a r t e d comprised about 40$ of the t o t a l O.D^gQ a p p l i e d , but l e s s than 5$ of the a c t i v i t y a p p l i e d . 5) S E - C e l l u l o s e Column Chromatography - Column 2 The most h i g h l y p u r i f i e d f r a c t i o n s e l u t e d from the two CM-cellulose columns and those from the S E - c e l l u l o s e column 39 40 were combined, concentrated and d i a l y z e d . They were then ap-p l i e d to a large S E - c e l l u l o s e column (3.3 x 90 cm) and washed i n with 0.05M ammonium acetate (pH 3.5) and e l u t e d with a 4 l i t r e l i n e a r pH and s a l t g r a d i e n t to .15M ammonium ac e t a t e , pH 4.5. The f r a c t i o n s of maximum s p e c i f i c a c t i v i t y were p o o l -ed and concentrated ( F i g . 4 ) . I t was t h i s f r a c t i o n which was used f o r the s y n t h e t i c substrate s t u d i e s . Determinations of the extent of p u r i f i c a t i o n and of the o v e r a l l y i e l d of t h i s enzyme;cannot be a c c u r a t e l y made be-cause of the f a r g r e a t e r q u a n t i t y of RNase T^ present i n the e a r l y stages. The most p u r i f i e d sample of t h i s enzyme pre-pared to date had a s p e c i f i c a c t i v i t y of 415 u n i t s per O.D^gQ u n i t . C h a r a c t e r i z a t i o n of RNase T^ A sample of high molecular weight yeast RNA was chro-matographed on Sephadex G-200 and that p o r t i o n which was e l u t e d a t the O.D.2£Q f r o n " f c w a s reserved f o r d i g e s t i o n with RNase T^. 18.5 ml of t h i s RNA ( c o n t a i n i n g 1650 O.D^^Q u n i t s ) were i n -cubated with 0.5 mg of RNase T^ at room temperature and the pH was maintained above 7.3 by the dropwise a d d i t i o n of 0.1N NaOH. A f t e r 9 hr at room temperature E. c o l i a l k a l i n e phosphatase was added to the s o l u t i o n and i n c u b a t i o n was continued at 37°C f o r 13 hours. At t h i s time h a l f of the m a t e r i a l was removed and the other h a l f l e f t to incubate f o r a f u r t h e r 24 hours. A f t e r each p o r t i o n was removed from the inc u b a t o r , potassium hydroxide was added to 0.3M and the s o l u t i o n s then 41 — A C T I V I T Y P R O T E I N — G R A D I E N T F R A C T I O N N U M B E R "S : :  F i g u r e 4. S E - c e l l u l o s e column chromatography of RNase 42 incubated f o r 24 hours at 37°C. Each was n e u t r a l i z e d with per-c h l o r i c a c i d and cooled to 0°C. Potassium p e r c h l o r a t e was cen-t r i f u g e d o f f and samples were submitted to high voltage e l e c -t r o p h o r e s i s on Whatman 31 paper. (0-.1M formate b u f f e r , pH 3.1, 83 vo l t s / c m ) . Standard samples of the four n u c l e o s i d e s and the four n u c l e o t i d e s were a l s o a p p l i e d as markers. The electrophoretograms showed the presence of AMP, CMP, and UMP as w e l l as guanosine i n the two d i g e s t i o n mixtures. These spots were cut out and e l u t e d , as were the regions where G-MP -and the 3 other nucleosides were expected to run. Spectra of a l l 8 spots i n both experiments showed no GMP, -no adenosine, c y t i d i n e or u r i d i n e . In contrast,, the spots corresponding to guanosine, AMP, UMP and CMP contained from 7.5 to 9 O.D. u n i t s each. On t h i s b a s i s i t can s a f e l y be assumed t h a t greater than 99$ of the h y d r o l y s i s c a t a l y z e d by the RNase T^ prepara-t i o n occured d i s t a l to guany l i c a c i d r e s i d u e s , and that greater than 99$ of a l l l i n k a g e s d i s t