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UBC Theses and Dissertations

Partial purification and characterization of dnase I from the intestinal mucosa of rat Frizell, John William 1973

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0 • • PARTIAL PURIFICATION AND CHARACTERIZATION OP DNASE I PROM THE INTESTINAL MUCOSA OP RAT by JOHN WILLIAM PRIZELL B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1969 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OP MASTER OP 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 f o r the degree of Master of Science THE UNIVERSITY OF BRITISH COLUMBIA September, 1973 In presenting 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 permission 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 T The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada i ABSTRACT DNase I a c t i v i t y has been found In the s m a l l i n t e s -t i n e o f the r a t . T h i s work i n v o l v e s a p a r t i a l p u r i f i -c a t i o n and c h a r a c t e r i z a t i o n o f t h i s enzyme. Crude enzyme e x t r a c t was prepared by u l t r a c e n t r l -f u g a t i o n o f the homogenate of washed mucosal s c r a p i n g s . The DNase I a c t i v i t y o f the crude enzyme p r e p a r a t i o n was n o t s t a b l e , i t c o u l d be s t a b i l i z e d by d i v a l e n t metal Ions, The crude enzyme e x t r a c t was chromatographed on DEAE c e l l u l o s e . i n phosphate b u f f e r and the adsorbed enzyme e l u t e d w i t h a phosphate g r a d i e n t . The crude enzyme was "shown to c o n t a i n p r o t e o l y t i c enzymes. The a c t i v e material.was f r e e z e - d r i e d , r e d i s s o l v e d f r e e d o f phosphate and chromatographed on Sephadex G-100. The a c t i v e m a t e r i a l e l u t e d from the Sephadex column was adsorbed on DEAE c e l l u l o s e and e l u t e d w i t h a l i n e a r g r a d i e n t o f phosphate. T h i s procedure gave a p u r i f i -c a t i o n o f 200-400 f o l d , r e l a t i v e to the crude enzyme e x t r a c t . The pro d u c t was not s t a b l e i n the absence o f C a + + and c o n t a i n e d p r o t e o l y t i c enzymes. The m o l e c u l a r weight of the enzyme was estimated as 3.05 x 10^ d a l t o n s by g e l f i l t r a t i o n on Sephadex G-100. The p r o p e r t i e s o f the r a t enzyme were compared to those o f the bovine enzyme. No s i g n i f i c a n t d i f f e r e n c e i l was found in molecular-weight, inhibition by Na"1", K , haemoglobin or iodoacetate, pH optimum, or metal ion requirements. The interactions of C a + + with the DNA-DNase system were explored. In the presence of Mg++, C a + + f i r s t activates and then inhibits DNase activity. ! I i i ACKNOWLEDGEMENTS The author would like to thank Dr. Rozanne Poulson for her advice and encouragement and Dr. George Krasny for placing many gradients on many columns. Sincere thanks are due to Dr. Zbarsky for advice throughout the course of this research project. iv TABLE OF CONTENTS INTRODUCTION A) Properties of DNase I 1 B) Kinetics of DNase 1 7 C) Linkage Specificity. 10 D) Protein Inhibitor. 11 E) Biological Role 12 MATERIALS AND METHODS A) DNase Assays 1) Kunitz Assay.... .....15 2) Acid Soluble Oligonucleotide Assay 16 3) Gel Assay 19 B) Preparation of Crude Enzyme Extract .21 C) Purification of DNase I 1) Column Chromatography on DEAE Cellulose....23 2) Removal of Phosphate 2k 3) Column Chromatography on G-100 Sephadex....25 h) Rechromatography on DEAE Cellulose.........26 D) Molecular Weight Determination on G-100 Sephadex 26 E) Polyacrylamlde Gel Electrophoresis 28 P) Determination of pH Optimum. .28 G) Ammonium Sulfate Fractionation. 28 V H) D e t e r m i n a t i o n o f t h e n a t u r e o f t h e p r o d u c t o f h y d r o l y s i s a) S e p a r a t i o n o f o l i g o n u c l e o t i d e 29 b) P h o s p h a t e a s s a y 31 I ) A s s a y f o r DNase I i n h i b i t o r RESULTS AND DISCUSSION A) P r e p a r a t i o n o f C r u d e Enzyme E x t r a c t 32 B) S t a b i l i t y o f DNase I P r e p a r a t i o n s . . . 33 C) P u r i f i c a t i o n o f DNase I a) C h r o m a t o g r a p h y o n DEAE C e l l u l o s e 38 b) R e m o v a l o f P h o s p h a t e 38 c) C h r o m a t o g r a p h y o n S e p h a d e x G-100. . .... . 38 d) R e c h r o m a t o g r a p h y o n DEAE C e l l u l o s e ....41 D) P r o p e r t i e s o f DNase I 44 E) P r o d u c t s o f h y d r o l y s i s 48 F) C h a r a c t e r i z a t i o n o f t h e Enzyme.......... 51 G) Ammonium S u l f a t e F r a c t i o n a t i o n 53 H) M o l e c u l a r W e i g h t 53 I ) G e l E l e c t r o p h o r e s i s 57 J ) I n t e r a c t i o n b e t w e e n c a l c i u m i o n a n d DNase I . » . . 64 CONCLUSION 73 BIBLIOGRAPHY 76 v i TABLES 1) Weight of ammonium s u l f a t e used i n f r a c t i o n a t i o n 29 2) T y p i c a l r e s u l t s of a t h r e e step p u r i f i c a t i o n of DNase I 42 3) D i g e s t i o n of n u c l e o t i d e s w i t h 5 ' n u c l e o t i d a s e . • • • • 50 4) DNase a c t i v i t y o f p r o t e i n s s u b j e c t e d t o g e l e l e c t r o p h o r e s i s 59 5) R e l a t i v e i n t e n s i t i e s o f p r o t e i n bands on g e l e l e c t r o p h o r e s i s 59 6) Values f o r K±, the i n h i b i t o r c o n s t a n t f o r Ca ++ a c t i n g as a c o m p e t i t i v e i n h i b i t o r ..69 v i i FIGURES 1) The complete amino acid sequence of Bovine pancreatic DNase I 6 2) Comparison of the Kunitz assay and the the acid soluble oligonucleotide assay... 18 3) Comparison of the gel assay and the acid acid soluble oligonucleotide assay..... 20 4) I n h i b i t i o n of DNase assays by phosphate 22 5) Time course of loss of DNase I a c t i v i t y from crude enzyme extract 34 6) Column chromatography on DEAE c e l l u l o s e 37 7) Column chromatography on Sephadex G-100......... 