a l to gu a n y l i c a c i d were h y d r o l -yzed. C h a r a c t e r i z a t i o n of RNase T^ Measurements were made of the rates of h y d r o l y s i s t i of adenosine 3 -phosphate p r o p y l e s t e r , u r i d i n e 3 -phosphate p r o p y l e s t e r and the two purine n u c l e o s i d e c y c l i c phosphates by the p r e v i o u s l y d e s c r i b e d chromatographic method. The spec-t r o p h o t o m e t r y method was used to f o l l o w the h y d r o l y s i s of the pyrimidine n u c l e o s i d e c y c l i c phosphates. In the l a t t e r case the r e a c t i o n s followed pseudo 43 f i r s t order k i n e t i c s but i n no case d i d the chromatographic data give an i n t e g r a l order. I t was shown that RNase 1^ would hydrolyze a l l four i » n u c l e o s i d e 2 ,3 - c y c l i c phosphates but comparative r a t e s and Km values have not y e t been determined. H y d r o l y s i s of u r i d i n e t i » » 2 ,3 - c y c l i c phosphate i s f a s t e r than c y t i d i n e 2 ,3 - c y c l i c phosphate and c y c l i c a d e n y l i c a c i d was hydrolyzed f a s t e r than i c y c l i c g u a n y l i c . Adenosine 3 -phosphate pro p y l e s t e r was t r a n s e s t e r i f i e d to the c y c l i c phosphate and then hydrolyzed i f a s t e r than u r i d i n e - 3 — p h o s p h a t e p r o p y l e s t e r . P r e l i m i n a r y s t u d i e s of d i v a l e n t c a t i o n i n h i b i t i o n have shown 0.001M c u p r i c s u l f a t e to be a very potent i n h i b i t o r i i of RNase T 2, at l e a s t f o r the h y d r o l y s i s of u r i d i n e 2 ,3 -c y c l i c phosphate. The same c o n c e n t r a t i o n of magnesium c h l o r i d e brought about l e s s than 15$ i n h i b i t i o n of the same r e a c t i o n . 44 DISCUSSION Substrate Syntheses t i A. Nucleoside 2 ,3 - c y c l i c Phosphates The method used i n the e a r l i e r work (41) gave s a t -i s f a c t o r y y i e l d s of a l l four c y c l i c phosphates but was incon-v e n i e n t i n that formamide was used as the s o l v e n t f o r the r e a c t i o n and could not be removed except by chromatography on DEAE-cellulose, or e l s e by p r e c i p i t a t i n g the c y c l i c phosphates from acetone with barium i o d i d e . The modified procedure of Smith and Khorana (42) d i d not use formamide and was t h e r e f o r e more convenient, but was of l i t t l e value f o r c y c l i z a t i o n of g u a n y l i c a c i d since t h i s compound was too i n s o l u b l e i n metha-n o l , the s o l v e n t used, to r e a c t with the DCC. B. Nucleoside 2 (3 )-phosphate P r o p y l E s t e r s Synthesis of the mixed isomers by the h y d r o c h l o r i c a c i d c a t a l y z e d t r a n s e s t e r i f i c a t i o n method of Tener and Khorana (40) was i n a l l cases s u c c e s s f u l and gave y i e l d s of greater t ! than 60%. S e p a r a t i o n o f t h e i s o m e r s o f a d e n o s i n e 2 (3 )-phos-phate p r o p y l e s t e r s was accomplished r e a d i l y on D E A E - c e l l u l o s e , but the s e p a r a t i o n of the guanosine d e r i v a t i v e s was more d i f -f i c u l t . The chromatographic methods used were incapable of s e p a r a t i n g the isomeric p r o p y l e s t e r s of c y t i d y l i c and u r i d y -i l i e a c i d s . However, the pure 3 -phosphate p r o p y l e s t e r s of the pyrimidine n u c l e o s i d e s could be synthesized e n z y m a t i c a l l y , but due to the absolute requirement f o r some water i n the i n -cubation mixture