40 8) Rechromatography on DEAE c e l l u l o s e 43* 9) pH optimum of r a t DNase I 45 10) Ac t i v a t i o n of DNase I by metal ions ..46 11) I n h i b i t i o n of DNase I a c t i v i t y by Na +, K + and haemoglobin 47 12) Time course of DNase I action on DNA. ...49 13) Ammonium su l f a t e f r a c t i o n a t i o n ...54 14) Four o p t i c a l density traces........... .....60 V l l l 15) Two o p t i c a l d e n s i t y t r a c e s .....63 16) Lineweaver Burk p l o t 66 17) P l o t o f a g a i n s t the c o n c e n t r a t i o n o f c a l c i u m 67 18) S t i m u l a t i o n o f DNase I by C a + + i n the presence o f 1 mM Mg + +.. .68 19) S t i m u l a t i o n o f DNase I by M g + + i n the presence of 5 mM C a + + 71 1 INTRODUCTION U n t i l r e c e n t l y , the p r o t e i n chemistry of DNase I was unknown because of the d i f f i c u l t i e s encountered i n pre-p a r i n g h i g h l y p u r i f i e d samples of the enzyme. Thus, as l a t e as 196? molecular weight estimates v a r i e d between 60,000 and 33,000 daltons w h i l e the amino a c i d composition of the enzyme remained unknown. Germann and Okada (1) estimated a minimum molecular weight of 60,000 d a l t o n s by amino a c i d a n a l y s i s , but the commercial DNase I p r e p a r a t i o n used was l a t e r shown (2) to be contaminated w i t h 4 o r 5 other p r o t e i n s . The f i r s t accurate amino a c i d a n a l y s i s was p u b l i s h e d by Lindberg (3) i n 1967 and confirmed by Moore and S t e i n (4-) i n 1969* L i a o et a l (11) have sequenced the enzyme. Based on the amino a c i d and carbohydrate composition the molecular weight has been e s t a b l i s h e d as 30,072 d a l t o n s . Moore, S t e i n and coworkers have made major c o n t r i -b u t i o n s towards e l u c i d a t i n g the p h y s i c a l and chemical p r o p e r t i e s of DNase I . Salnikow, Moore and S t e i n (5) chromatographed p a n c r e a t i c DNase I on phosphocellulose i n a c e tate b u f f e r a t pH 4.7 and separated DNase I into three f r a c t i o n s ! A, B, and C. These f r a c t i o n s d i f f e r e d i n number and type of sugar groups attached and i n amino 2 a c i d content. DNase C contains one l e s s h i s t i d i n e and one more p r o l i n e than DNase A o r B. The same workers have a l s o detected a DNase D i h sm a l l amounts. Moore, S t e i n and coworkers (4) have demonstrated tha t the f o u r h a l f c y s t i n e s l n DNase I are present as S-S not S-H. They showed t h a t the enzyme a l s o contains sugar residues and i s t h e r e f o r e a g l y c o p r o t e i n . The two d i s u l p h i d e bonds were found (6) to be extremely s u s c e p t i b l e to r e d u c t i o n . A b r i e f exposure to 0.05 M mercaptoethanal a t room temperature caused complete r e d u c t i o n and i n a c t i v a t i o n of the enzyme. To achieve a s i m i l a r s t a t e of r e d u c t i o n w i t h most p r o t e i n s , a denaturing agent such as 7 M urea i s r e q u i r e d . Once formed, the reduced enzyme i s very stable? l e s s than $% of the reduced enzyme i s r e a c t i v a t e d when i t i s st o r e d f o r 24 hours under ae r o b i c c o n d i t i o n s . Addi-t i o n of calcium i o n to the reduced enzyme p r e p a r a t i o n r e s u l t e d i n the complete recovery of a c t i v i t y w i t h i n a few minutes, although only one S-S bond was reformed. However, o n l y one S-S bond i s necessary f o r a c t i v i t y , s i n c e the f r e e S-H groups can be carboxymethylated without l o s s of enzyme a c t i v i t y . I t has been demonstrated th a t calcium i o n has a str o n g s t a b i l i z i n g e f f e c t on the enzyme, p r o t e c t i n g i t from the a c t i o n o f p r o t e o l y t i c enzymes and h i g h tempera-3 tures ( 4 ) . Moore and S t e i n have p o s t u l a t e d t h a t calcium may p r o t e c t the DNase I found i n p a n c r e a t i c Juice from the a c t i o n of p r o t e o l y t i c enzymes which are present i n l a r g e amounts. Although calcium i o n p l a y s an important r o l e i n determining the three dimensional s t r u c t u r e of the enzyme there i s evidence t h a t i t i s not s t r o n g l y bound to the enzyme. Incubation of a sample of DNase I w i t h f o l l o w e d by g e l f i l t r a t i o n a t pH 7 removed a l l the ^ C a from the enzyme p r o t e i n . Poulas and P r i c e ( 7 ) have shown tha t the a d d i t i o n of calcium i o n changes the o p t i c a l r o t a t o r y d i s p e r s i o n spectrum. They examined the o p t i c a l r o t a t o r y d i s p e r s i o n and c i r c u l a r d i c h r o i s m s p e c t r a of DNase I i n the presence and absence of calcium ions and concluded t h a t calcium i o n b i n d i n g causes s i g n i f i c a n t p e r t u r b a t i o n s of tryptophan and t y r o s i n e r e s i d u e s . As the calcium i o n bin d s , the environment of these residues becomes i n c r e a s i n g l y non p o l a r . This change has ben i n t e r -p r e ted as i n d i c a t i n g t h a t the enzyme i s more r i g i d i n the presenoe of calcium i o n . P r i c e ( 8 ) has shown t h a t DNase IA a t pH 7.5 has two s i t e s which bind calcium i o n s t r o n g l y and three weak b i n d i n g s i t e s . One of the stro n g b i n d i n g s i t e s i s calcium s p e c i f i c , s i n c e a 1000 f o l d excess of manganese or magnesium ions does not a f f e c t calcium b i n d i n g to t h i s s i t e . However manganese and magnesium can com-pete w i t h calcium f o r the second s i t e . I t seems l i k e l y , t h e r e f o r e , t h a t the b i n d i n g of calcium to the the f i r s t s i t e i s r e s p o n s i b l e f o r a change i n conformation of the DNase I molecule which b u r i e s the t y r o s i n e and tryptophan w i t h i n the molecule and thereby confers s t a b i l i t y on the molecule l n the presence of p r o t e o l y t i c enzymes. H i s t i d i n e has been i m p l i c a t e d as p a r t of the a c t i v e s i t e . P r i c e , Moore and S t e i n (9 ) found that DNase I co u l d o n l y be i n a c t i v a t e d by iodoacetate a t pH 7 . 2 i f d i v a l e n t ions were present. Although there are 6 h i s t i d i n e residues i n DNase I , a l l e v i a t i o n w i t h l i fC iodoacetate gave o n l y one l a b e l l e d h i s t i d i n e . The i n a c t i v a t e d p r o t e i n was d i g e s t e d and found to con-t a i n one residue of 3 carboxymethyl h i s t i d i n e per molecule. Hugle and S t e i n (10) s e l e c t i v e l y n i t r a t e d a s i n g l e t y r o s i n e residue and i n a c t i v a t e d DNase I . The n i t r a t e d enzyme co u l d not be s t a b i l i z e d a g a i n s t chymotryptic d i g e s t i o n by calcium. A d d i t i o n of c a l -ciums i o n no longer Induced change i n the O.R.D. spectrum. Although caloium i o n d i d not s t a b l l z e the 5 n i t r a t e d enzyme a g a i n s t mercaptoethanal r e d u c t i o n , removal of excess mercaptoethanal f o l l o w e d by a d d i t i o n of calcium r e s u l t e d i n the r a p i d r e f o r -mation of the S-S bonds. This suggests the presence of two calcium i o n b i n d i n g s i t e s , o n l y one of which i s a f f e c t e d by n i t r a t i o n . The complete amino a c i d sequence of DNase IA p u b l i s h e d by Moore and S t e i n (11) i s shown i n f i g u r e 1. The d i s u l p h i d e bond necessary f o r enzymic a c t i v i t y l i n k s r e s i d u e s 170 and 206, w h i l e the non e s s e n t i a l d i s u l p h i d e bond forms a s m a l l loop between residues 98 and 101. The h l s t i d l n e carboxymethylated by P r i c e , Moore and S t e i n i n the presence of d i v a l e n t metal ions was found to be h l s t i d l n e 131. The s e l e c t i v e l y n i t r a t e d t y r o s i n e d e s c r i b e d by Hugle and S t e i n was found to be t y r o s i n e 62. The sequence shows no r e p e a t i n g s e c t i o n s and there i s no evidence to suggest the ex1stance of two a c t i v e s i t e s . 