only r e l a t i v e l y low y i e l d s of e s t e r were ob-45 t a i n e d . RNA P r e p a r a t i o n The method of C r e s t f i e l d e_t a l . (43) i s adequate f o r p r e p a r i n g RNA s u i t a b l e f o r an a n a l y t i c a l s u b s t r a t e . The i n -t r o d u c t i o n of a phenol e x t r a c t i o n step ensures the removal of a l l r i b o n u c l e a s e s which may contaminate and degrade the pre-p a r a t i o n (22). In a d d i t i o n i t was found that combining t h i s phenol e x t r a c t i o n step with f i l t r a t i o n through a C e l i t e bed makes u l t r a c e n t r i f u g a t i o n unnecessary. P a n c r e a t i c RNase The p r e l i m i n a r y s t u d i e s c a r r i e d out on p a n c r e a t i c RNase using n u c l e o s i d e 2 (3 )-phosphate pro p y l e s t e r s showed 1 the need f o r pure 3 -isomers. In no case was i t p o s s i b l e to o b t a i n zero or f i r s t order k i n e t i c s f o r the disappearance of the s u s c e p t i b l e isomer. This was due to competitive i n h i b i -t i o n by the 2 -isomer which was present as about 36$ of the 1 t o t a l e s t e r c o n c e n t r a t i o n , and by the product, n u c l e o s i d e 3 -phosphate. The k i n e t i c s of the t r a n s e s t e r i f i c a t i o n r e a c t i o n are d i f f i c u l t to f o l l o w because of the presence of the com-p e t i n g h y d r o l y t i c r e a c t i o n . F u r t h e r , t h i s competing h y d r o l y t i c 1 i r e a c t i o n makes determination of nu c l e o s i d e 2 ,3 - c y c l i c phos-phate not meaningful f o r k i n e t i c s t u d i e s so a l l t h a t can be measured i s the o v e r a l l two step r e a c t i o n . The i n a b i l i t y of both the A and B f r a c t i o n s of p a n c r e a t i c RNase to c a t a l y z e hy-d r o l y s i s of the p r o p y l phosphates of adenosine and guanosine d i s a g r e s s with the repo r t e d n o n - s p e c i f i c i t y . o f these f r a c t i o n s 46 (6) and i s i n agreement with the g e n e r a l l y accepted pyrimidine s p e c i f i c i t y of p a n c r e a t i c RNase. Mann Diastase The nature of the enzyme or enzymes present i n the A. oryzae d i a s t a s e p r e p a r a t i o n from Mann L a b o r a t o r i e s has not y e t been e s t a b l i s h e d . The p o s s i b i l i t y t h a t RNase 1^ or a very s i m i l a r enzyme i s present i s s t r o n g l y i n d i c a t e d by the obser-v a t i o n t h at a chromatographically p u r i f i e d p r e p a r a t i o n was able t i to break down a l l four n u c l e o s i d e 2 (3 )-phosphate p r o p y l es-t e r s . Compared with Japanese Takadiastase t h i s d i a s t a s e pre-p a r a t i o n has a very low l e v e l of RNase a c t i v i t y . In c o n t r a s t to the l a c k of s p e c i f i c i t y of the p a r t i a l l y p u r i f i e d enzyme i i t was observed t h a t a crude e x t r a c t hydrolysed adenosine 2 (3 )-phosphate p r o p y l e s t e r s more r a p i d l y than the correspond-i n g pyrimidine d e r i v a t i v e s while not causing d e t e c t a b l e break-down of the guanosine e s t e r s . This suggested that an i n h i b i -t o r may have been present which was removed e i t h e r by the ad-d i t i o n of EDTA or by the DEAE-cellulose chromatography. Such an i n h i b i t o r might be very u s e f u l f o r s t r u c t u r a l s t u d i e s of s-RNA i f i t could be shown to completely i n h i b i t h y d r o l y s i s d i s t a l to one or more d i f f e r e n t n u c l e o t i d e