6 10 Carb. U u - L y j - I I « - A l o - A l f l - P h e - A j n - I I « - A r g - T h r - P h « - G l y - G l u - T h r - L y s - M e 1 - S e r - A j n -20 30 Alo - T h f L t u - A l a - S e r - T y r - I l e - V o l - A r g - A r g - T j r r - A i p - I I « - V o l - L « u - I I « - G l u - G l f l - V o l -10 50 Arg -Asp -Ser -H is -Leu -Vo l -A lo -Vo l -G ly -Ly » -Leu -L iBu -Asp -Ty r -Leu -Asn -G ln -Asp -Asp -60 70 Pro-Asn-Thf-Tyr-His-Tyf-Vol-Vol-Ser-Glu-Pto-Leu-Gly-Arg-Asn-Ser-Tyi-tys-Glu-60- 90 A f g - T y r - L e u - P h e - L e u - P h e - A r g - P f O - A s n - L y s ; V o l - S e r - V a l - L « u - A $ p - T h r - T y r - G l n - T y r -100 110 Asp-Asp-Gly-Cys-Glu-Ser-Cys-Gly-Asn-Asp-Ser-Phe-Ser-Arg-Glu-Pro-Alo-Vol-Vol-1 1 ,30 Lys-Pht-Str-SefHit-Ser-Thr-Lys-Vol-Lyj-Glu-Phe-Alo-lle-Vol-Alo-Leu-His-Ser-140 150 Ala-PfO-Ser-As(rAlo-Vol-Alo-Glu-lle-Asn-Ser-Leu-Tyr-Asp-Vol-Tyr-Leu-Asp-Vol-160 170 G ln -G ln -Lys -T fp -H is -Leu -Asn -A$p -Vo l -Me l -Leu -Me l -G ly -Asp -Ph« -Am-A lo -Asp -Cys -180 ' S c r - T y r - y o l - T h f - S « r - S e r - G l n - T f p - S e r - S e r - I l e - A r g - L e u - A r g - T h r - S e r - S e r - T h r - P h e -190 200 | G l n - T r p - L « u - I I « - P r o - A s p - S e r - A l o - A s p - T h r - T h f - A l o - T h f - S « f - T h f - A » n - C y j - A l o - T y r -210 220 Asp-Arg-lle- Vol-Vol- Alo-Gly-Ser-Leu-Leu-Gln-Ser-Ser-Val-Vol-Gly-Pro-Ser-Alo-230 240 A l o - P f o - P h e - A s p - P h e - G l n - A l o - A l a - T y r - , G l y - L e u - S « r - A i n - G l u - M e l - A l o - L e u - A l o - I l e -250 257 S e r - A s p - H i s - T y r - P f o - V a l - G l u - V o l - T h f - L « u - T h f Figure i . The above sequence and the pa i r i n g of the hal f - c y s t i n e residues has been derived for bovine pancreatic DNase I A by Moore, Stein and coworkers. 7 KINETICS OF DNase 1 The mechanism of degradation of DNA by DNase and the r o l e of metal ions i n the r e a c t i o n are p o o r l y understood. Erkama and Suutarinen (12) proposed t h a t d i v a l e n t metal ions combine w i t h DNA to form a metalo-s u b s t r a t e . They used Mg-DNA as a su b s t r a t e and found th a t added M g + 2was r e q u i r e d f o r maximal a c t i v i t y , suggesting t h a t a metalo-enzyme was a l s o necessary. Other i n v e s t i g a t o r s have reached s i m i l a r c o n c l u s i o n s but the d e t a i l s of the mechanism remain unresolved ( 2 ) . Without s p e c u l a t i n g as to i t s s i g n i f i c a n c e Lee (13) and Melgar and Goldthwait (14) demonstrated non l i n e a r behavior of DNase I u s i n g the Llneweaver-Burk p l o t (31). P e r l g u t and Hernondez (15)» u s i n g p u r i f i e d Mg-DNA as a s u b s t r a t e , observed a b i p h a s i c curve f o r the a c t i -v a t i o n of the enzyme by magnesium i o n . B e p l o t t i n g the two p o r t i o n s of t h i s curve as Lineweaver-Burk p l o t s they obtained upward curved p l o t s . The magnesium i o n a c t i v a t i o n curve was sl g m o l d a l i n form and was th e r e f o r e analyzed by P e r l g u t and Hernondez u s i n g the equation f i r s t developed by H i l l to i n v e s t i g a t e the uptake of oxygen by haemoglobin. They used the formt v = Mg++ 8 and found n = 2 f o r both s e c t i o n s of the curve. This suggests t h a t two metal ions combine s e q u e n t i a l l y a t each s i t e . However, the experiments performed by P e r l g u t and H e r n a n d e z were done a t pH 5.0 and recent work (8) has e s t a b l i s h e d t h a t DNase 1 has a d i f f e r e n t number of metal ion b i n d i n g s i t e s a t pH 5.0 than a t pH 6 .8. Melgar and Goldthwalt found t h a t the mechanism of a c t i o n of DNase 1 on DNA may be switched from a s i n g l e h i t mode to a double h i t mode. The s i n g l e h i t mode i s so named s i n c e a s i n g l e event breaks both strands of the s u b s t r a t e DNA a t the same l o c u s ; the double h i t mode i n v o l v e s random h i t s w i t h each h i t breaking o n l y a s i n g l e s t r a n d . Double s t r a n d breaks thus occur as a r e s u l t of accumulating random s i n g l e s t r a n d breaks. P e r l g u t and H e r n a n d e z , u s i n g l a b e l l e d DNA trapped i n an acrylamlde g e l as an assay system, showed t h a t there was an i n i t i a l l a g i n the r a t e of r e l e a s e of l a b e l l e d DNA fragments from the g e l when magnesium i o n alone was used as the a c t i v a t o r . This i n i t i a l l a g was e l i m i n a t e d when manganese i o n , c o b a l t i o n , calcium i o n , o r a mixture of calcium i o n and magnesium i o n was used as an a c t i v a t o r . The a d d i t i o n of sodium i o n or potassium i o n r e e s t a b l i s h e d the l a g . Melgar and Goldthwalt (14) p o s t u l a t e d t h a t the presence of the la g c i n d i c a t e d a double h i t mechanism 9 w h i l e i t s abscence i n d i c a t e d a s i n g l e h i t mechanism. This i n t e r p r e t a t i o n has been confirmed by v l s c o m e t r l c experiments analyzed by the method of Bernard! and Sadron (16) and by u l t r a c e n t r i f u g a t l o n a n a l y s i s ( 1 7 ) . Three p o s s i b l e mechanisms f o r s i n g l e h i t k i n e t i c s have been considered by Melgar and Goldthwaitt 1) The enzyme may a s s o c i a t e to form a dlmer which then a t t a c k s both strands simultaneously. This i s very u n l i k e l y as no dlmeric form of DNase I has been reporte d d e s p i t e i n t e n s i v e i n v e s t i g a t i o n . 2) The enzyme may have two a c t i v e s i t e s . This p o s s i b i l i t y has been e l i m i n a t e d by the recent work of L l a o e t a l (11) who e l u c i d a t e d the e n t i r e amino a c i d sequence of the enzyme. 3) B i n d i n g of one DNase I molecule to DNA may f a c i l i t a t e the b i n d i n g of a second enzyme molecule a t the same s i t e . No experimental evidence f o r t h i s hypothesis e x i s t s i indeed no experiments have been reporte d which attempt to t e s t t h i s suggestion. 