r e s i d u e s . Future work on the p u r i f i c a t i o n of t h i s enzyme and on the nature of i t s i n h i b i t o r s i s proposed, RNase T^ P u r i f i c a t i o n RNase T^ i s a very s t a b l e enzyme and can be q u i t e s a f e l y subjected to heat (80°C), a c i d (below pH l ) , phenol, 47 and c o n c e n t r a t i o n on a r o t a r y evaporator at 30°C with l i t t l e d e t e c t a b l e l o s s of a c t i v i t y . In i t s p u r i f i c a t i o n use was made of these s t a b i l i t y p r o p e r t i e s . In the f i r s t p r e p a r a t i o n of RNase T^, u s i n g a pro-cedure d e r i v e d from t h a t of Takahashi (10), a y i e l d of 15.5 mg of RNase T^ was obtained from 80 g of Takadiastase. This compares q u i t e f a v o r a b l y with h i s r e p o r t e d y i e l d of about 100 mg from a kilogram of Takadiastase (10). The two preparations had approximately the same s p e c i f i c a c t i v i t y . In a more r e -cent p u b l i c a t i o n Takahashi rep o r t e d an increase i n h i s average y i e l d of RNase T^ to about 150 mg per k i l o ( l l ) . M o d i f i c a t i o n s of Takahashi's procedure i n c l u d e d the s u b s t i t u t i o n of acetone f r a c t i o n a t i o n f o r ammonium s u l f a t e f r a c t i o n a t i o n , a step i n which he l o s t between 20 and 25$ of h i s t o t a l RNase a c t i v i t y . The use of phenol to e x t r a c t RNase T^ from aqueous s o l u t i o n was reported by Rushizky e_t a l . (22) to be an e f f e c t i v e method f o r c o n c e n t r a t i n g and d e s a l t i n g t h i s , and other enzymes. In the present work phenol e x t r a c t i o n was used i n s t e a d of d i a l y s i s to avoid the l o s s e s of a c t i v i t y encountered i n d i a l y s i s as a r e s u l t of the small s i z e of the p r o t e i n . Since no l a r g e s c a l e method has y e t been d e s c r i b e d f o r the removal of RNase T^ from the phenol the use of ether to f o r c e the enzyme i n t o a s l i g h t l y b a s i c s o l u t i o n was i n v e s t i g a t e d . In general t h i s was f a i r l y e f f e c t i v e and gave a good recovery of a c t i v i t y when the d i e t h y l ether used was p r e v i o u s l y t r e a t e d with calcium hydride to des-t r o y peroxides (44). The t h i r d DEAE-cellulose column chromat-48 ography was not c a r r i e d out by Takahashi but i n t h i s work doubled the s p e c i f i c a c t i v i t y of the RNase T^ p r e p a r a t i o n . A l s o the s i z e s of columns and the volumes of e l u t i o n gradients used by Takahashi were much smaller than those r e p o r t e d here. The second RNase T^ - p r e p a r a t i o n was c a r r i e d out by a procedure modified from that of Rushizky and Sober (12). This p r e p a r a t i o n r e s u l t e d i n the recovery of about 50 mg of RNase T^ with a lower s p e c i f i c a c t i v i t y than the f i r s t . T h i s y i e l d i s about 75% of that r e p o r t e d by Rushizky and Sober but due to d i f f e r e n t RNase assay procedures the s p e c i f i c a c t i v i -t i e s cannot be compared. A d e f i n i t e improvement over Taka-h a s h i ' s procedure i s the low temperature a c i d e x t r a c t i o n used by the former workers. E l i m i n a t i o n of the heat step and the use of even lower pH produces an i n c r e a s e i n the.