10 THE LINKAGE SPECIFICITY OF DNase I The l i n k a g e s p e c i f i c i t y of DNase I i s of great i n t e r e s t to those who wish to sequence DNA. I f an unequivocal preference c o u l d be demonstrated the enzyme co u l d be used as an a n a l y t i c a l t o o l . Many groups have i n v e s t i g a t e d t h i s problem and c o n t r a d i c t o r y data have accumulated,(18). The e a r l y experiments i n v o l v e d the d i g e s t i o n of DNA u n t i l the r e a c t i o n reached e q u i l i b r i u m and sub-sequent a n a l y s i s of the products. Such experimental design assumes a constant l i n k a g e s p e c i f i c i t y through-out the r e a c t i o n . This assumption remains unproven. L a t e r work (19) has shown t h a t the DNase I r e a c t i o n e x h i b i t s a u t o r e t a r d a t i o n . The d i s c r e p a n c i e s between d i f f e r e n t workers* r e s u l t s were f u r t h e r e x p l a i n e d by Bollum (20) who discovered t h a t the nature of the a c t i v a t i n g i o n a f f e c t s the s p e c i f i c i t y of r e a c t i o n . He d i g e s t e d an a r t i f i c i a l homopolymer d l i d C w i t h DNase I i n the presence of magnesium i o n and found t h a t the dC chain was completely r e s i s t a n t to a t t a c k . A d d i t i o n of a t r a c e of calcium i o n o r changing the d i v a l e n t i o n to manganese r e s u l t e d i n complete d i g e s t i o n o f both s t r a n d s . 11 I t has been suggested (22) th a t the f i r s t few s c i s s i o n s made by DNase I are h i g h l y s p e c i f i c and tha t s p e c i f i c i t y decreases d u r i n g the r e a c t i o n . S i c a r d et a l (22) showed by a n a l y t i c a l u l t r a c e n t r l -f u g a t i o n t h a t the s c i s s i o n s made i n DNA by the enzyme tend to c l u s t e r ; DNase I behaved as a region s p e c i f i c enzyme i n these experiments. U n f o r t u n a t e l y the e a r l y s p e c i f i c i t y e x h i b i t e d by DNase I cannot be v e r i f i e d u n t i l some means of sequencing the t e r m i n a l bases of lon g o l i g o n u c l e o t i d e s i s developed. Since the reason f o r determining the base s p e c i f i c i t y of DNase I was to use i t i n sequencing, i t seems tha t data on the s p e c i -f i c i t y of DNase I must await the development of some new technique f o r sequencing DNA. PROTEIN INHIBITOR In 19^5 Laskowski (23) f i r s t r e p o r t e d a p r o t e i n i n h i b i t o r of DNase I . I t was p u r i f i e d from the hypertrophic e p i t h e l i u m of the pigeon crop gland. P r o t e i n i n h i b i t o r s have subsequently been found i n other t i s s u e s (24). Lindberg (25) p u r i f i e d two DNase I i n h i b i t o r s from bovine spleen. He deter-mined the molecular weight and amino a c i d composition of one which he designated "spleen i n h i b i t o r I I M . 12 Spleen I n h i b i t o r I I was shown by g e l f i l t r a t i o n to form a l i l molecular complex w i t h DNase I . A sample of DNase I was chromatographed on Sephadex G-100 and e l u t e d a t the expected p o s i t i o n . A d d i t i o n of excess i n h i b i t o r to DNase I a b o l i s h e d the peak of enzyme a c t i v i t y which was r e p l a c e d by a peak e l u t i n g a t the p o s i t i o n expected f o r 1«1 enzyme-inhibitor complex. This complex could be d i s s o c i a t e d i n 3 M urea to gi v e DNase I and an i r r e v e r s i b l y denatured i n h i b i t o r . The i n h i b i t o r was s p e c i f i c f o r DNase I . I t d i d not i n -h i b i t DNase I I , endonuclease I from E. C o l l o r BNase. BIOLOGICAL ROLE OF DNase The p h y s i o l o g i c a l r o l e of DNase I remains uncer-t a i n although there i s much i n d i r e c t evidence which suggest t h a t DNases are Involved i n DNA s y n t h e s i s . A l l f r e y and Mirsky (26) found a p o s i t i v e c o r r e l a t i o n between the DNase content of a v a r i e t y of animal t i s s u e s and the r a t e of t i s s u e p r o l i f e r a t i o n o r reg e n e r a t i o n . Other i n v e s t i g a t o r s have extended these observations to p l a n t s (27) and b a c t e r i a (28). 13 Although there i s much c i r c u m s t a n t i a l evidence, no experimental data e x i s t which I n d i c a t e p r e c i s e l y how DNase i s i n v o l v e d i n DNA s y n t h e s i s . I t i s known t h a t some b a c t e r i a l c e l l s possess r e s t r i c t i o n enzymes which destroy DNA not l a b e l l e d w i t h the c e l l s "tag" -a sugar group oh the 5-hydroxymethylcytoslne ( 2 ° ) . DNases co u l d serve a s i m i l a r f u n c t i o n i n the mammalian c e l l , however, "tagged" mammalian DNA has never been observed. A l t e r n a t i v e l y , the enzyme could serve as a r e p a i r enzyme by removing mismatched bases and unordered loops of s i n g l e stranded DNA caused by u l t r a v i o l e t r a d i a t i o n damage. However, t h i s i s an u n l i k e l y f u n c t i o n of the DNase I s i n c e i t p r e f e r s n a t i v e to s i n g l e stranded DNA. DNases w i t h the opposite preference have been re p o r t e d (59). Richardson, S c h i l d k r a u t and Kornberg (30) showed t h a t s m a l l amounts of endonuclease I added to a DNA polymerase system g r e a t l y i n c r e a s e d the r a t e of s y n t h e s i s by " n i c k i n g " the B. S u b t i l e s DNA primer-template and producing a d d i t i o n a l y OH groups. I t i s a t t r a c t i v e to speculate t h a t DNase I f u n c t i o n s by " n i c k i n g " the DNA primer-template, e s p e c i a l l y as i t has been suggested t h a t the f i r s t few breaks made by the enzyme are h i g h l y s p e c i f i c . 1 4 The presence of DNases i n the c e l l throughout the e n t i r e c e l l c y c l e i m p l i e s t h a t the c e l l possesses a mechanism f o r the c o n t r o l of these powerful nucleo-l y t i c agents. P r o t e i n i n h i b i t o r s of DNase I are w e l l c h a r a c t e r i z e d but t h e i r b i o l o g i c a l f u n c t i o n i s not w e l l d e l i n e a t e d . I t has not been p o s s i b l e to d i s -s o c i a t e the i n h i b i t o r enzyme complex under physio-l o g i c a l c o n d i t i o n s . While i t seem l i k e l y t h a t DNases are i n v o l v e d i n s y n t h e s i s , the mechanism of t h e i r involvement remains obscure. 15 METHODS AND MATERIALS A wide v a r i e t y of methods have/been employed to assay DNase I a c t i v i t y . These Include l o s s of v i s c o s i t y by DNA s o l u t i o n s (31)» r e l e a s e of a c i d s o l u b l e o l i g o n u c l e o t i d e s (32), r e l e a s e of bound DNA from a polyacrylamide m a t r i x (33), h y d r o l y s i s of DNA i n a DNA g e l (34), the l o s s of b i o l o g i c a l a c t i v i t y of c i r c u l a r phage DNA (35)» and immuno-l o g i c a l methods (36). Three assays have been used i n the present work. F i r s t , the e s t i m a t i o n of the hyperchromicity of a DNA s o l u t i o n , second the measurement of the amount of a c i d s o l u b l e o l i g o -n u c l e o t i d e s r e l e a s e d , and t h i r d the determination of the amount of DNA hydrolysed l n a DNA agar g e l by measuring the zone of c l e a r i n g . KUNITZ ASSAY The K u n i t z assay measures the increase i n absorbance a t 260 nm of a DNA s o l u t i o n a f t e r the a d d i t i o n of enzyme. In the present work, the assay was m o d i f i e d by r e p l a c i n g the acetate b u f f e r , pH 5 . 