-specif i c ac-t i v i t y of the p r e p a r a t i o n without undue l o s s of t o t a l a c t i v i t y . Gel f i l t r a t i o n on Sephadex G-75 p r i o r to DEAE-cellulose chro-matography was i n t r o d u c e d to e l i m i n a t e low molecular weight m a t e r i a l s and to separate RNase from T^. This now seems' to be i m p r a c t i c a l at t h i s stage i n the p u r i f i c a t i o n because of the bulk of the s o l u t i o n a p p l i e d and i t s v i s c o s i t y . Phenol e x t r a c t i o n a f t e r the f i r s t DEAE-cellulose column chromatography was l e s s s u c c e s s f u l than i n the previous experiment, and consequently was the p o i n t of major l o s s of a c t i v i t y during t h i s p r e p a r a t i o n . The homogeneity of the most p u r i f i e d p r e p a r a t i o n of RNase T, i s i n d i c a t e d by the narrow band of a c t i v i t y e l u t e d 49 from DEAE-cellulose which c o i n c i d e d with a sharp peak of pro-t e i n ( F i g . 1). Future p r e p a r a t i o n s of t h i s enzyme would probably be best c a r r i e d out by using column chromatography on Sephadex G-25 to remove s a l t s between DEAE-cellulose chromatography steps. RNase P r e p a r a t i o n The p u r i f i c a t i o n of RNase T 2 n a S n o ^ progressed to the s t a t e where the p r e p a r a t i o n can be considered as c o n s i s t -i n g p r i m a r i l y of a s i n g l e molecular s p e c i e s . The presence of an overwhelming amount of RNase T^ i n the s t a r t i n g m a t e r i a l makes e s t i m a t i o n of the p u r i f i c a t i o n f a c t o r impossible. The use of a d i f f e r e n t assay procedure by Rushizky and Sober (14) makes comparison of s p e c i f i c a c t i v i t i e s d i f f i c u l t . The phenol e x t r a c t i o n technique, used s u c c e s s f u l l y f o r d e s a l t i n g of RNase T^ pr e p a r a t i o n s could not be a p p l i e d to the T 2 p u r i f i c a t i o n because i t caused complete l o s s of ac-t i v i t y . However, g e l f i l t r a t i o n of RNase T 2 preparations concentrated from i o n exchange column e f f l u e n t s proved very s a t i s f a c t o r y , both f o r d e s a l t i n g and f o r removal of lower molecular weight m a t e r i a l (when Sephadex G-75 was used). As mentioned p r e v i o u s l y the e l u t i o n p a t t e r n of RNase T 2 obtained on DEAE-cellulose chromatography showed the enzyme to be e l u t e d as a double peak of a c t i v i t y . T h i s has been ob-served p r e v i o u s l y but as y e t no explanations have been ad-vanced (10,14). S i m i l a r observations have been made with pan-c r e a t i c RNase when chromatographed on CM-cellulose or XE-64 50 ion-exchange r e s i n . This has r e c e n t l y been shown to be due to sev.eral forms of the enzyme, a l l but one of which co n t a i n s e v e r a l g l y c o s y l residues (45). CM-cellulose chromatography of RNase T 2, using es-s e n t i a l l y the same c o n d i t i o n s as those reported by Rushizky and Sober (14) was u n s u c c e s s f u l i n every attempt made. In each case most or a l l of the a c t i v i t y passed through the c o l -umn without being adsorbed. In two cases small q u a n t i t i e s were adsorbed and e l u t e d i n a much more h i g h l y p u r i f i e d form but the q u a n t i t i e s adsorbed were too small to make the procedure p r a c t i c a l . The reason f o r i t s non-adsorption may have been i o n i c bonding to an a c i d i c m a t e r i a l present i n the mixture. To circumvent t h i s p o s s i b i l i t y a lower pH was t r i e d . The pKa of p r o t e i n carboxyl groups i s s u f f i c i e n t l y high that at pH 3.5 t h e i r