0 , by M.E.S. b u f f e r , pH 6 .8. Cu-v e t t e s c o n t a i n i n g 0.15 mg of DNA i n 2.9 ml o f 16 0.1 M M.E.S. b u f f e r , pH 6 .8, 10 mM i n manganese io n were prepared. The r e a c t i o n was s t a r t e d by the a d d i t i o n of enzyme s o l u t i o n . The mixture was s t i r r e d q u i c k l y and a continuous record of the change i n absorbance a t 260 nm w i t h time was obtained u s i n g a Cary 15 spectrophotometer. One u n i t of enzyme a c t i v i t y was d e f i n e d as the amount produ-c i n g an Increase i n A260 o f 0.001 per minute under the assay c o n d i t i o n s used. This assay i s very time consuming and, i n the presence of l a r g e amounts of p r o t e i n , p r e c i p i t a t i o n can g i v e f a l s e r e s u l t s . ACID SOLUBLE OLIGONUCLEOTIDE ASSAY The a c i d s o l u b l e o l i g o n u c l e o t i d e assay used here i s a m o d i f i c a t i o n of the method de s c r i b e d by Bern a r d i (37). E x a c t l y the same s o l u t i o n s were used as des c r i b e d f o r the K u n i t z assay. The assay mixtures were incubated i n S o r v a l l c e n t r i f u g e tubes a t 37° C and 0.1 ml of enzyme s o l u t i o n was added to each mixture to s t a r t the r e a c t i o n . A f t e r 15 minutes each r e a c t i o n was stopped by the a d d i t i o n of 0.5 ml of 12# p e r c h l o r i c a c i d to g i v e a f i n a l volume o f 3*5 ml. The tubes were cooled i n an 17 i c e water bath f o r 10 minutes, and then c e n t r i -fuged a t 10,000 x g f o r 10 minutes. The absor-bance a t 260 nm of the supernatants was measured u s i n g a Cary 15 spectrophotometer. One u n i t of enzyme a c t i v i t y was d e f i n e d as the amount pro-ducing an absorbance change a t 260 nm of 1.0 i n 15 minutes under the assay c o n d i t i o n s . This assay i s much l e s s time consuming than the K u n i t z assay s i n c e a l a r g e number of assays can be per-formed simultaneously. In a d d i t i o n , i n t e r f e r i n g p r o t e i n s are removed by p r e c i p i t a t i o n w i t h per-c h l o r i c a c i d . The s e n s i t i v i t y of the K u n i t z assay and the a c i d s o l u b l e o l i g o n u c l e o t i d e assay were compared. A s e r i e s of d i l u t i o n s of crude enzyme e x t r a c t were prepared and assayed by both methods. Figu r e 2 i l l u s t r a t e s t h a t the a c i d s o l u b l e o l i g o n u c l e o t i d e assay i s i n s e n s i t i v e to low enzyme c o n c e n t r a t i o n s . I n s e n s i t i v i t y to low concentrations of enzyme i n the e a r l y stage of the r e a c t i o n are c h a r a c t e r i s t i c s of a l l assays which depend on the l i b e r a t i o n of a c i d s o l u b l e o l i g o n u c l e o t i d e s . This i s because the f i r s t breaks i n the DNA str a n d caused by DNase I are u n l i k e l y to produce o l i g o n u c l e o t i d e s s m a l l % Enzyme added P i g . 2. A comparison of the s e n s i -t i v i t i e s o f the K u n i t z assay and the a c i d s o l u b l e o l i g o n u c l e o t i d e assay. Dotted l i n e r e p r e s e n t s the K u n i t z assay, the s o l i d l i n e r e p r e s e n t s the a c i d s o l u b l e o l i g o n u c l e o t i d e assay. A l l assays were done i n 0.1 M T r i s + b u f f e r , pH 6.8, c o n t a i n i n g 10 mM Mn 19 enough to be a c i d s o l u b l e . l a r g e r a c i d i n s o l u b l e o l i g o n u c l e o t i d e s are not detected, by t h i s assay. GEL ASSAY The DNA g e l assay developed by J a r v i s and Lawrence (34) was a l s o used. Agar was d i s s o l v e d i n a mixture of 5 ml of water and 10 ml of 0.1 M T r i s - H C l b u f f e r , pH 7.8i w i t h h e a t i n g a b o i l i n g water bath. The hot s o l u t i o n was allowed to c o o l to approximately 60° C and 20 mg of DNA d i s s o l v e d i n 5 ml of 0.01 M NaCI were added to g i v e a f i n a l c o n c e n t r a t i o n of 1 mg per ml of DNA. The s o l u t i o n was made 0.01 M w i t h respect to manganese i o n . The s o l u t i o n was p i p e t t e d w i t h a warm p l p e t onto g l a s s s l i d e s o u t l i n e d w i t h p l a s t i c tape. A hole 3 mm i n diameter was punched i n the centre of the agar coated s l i d e . The s l i d e s were incubated a t 37° C f o r 20 hours and developed by immersion i n 0.5 M HC1 f o r 1 minute. Enzyme a c t i v i t y was i n -d i c a t e d by the appearance of a c l e a r c i r c l e sur-rounding the centre w e l l . The area of t h i s c i r c l e was r e l a t e d to the l e v e l of DNase I a c t i v i t y . The g e l assay was s e v e r a l f o l d more s e n s i t i v e than e i t h e r of the two assays d e s c r i b e d p r e v i o u s l y . F i g u r e 3 shows a comparison of the g e l and a c i d i ' ' I I • t I I J . .10 . A c t i v i t y ( A S O A ) F i g . 3« A comparison of the s e n s i -t i v i t i e s of the g e l assay and the a c i d s o l u b l e o l i g o n u c l e o t i d e assay. Samples were assayed l n d u p l i c a t e by both assays and the r e s u l t s p l o t t e d . 21 s o l u b l e o l i g o n u c l e o t i d e assay. Since the g e l assay system was sa t u r a t e d a t r e l a t i v e l y low l e v e l s of DNase I a c t i v i t y , i t cannot be used f o r q u a n t i t a -t i v e a n a l y s i s . However, i t i s i d e a l f o r q u a l i t a -t i v e s t u d i e s and was f r e q u e n t l y used to de t e c t enzyme a c t i v i t y e l u t i n g from columns as i t was the only assay used here which was not i n h i b i t e d by phosphate Ion (Figure ). I t was p a r t i c u l a r l y v a l u a b l e f o r the d e t e c t i o n of enzyme a c t i v i t y which e l u t e d from columns w i t h phosphate g r a d i e n t s . PREPARATION OF THE CRUDE ENZYME EXTRACT Fi v e male Wlstar r a t s , each weighing'20(5-250 grams, were stunned by a blow on the head and then d e c a p i t a t e d . The sm a l l i n t e s t i n e was r e -moved immediately and f l u s h e d w i t h c o l d 0.02 M phosphate b u f f e r c o n t a i n i n g 10 mM Mg + +. The i n -t e s t i n e was placed on a c o l d s u r f a c e and s p l i t l engthwise. The i n t e s t i n a l mucosa c e l l s were then scraped o f f w i t h a g l a s s s l i d e . The mucosal t i s s u e was homogenized a t 700 rpm In a S o r v a l l Omnlmix f o r 5 minutes. The r e s u l t i n g foamy homogenate was d e n t r i f u g e d a t 10,000 x g f o r 5 minutes. The supernatant was r e c e n t r l f u g e d F i g . 4 . I n h i b i t i o n o f t h e g e l a n d a c i d s o l u b l e o l i g o n u c l e o t i d e assay-b y p h o s p h a t e . D o t t e d l i n e r e p r e -s e n t s a c t i v i t y b y t h e g e l a s s a y , s o l i d l i n e r e p r e s e n t s a c t i v i t y b y t h e a c i d s o l u b l e o l i g o n u c l e o t i d e a s s a y . 