i o n i z a t i o n should be depressed and bi n d i n g of a c i d i c p r o t e i n s to RNase T 2 should be reduced. Since t h i s pH would a l s o depress the i o n i z a t i o n of CM-cellulose the more s t r o n g l y a c i d i c i o n - e x c h a n g e r , S E - c e l l u l o s e , was used. A d s o r p t i o n of n e a r l y a l l of the RNase T 2 a c t i v i t y a p p l i e d was accompanied by e l i m i n a t i o n of a s i g n i f i c a n t q u a n t i t y of i n a c t i v e contam-i n a t i n g m a t e r i a l . Further work on the p u r i f i c a t i o n of t h i s enzyme w i l l be c a r r i e d out u s i n g s t a r c h - g e l e l e c t r o p h o r e s i s (46). Com-p l e t e s e p a r a t i o n of the two isomeric forms of RNase T 2 ' w i l l ' a l s o be attempted u s i n g DEAE-cellulose column chromatography. 51 C h a r a c t e r i z a t i o n of RNase The method used for. t h i s determination of s p e c i f i c i t y of RNase T^ i n v o l v e d complete d i g e s t i o n of high molecular weight yeast RNA with RNase T^, followed by E. c o l i phospho-monoesterase treatment and then complete h y d r o l y s i s with a l -k a l i . The f i n d i n g of guanosine as the only n u c l e o s i d e present and no guanylic a c i d i n d i c a t e s complete d i g e s t i o n by the enzyme and confirms the r e p o r t e d absolute s p e c i f i c i t y f o r those l i n k -ages d i s t a l to g u a n y l i c a c i d r esidues (12). A s i m i l a r method was used by McCully and Cantoni (38) f o r determination of sus-c e p t i b l e bonds i n s-RNA which contains 1-methyl gua n y l i c a c i d and N,N-dimethyl-2-amino-6-hydroxy purine n u c l e o t i d e as minor c o n s t i t u e n t s . They found that both of these were present i n the complete d i g e s t as the n u c l e o t i d e s . Prom t h i s they con-cluded that RNase T^ would not a t t a c k linkages i n v o l v i n g these methylated bases. T h i s experiment was not c o n c l u s i v e though because i f the t r a n s e s t e r i f i c a t i o n step had occured to produce a 2 ,3 - c y c l i c phosphate which was r e s i s t a n t to h y d r o l y s i s then the same r e s u l t s would be found. The p o s s i b i l i t y t h a t t h i s may have been the case f o l l o w s from experiments of ¥hitfeld and "Witzel who showed t r a n s e s t e r i f i c a t i o n of d i n u c l e o s i d e phosphates i n v o l v i n g g l y o x a l guanosine but no h y d r o l y s i s of the r e s u l t i n g n u c l e o s i d e 2 ,3 - c y c l i c phosphate (36). The non-c r i t i c a l nature of McCully and Cantoni's experiments makes necessary a re-examination of t h i s problem. More meaningful i n f o r m a t i o n would be obtained i f the phosphomonoesterase d i -g e s t i o n were c a r r i e d out i n the presence of an enzyme such as t h a t d e s c r i b e d by Drummond e_t a J . (47) which hydrolyzes a l l nu-• i i c l e o s i d e 2 ,3 - c y c l i c phosphates forming the 2 -phosphates. In i i t h i s case r e s i s t a n c e of a 2 ,3 - c y c l i c ended o l i g o n u c l e o t i d e to phosphomonoesterase would be overcome and a f t e r a l k a l i n e h y d r o l -y s i s an a d d i t i o n a l q u a n t i t y of n u c l e o s i d e would be present. T h i s experiment w i l l be attempted soon because a d e f i n i t e ans-wer to t h i s problem w i l l be r e q u i r e d before the T^ enzyme can be used f o r s t r u c t u r a l s t u d i e s of s-RNA. Accurate q u a n t i t a t i v e measurements