23 a t 100,000 x g f o r 1 hour i n a Beckman model L u l t r a c e n t r l f u g e . The p e l l e t was di s c a r d e d and the supernatant s o l u t i o n was designated the crude enzyme p r e p a r a t i o n . In g e n e r a l , 500-700 u n i t s of a c t i v i t y ( a c i d s o l u b l e o l i g o n u c l e o t i d e assay) l n a t o t a l volume of 100-125 ml were obtained. PlTftTPTCATTON OP DNase I ON DEAE CELLULOSE DEAE c e l l u l o s e , DE-32, was suspended i n 0.5 M HC1 f o r 1 hour and then washed w i t h d i s -t i l l e d water on a Buchner f u n n e l u n t i l the f i l -t r a t e reached a pH of 4. The cake:of c e l l u l o s e was suspended i n 0.5 M NaOH f o r 1 hour. I t was then f i l t e r e d on a Buchner f u n n e l and resuspended i n 0.5 M NaOH f o r a f u r t h e r hour. The c e l l u l o s e was washed^with d i s t i l l e d water u n t i l the pH of the f i l t r a t e was between 7 and 8. The r e c y c l e d c e l l u l o s e was s t o r e d a t room temperature as a suspension i n 0.5 M potassium phosphate b u f f e r u n t i l used. The DEAE c e l l u l o s e s l u r r y was poured i n a column (2.5 x 20 cm) and allowed to s e t t l e under g r a v i t y . The column was washed w i t h e q u i l i b r a t i n g b u f f e r u n t i l the con-d u c t i v i t i e s of el u a n t and e f f l u a n t were i d e n t i c a l . 24 The crude enzyme p r e p a r a t i o n (approximately 1500 mg p r o t e i n ) was a p p l i e d to the e q u i l i b r a t e d column and e l u t e d w i t h the e q u i l i b r a t i n g b u f f e r u n t i l the absorbance a t 280 nm of the washings dropped below 0.2. A l i n e a r g r a d i e n t of 0.02 M to 0.25 M phosphate was a p p l i e d and 10 ml f r a c -t i o n s c o l l e c t e d . The f r a c t i o n s were assayed by the a c i d s o l u b l e o l i g o n u c l e o t i d e assay and those c o n t a i n i n g DNase I a c t i v i t y were pooled and f r e e z e d r i e d Immediately a f t e r e l u t l o n . The used columns were washed w i t h 0 .5 M potassium phosphate b u f f e r , pH 6.8, to remove as much p r o t e i n as p o s s i b l e and the top 2-3 cm of DEAE 32 c e l l u l o s e were di s c a r d e d . The DEAE c e l l u l o s e was r e c y c l e d each time; i t could be r e e q u i l i b r a t e d and used d i r e c t l y but the y i e l d and p u r i f i c a t i o n of the enzyme decreased. REMOVAL OF PHOSPHATE The f r e e z e d r i e d enzyme p r e p a r a t i o n obtained from the DEAE c e l l u l o s e chromatography step con-t a i n e d l a r g e amounts of phosphate. ItT^was neces-sary to remove t h i s phosphate before chromatography 25 on Sephadex G-100 to a v o i d the f o r n a t i o n of c a l -cium phosphate. The f r e e z e d r i e d m a t e r i a l was d i s s o l v e d l n a minimum volume of d i s t i l l e d water and 1 M calcium c h l o r i d e s o l u t i o n was added dropwise to p r e c i p i t a t e most of the phosphate present. The white p r e c i p i -t a t e was removed by c e n t r i f u g a t i o n . This p r e c i p i -t a t e could not be r e d i s o l v e d i n d i s t i l l e d water and possessed no DNase I a c t i v i t y . I t d i s s o l v e d i n d i l u t e a c i d and was probably Ca^PO^^. I t was e s s e n t i a l to add the calcium c h l o r i d e s o l u t i o n c a r e f u l l y to avo i d p r e c i p i t a t i n g a l l the phosphate and thereby removing the b u f f e r . In some experiments the fr e e z e d r i e d powder was d i s -s o l v e d i n T r l s b u f f e r i n s t e a d of water so t h a t an excess of calcium i o n could be added to remove a l l the phosphate b u f f e r . CHROMATOGRAPHY ON SEPHADEX G-100 A column 2.5 x 90 cm was f i l l e d w i t h Sephadex G-100 and allowed to s e t t l e overnight a t >° C. I t was washed w i t h e q u i l i b r a t i n g b u f f e r (0.025 M T r i s - H C l , pH 7.5 c o n t a i n i n g 5 mM C a + + ) u n t i l the A280 o f t n e e f f l u e n t was zero. The enzyme 26 p r e p a r a t i o n , which had been t r e a t e d to remove phos-phate, was loaded onto the column and subsequently e l u t e d w i t h e q u i l i b r a t i n g b u f f e r . The e l u t i o n p o s i t i o n s of the p r o t e i n s were determined by measuring the a b s o r p t i o n of each f r a c t i o n a t 280 nm and enzyme a c t i v i t y was detected by the g e l assay and by the a c i d s o l u b l e o l i g o -n u c l e o t i d e assay. RECHROMATOGRAPHY ON DEAE 32 A DEAE column (1 x 10 cm) was packed and e q u i l i b r a t e d as d e s c r i b e d f o r the 2.5 x 20 cm column. The p a r t l y p u r i f i e d DNase I p r e p a r a t i o n obtained from the Sephadex chromatography step was a p p l i e d d i r e c t l y to the column. The column was e l u t e d w i t h a g r a d i e n t of 0.02 M to 0.1 M phosphate b u f f e r , pH 6.8. Loss of DNase I a c t i v i t y was prevented by c o l l e c t i n g 2 ml f r a c t i o n s i n tubes which contained 1.0 ml of 0.1 M T r i s - H C l b u f f e r , pH 7.5, 0.25 M i n CaCl2. This removed a l l the phosphate and l e f t the enzyme i n a b u f f e r c o n t a i n i n g calcium Ion. MOLECULAR WEIGHT DETERMINATION ON SEPHADEX G-100 A 2.5 cm x 1 m Pharmacia column, equipped w i t h flow adaptors, was f i l l e d w i t h swollen Sephadex 27 G-100 beads. The o u t l e t was maintained no more than 20 cm below the l i q u i d surface u n t i l the column was packed. The f i n a l h e i g ht of the column was 90 cm. A continuous flow of e q u i l i b r a t i n g b u f f e r (0.1 M T r i s - H C l , pH 7.5, 5 mM i n CaCl2) was main-t a i n e d through the column a t a l l times. Samples were d i s s o l v e d i n no more than 5 ml of the e q u i l i -b r a t i n g b u f f e r , which was a l s o used f o r e l u t i o n . F i v e ml f r a c t i o n s were c o l l e c t e d . The marker p r o t e i n s used were BNase A, Ovalbumin, and chymotrypsinogen A. BNase was detected by i t s enzyme a c t i v i t y (38) w h i l e chymo-trypsinogen and ovalbumin were detected by measuring t h e i r absorbance a t 280 nm u s i n g a Cary 15 spectrophotometer. Samples of p a r t l y p u r i f e d DNase I were d e s a l t e d on Sephadex G-15. The d e s a l t e d enzyme p r e p a r a t i o n was made 5 ml i n volume w i t h e q u i l i -b r a t i o n b u f f e r and a p p l i e d to the c a l i b r a t e d Sephadex G-100 column. Enzyme a c t i v i t y was detected by the a c i d s o l u b l e o l i g o n u c l e o t i d e assay. Commercial DNase I was d i s s o l v e d i n the e q u i l i -b r a t i n g b u f f e r and chromatographed to check the c a l i b r a t i o n of the column. 28 " POLYACRYLAMIDF. GEL ELECTROPHORESIS p . o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s was performed a c c o r d i n g to Davis (39). Gels were run i n batches o f e i g h t . DETERMINATION OF pH OPTIMUM Three b u f f e r systems were used to cover the range from pH 4.0-to 8.0. Each b u f f e r was used a t a c o n c e n t r a t i o n o f 0.1 M; they were prepared and used the same day. A l l pH v a l u e s were checked w i t h a pH meter d u r i n g the experiment. The.enzyme s o l u t i o n used was a 10 f o l d p u r i f i e d p r e p a r a t i o n o b t a i n e d from a DEAE 32 c e l l u l o s e column. AMMONIUM SULPHATE FRACTIONATION Ammonium s u l f a t e f r a c t i o n a t i o n s were performed on samples of crude enzyme e x t r a c t . Ten ml o f crude enzyme e x t r a c t were p i p e t t e d i n t o a S o r v a l l c e n t r i f u g e tube and s o l i d ammonium s u l f a t e was added to g i v e the r e q u i r e d c o n c e n t r a t i o n . The weight o f s a l t n e c e s s a r y was determined from the nomogram p u b l i s h e d by Dixon (40). The amounts o f ammonium s u l f a t e added a r e shown i n the t a b l e below» 29 Table I . The amounts of s o l i d ammonium s u l f a t e added to 10 ml of 0.02 M T r i s - H C l b u f f e r to give the r e q u i r e d percentage s o l u t i o n s . % SAT g/10 ml 25 1.44 30 1.75 35 2.09 40 2.42 45 2.78 50 3.12 55 3.50 60 3-90 The mixture was s t i r r e d to d i s s o l v e a l l the s o l i d ammonium s u l f a t e and then allowed to stand a t 4° C f o r 30 minutes. The p r e c i p i t a t e was c o l l e c t e d by c e n t r i f u g a t l o n a t 10,000 x g f o r 10 minutes i n a S o r v a l l RC-2B. The supernatant was removed and the p r e c i p i t a t e was r e d i s s o l v e d i n 10.0 ml of T r i s - H C l b u f f e r . The p r o t e i n c o n c e n t r a t i o n of the super-natant and p r e c i p i t a t e s o l u t i o n s was determined by measuring the absorbance a t 280 nm. DNase I a c t i v i t y was measured by g e l assay and by the a c i d s o l u b l e o l i g o n u c l e o t i d e assay. DETERMINATION OF THE NATURE OF THE PRODUCTS OF HYDROLYSIS Ten mg of DNA were d i s s o l v e d i n 10 ml o f M.E.S. b u f f e r , pH 6.8, and mixed w i t h 0.5 ml o f 200 f o l d 30 p u r i f i e d enzyme i n a t e s t tube. The mixture was incubated a t 37° C f o r 3 hours. The r e a c t i o n was stopped by immersing the t e s t tube i n a b o i l i n g water bath f o r 10 minutes. The r e s u l t i n g cloudy s o l u t i o n was c l a r i f i e d by c e n t r i f u g a t l o n . A column of DEAE c e l l u l o s e , DE-32, (1 x 10 cm), was packed a t atmospheric pressure In 0.5 M ammonium carbonate and washed w i t h d i s t i l l e d water accor d i n g to the method of Tomlinson and Tener (41). F i v e ml of the c l a r i f i e d d i g e s t were added and washed i n w i t h 5 ml of d i s t i l l e d water. A l i n e a r g r a d i e n t of 250 ml of 7 M urea c o n t a i n i n g 25 ml of 0.1 M T r i s -HC1 and 250 ml of 7 M urea c o n t a i n i n g 0.3 M NaCI and T r l s b u f f e r was a p p l i e d . The e l u t i o n of nucleo-t i d e s was f o l l o w e d by meausring the A260 o f t n e e l u t e d f r a c t i o n s . S e l e c t e d peaks were d e s a l t e d by Ion exchange on DEAE c e l l u l o s e as des c r i b e d by Tomlinson and Tener. The n u c l e o t i d e was e l u t e d w i t h 2 M ammonium carbonate and the pooled m a t e r i a l was repeatedly taken to dryness on a r o t a r y evaporator and r e d i s -s o l v e d u n t i l no ammonium carbonate remained. The d e s a l t e d , p u r i f i e d m a t e r i a l was assumed to be mononucleotide on the b a s i s of i t s e l u t l o n p o s i t i o n . 31 I t was d i g e s t e d w i t h a p r e p a r a t i o n of 5 ' nucleo-t i d a s e d i s s o l v e d i n 0.1 M sodium b a r b i t a l b u f f e r , pH 9.0 ( 4 2 ) . The phosphate l i b e r a t e d was assayed by a m o d i f i c a t i o n of the method de s c r i b e d by F l s k e and Subba Row ( 4 3 ) . PHOSPHATE ASSAY Each sample was made 2 ml i n volume w i t h g l a s s d i s t i l l e d water and an equal volume of the assay r e -agent (6N ^ S O J L J H2O1 2.5% ammonium molybdatei 10# a s c o r b i c a c i d , 1 « 2 1 1 1 1 , v/v/v/v) was added. The tubes were covered w i t h p a r a f l l m , shaken, and i n c u -bated a t 4 5 ° C f o r 20 minutes. The blue c o l o u r which appeared was estimated a t 8 0 0 nm, ASSAY FOR DNase I INHIBITOR Commercial DNase I was d i s s o l v e d i n T r l s b u f f e r , pH 7.5, and d i l u t e d to an a c t i v i t y of 5 u n i t s per ml as determined by the a c i d s o l u b l e o l i g o n u c l e o t i d e assay. P r o t e i n s o l u t i o n s suspected to c o n t a i n i n h i b i -t o r s of DNase I were p i p e t t e d i n t o S o r v a l l c e n t r i f u g e tubes c o n t a i n i n g the s o l u t i o n s f o r the a c i d s o l u b l e o l i g o n u c l e o t i d e assay. The standardized DNase I s o l u t i o n was added and the tubes were incubated and processed as d e s c r i b e d p r e v i o u s l y . 32 RESULTS AND DISCUSSION PREPARATION OF CRUDE ENZYME EXTRACT Two d i f f e r e n t procedures have been adopted to d i s r u p t the I n t e s t i n a l mucosa c e l l s and s o l u b l l i z e the DNase a c t i v i t y . In the one case enzyme prepa-r a t i o n s were obtained by homogenizing the t i s s u e f o r l o n g p e r i o d s of time a t low speeds i n an attempt to a v o i d foaming and p o s s i b l e d e n a t u r a t i o n . The second method i n v o l v e d s h o r t homogenization times and h i g h speeds to e f f e c t c e l l breakage as q u i c k l y as p o s s i b l e . Dixon and Webb (44) s t a t e t h a t many enzymes are denatured a t s u r f a c e s , f o r t h i s reason i t i s important to a v o i d the formation of foam. Large amounts of foam were produced i n the second method where hig h speed homogenization was employed? indeed more than h a l f of the crude enzyme e x t r a c t was present as a t h i c k pink foam which took approximately f i f t e e n minutes to s e t t l e . Denaturation occurs when the p r o t e i n molecule i s opened e i t h e r by u n f o l d i n g o r s e p a r a t i o n of the adjacent p o r t i o n s of polypeptide c h a i n s . This i s 33 u s u a l l y caused by the breaking of a l a r g e number of hydrogen bonds. Bovine DNase I has been shown (11) to possess a d i s u l f i d e bond which i s e s s e n t i a l f o r a c t i v i t y and Joins r e s i d u e s 170 and 206. Separation of the adjacent p o r t i o n s of the polypeptide chain i n the c a r b o x y l t e r m i n a l end of DNase I cannot occur without p r i o r r e d u c t i o n of the d i s u l f i d e b r i d g e . I t i s not known whether DNase I can be denatured a t a sur f a c e i f the e s s e n t i a l d i s u l f i d e b ridge i s i n -t a c t . However, samples of acetone powder i n b u f f e r e d 7 M urea e x h i b i t e d c o n s i d e r a b l e DNase I a c t i v i t y suggesting t h a t the enzyme i s not e a s i l y denatured by u n f o l d i n g . The crude enzyme e x t r a c t s prepared by low speed homogenization possessed both lower t o t a l a c t i v i t y and lower s p e c i f i c a c t i v i t y than the enzyme prepa-r a t i o n s obtained by the sh o r t h i g h speed homogeni-z a t i o n procedure. I t seems l i k e l y t h e r e f o r e , t h a t p r o t e o l y s i s r a t h e r than d e n a t u r a t l o n i s the major cause of l o s s of DNase I a c t i v i t y i n the p r e p a r a t i o n s obtained by low speed homogenization. STABILITY OF DNase I PREPARATIONS Figur e 5 shows a time course of the l o s s of T i m e i n H o u r s F i g . 