of the r a t e of hy-t i d r o l y s i s of guanosine 2 ,3 - c y c l i c phosphate must a l s o be made si n c e i t has been r e p o r t e d t h a t t h i s h y d r o l y t i c step i s much slower than the t r a n s e s t e r i f i c a t i o n step. T i t r a t i o n measure-ments of t h i s h y d r o l y s i s may be not only a simpler but a l s o a more accurate method f o r f o l l o w i n g t h i s r e a c t i o n than the chro-matographic method. C h a r a c t e r i z a t i o n of RNase T^ The e a r l i e r reported s p e c i f i c i t y of RNase f o r bonds d i s t a l to a d e n y l i c a c i d residues (13) has been more r e c e n t l y shown to be i n c o r r e c t (14). I t i s now thought t h a t RNase T 2 i d i g e s t i o n of RNA br i n g s about complete h y d r o l y s i s to the 3 -mononucleotide l e v e l . The present s t u d i e s with the most h i g h l y p u r i f i e d p r e p a r a t i o n of RNase T 2 confirmed t h i s l a c k of base i i s p e c i f i c i t y by demonstrating that a l l four nucleoside 2 ,3 -i c y c l i c phosphates are hydrolyzed to the 3 -phosphates. In ad-i d i t i o n i t has been demonstrated that adenosine 3 -phosphate i p r o p y l e s t e r and u r i d i n e 3 -phosphate pro p y l e s t e r are broken 53 • i t down, v i a the 2 ,3 - c y c l i c phosphates, to 3 -mononucleotides. This ob s e r v a t i o n i s contr a r y to t h a t of Naoi-Tada ejb a l . (13) who were unable to demonstrate the fornmtion of c y c l i c phos-phate intermediates i n T 2 c a t a l y z e d h y d r o l y s i s of p o l y - a d e n y l i c a c i d . In p r e l i m i n a r y s t u d i e s of r a t e s of h y d r o l y s i s of nuc-i t l e o s i d e 2 ,3 - c y c l i c phosphates the adenosine d e r i v a t i v e was found to be opened at l e a s t twice as f a s t as the guanosine t d e r i v a t i v e . In d i g e s t i o n s of adenosine 3 -phosphate propyl e s t e r the c y c l i c intermediate was found, but i t d i d not accum-u l a t e to the same extent ad the corresponding c y c l i c phosphate i n the d i g e s t i o n s of u r i d i n e 3 -phosphate pro p y l e s t e r . As d i s c u s s e d e a r l i e r , there are a number of l i m i t a -t i o n s to procedures f o r f o l l o w i n g these r e a c t i o n s . I n h i b i t i o n by the 2 - e s t e r was e l i m i n a t e d from these s t u d i e s by using the i pure 3 - p r o p y l e s t e r s which were obtained e i t h e r by p u r i f i c a -t i o n on chromatographic columns or by enzymatic s y n t h e s i s . At present there i s no means of a c c u r a t e l y measuring the rate of the t r a n s e s t e r i f i c a t i o n step i n d e p e n d a n t of the h y d r o l y t i c step. E l e c t r o p h o r e t i c s e p a r a t i o n of d i n u c l e o s i d e phosphate d i g e s t i o n s , as repo r t e d by W h i t f e l d and "Witzel (36) f o r RNase T^, could give some in f o r m a t i o n on the f i r s t step r e a c t i o n , but only i f the r a t e of t h i s r e a c t i o n were much grea t e r than the r a t e of the second step. Even i n t h i s case the only v a l i d r e a c t i o n r a t e measurements would be those made very e a r l y i n the r e a c t i o n , before the h y d r o l y s i s of the c y c l i c phosphates formed becomes an important competing r e a c t i o n . I f the hydro-54 l y t i c r e a c t i o n was o c c u r r i n g simultaneously with the t r a n s e s -t e r i f i c a t i o n r e a c t i o n the r e s u l t would be a decreased q u a n t i t y of enzyme a v a i l a b l e f o r c a t a l y s i s of t r a n s e s t e r i f i c a t i o n . The reason f o r t h i s decrease would be the existence of two d i f f e r -ent types of enzyme-substrate-complex. In the h y d r o l y t i c step p r e l i m i n a r y experiments i n d i -cate t h a t r e l i a b l e q u a n t i t a t i v e data may be obtained by the d i r e c t spectrophotometric method, at l e a s t f o r the measurement i i of h y d r o l y s i s of the pyrimidine n u c l e o s i d e 2 ,3 - c y c l i c phos-i phates. The s p e c t r a l d i f f e r e n c e s between the 3 -phosphates and i t 2 ,3 - c y c l i c phosphates of purine n u c l e o s i d e s are too small to i be a c c u r a t e l y measured. In the case of the pyrimidine 3 -nuc-l e o t i d e s the s p e c t r a l d i f f e r e n c e i s probably due to hydrogen t bonding between the 2 -hydroxyl and the 2-oxygen s u b s t i t u e n t on the p y r i m i d i n e r i n g . This b i n d i n g cannot occur i n the c y c l i c phosphates. S i m i l a r s p e c t r a l d i f f e r e n c e s should also occur be-i t t tween the pyrimidine n u c l e o s i d e s and.their 2 - or 2 ,3 - c y c l i c p h o s p h a t e s . U n f o r t u n a t e l y , t i t r a t i o n cannot be used to f o l l o w the h y d r o l y t i c r e a c t i o n because the pH optimum f o r RNase T^ i s 4.5 and at t h i s pH the l i b e r a t e d phosphate groups are u n d i s s o c i a t e d (pKa 6-7). F i n a l l y , a number of d i v a l e n t c a t i o n s should be t e s t -ed f o r t h e i r a b i l i t y to i n h i b i t the t r a n s e s t e r i f i c a t i o n and hy-i d r o l y s i s of e s t e r s of a l l f o u r 3 - n u c l e o t i d e s . I f a d i f f e r e n -t i a l i n h i b i t i o n could be demonstrated then p o s s i b l y the T„ en-zyme would be of value i n s-RNA s t r u c t u r a l s t u d i e s . The pre-l i m i n a r y observations on the e f f e c t of EDTA on the crude enzyme from the Mann d i a s t a s e o f f e r some hope f o r the success of these s t u d i e s . As a r e s u l t of time l i m i t a t i o n s more extensive stud-i e s were not c a r r i e d out but the p r e l i m i n a r y r e s u l t s reported here are to be extended. 56 SUMMARY I I t 1. Nucleoside 2 ,3 - c y c l i c phosphates and nucl e o s i d e 3 -phosphate p r o p y l e s t e r s have been synthesized f o r use as substrates i n s t u d i e s on r i b o n u c l e a s e s . 2. Ribonuclease has been h i g h l y p u r i f i e d from Takadiastase and s t u d i e s on i t s s p e c i f i c i t y have been c a r r i e d out. 3. Ribonuclease T 2 has been e x t e n s i v e l y p u r i f i e d and p r e l i m i n a r y s t u d i e s have been c a r r i e d out on i t s r e l a v i v e a c t i v i t i e s on d i f f e r e n t s y n t h e t i c s u b s t r a t e s . 4. A r i b o n u c l e a s e has been p a r t i a l l y p u r i f i e d from an A. oryzae d i a s t a s e e x t r a c t and some of i t s p r o p e r t i e s are r e p o r t e d . 57 BIBLIOGRAPHY M. K u n i t z , J . Gen. P h y s i o l . 24, 15 (1940). G. Schmidt, R. C u b i l e s , N. Z o l l n a r , L. Hecht, N. S t r i c k -l e r , K. S a r a i d a r i a n , M. S e r a i d a r i a n , and S. J . Thannhauser J . B i o l . Chem. 192, 715 (1951). R. Markham and J . D. Smith, Biochem. J . 52, 552 (1952). D. M. Brown and A. R. Todd, J . Chem. Soc. 1953, 2040. E. V o l k i n and W. E. Cohn, J . B i o l . 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