5. Time course of the l o s s o f DNase I a c t i v i t y from a crude enzyme p r e p a r a t i o n l n 0.02 M T r i s - H C l b u f f e r , pH 7.2, c o n t a i n i n g no d i v a l e n t metal i o n s . A c t i v i t y was determined by the a c i d s o l u b l e o l i g o n u c l e o t i d e assay. 35 a c t i v i t y of a crude enzyme p r e p a r a t i o n s t o r e d a t 4° C i n 0.02 M T r i s b u f f e r . Enzyme a c t i v i t y de-c l i n e d r a p i d l y and h a l f the enzyme a c t i v i t y had disappeared w i t h i n 8 hours. The crude enzyme pre-p a r a t i o n remained c l e a r throughout the experiment and there was no evidence of p r e c i p i t a t i o n . The a d d i t i o n of d i v a l e n t metal ions helps maintain enzymic a c t i v i t y of the crude p r e p a r a t i o n ( 4 ) . In the presence of 10 mM Mg + +, DNase I a c t i v i t y was s t a b l e f o r a t l e a s t 2 days a t 4° C. However, storage of crude enzyme pr e p a r a t i o n s a t 4° C f o r more than 2 days g e n e r a l l y r e s u l t e d i n b a c t e r i a l contamination and subsequent l o s s of between 30$ and 50# of the a c t i v i t y of the f r e s h p r e p a r a t i o n s . For t h i s reason the crude enzyme pr e p a r a t i o n s were not s t o r e d p r i o r to p u r i f i c a t i o n . With i n c r e a s i n g p u r i f i c a t i o n the enzymio a c t i v i t y was no longer s t a b i l i z e d by magnesium i o n . A d d i t i o n of 5 mM calcium i o n , however, pre-served a c t i v i t y f o r s e v e r a l days a t 4° C. The more h i g h l y p u r i f i e d enzyme p r e p a r a t i o n s , t h e r e f o r e , were s t o r e d i n 0.1 M T r l s b u f f e r , pH 7 . 5 , c o n t a i n i n g 5 mM calcium i o n a t 4° C o r as a f r e e z e d r i e d powder a t -20° C. 36 CHROMATOGRAPHY ON DEAE 32 Ion exchange chromatography was adopted as the f i r s t step i n the p u r i f i c a t i o n of DNase I . Crude enzyme e x t r a c t (approximately 1500 mg of p r o t e i n ) was loaded onto a DEAE c e l l u l o s e column, 2 x 25 cm, prepared as desc r i b e d p r e v i o u s l y . A l a r g e amount of p r o t e i n con-t a i n i n g 5% to 10$ of the t o t a l e l u t e d enzyme a c t i v i t y washed through the column without being r e t a i n e d . The absorbed p r o t e i n was e l u t e d w i t h a l i n e a r phos-phate g r a d i e n t of 0.02 M to 0.25 M (Figure 6 ) . Two major peaks of p r o t e i n were f r a c t i o n a t e d . The f i r s t of these contained 90% to 95%,of the e l u t e d DNase a c t i v i t y . The second peak contained much p r o t e i n but was devoid of enzyme a c t i v i t y . Figure 6 shows the r e l a t i v e areas of these three p r o t e i n peaks. The amount of p r o t e i n i n the f i r s t and t h i r d peaks represents 90% of the t o t a l e l u t e d p r o t e i n . This p r o t e i n was dis c a r d e d . The t h e o r e t i c a l p u r i f i c a -t i o n based on 100% recovery of the enzyme was 10 f o l d . In ge n e r a l , however, the DEAE c e l l u l o s e chromatography step a f f o r d e d o n l y a 6-8 f o l d p u r i -f i c a t i o n , i n d i c a t i n g t h a t some enzyme a c t i v i t y was l o s t d u r i n g the procedure. Co 2 0 4 0 6 0 Fraction N o . F i g . 6. Chromatography o f a crude enzyme e x t r a c t of DNase I on DEAE c e l l u l o s e column e q u i l i b r a t e d w i t h 0.02 M phosphate b u f f e r , pH 6.8, c o n t a i n i n g 10 mM Mg + +. E l u t i o n w i t h a l i n e a r phosphate grad-i e n t 0.02 M to 0.25 M a l s o c o n t a i n i n g 10 mM Mg + +. Dotted l i n e r e p r e s e n t s a b s o r p t i o n a t 280 nm, s o l i d l i n e r e p r e s e n t s u n i t s o f DNase I a c t i v i t y , d o t t e d and dashed l i n e r e p r e s e n t s c o n d u c t i v i t y o f e l u t i o n g r a d i e n t . 38 REMOVAL OF PHOSPHATE The p a r t l y p u r i f i e d enzyme from the DEAE c e l l u -l o s e step was f r e e z e - d r i e d immediately a f t e r e l u t i o n . T h i s y i e l d e d a white powder c o n t a i n i n g l a r g e amounts of phosphate. The phosphate was removed by p r e c i p i -t a t i o n w i t h c a l c i u m i o n . T h i s was n e c e s s a r y as c a l -cium i o n i s r e q u i r e d to p r e s e r v e the a c t i v i t y o f the more h i g h l y p u r i f i e d enzyme p r e p a r a t i o n s . The amount of c a l c i u m i o n added as a p r e c i p i t a t i n g agent was c r u c i a l . A d d i t i o n o f excess c a l c i u m i o n p r e c i p i t a t e d a l l the phosphate and removed the b u f f e r i n g c a p a c i t y , which r e s u l t e d l n p r e c i p i t a t i o n o f p r o t e i n and DNase I. Th i s c o u l d be avoided by adding T r i s b u f f e r to the p a r t l y p u r i f i e d enzyme p r e p a r a t i o n b e f o r e the a d d i t i o n of c a l c i u m i o n . The a d d i t i o n o f too l i t t l e c a l c i u m i o n removed i n s u f f i c i e n t phosphate. When samples t r e a t e d i n t h i s way were chromatographed on Sephadex G-100, a white p r e c i p i t a t e formed which b l o c k e d the g e l column. Such columns had a v e r y poor flow r a t e and the r e s u l t s o b t a i n e d were v a r i a b l e . CHROMATOGRAPHY ON G-100 F o l l o w i n g the removal o f phosphate, the enzyme 39 p r e p a r a t i o n was f u r t h e r p u r i f i e d on Sephadex G-100. DNase I was retarded by the g e l and separated from the bulk of the p r o t e i n as ^ i l l u s t r a t e d i n Figure 7. A l a r g e peak of m a t e r i a l absorbing a t 280 nm was e l u t e d j u s t before the s a l t peak. Since t h i s m a t e r i a l was e l u t e d s l i g h t l y before the s a l t i t was probably r e t a i n e d because of i t s molecular dimensions and not because of i o n i c i n t e r a c t i o n s w i t h the g e l . The average molecular weight of t h i s f r a c t i o n must be approximately 5,000 dal t o n s o r l e s s s i n c e i t s e l u t i o n volume i s o n l y s l i g h t l y l e s s than the t o t a l volume of the column. This m a t e r i a l probably c o n s i s t s of amino a c i d s and short p o l y p e p t i d e s . The v o i d volume (V0) of the p r e p a r a t i v e Sephadex G-100 column was determined u s i n g Blue Dextran 2000. The p a r t i t i o n c o e f f i c i e n t K a v was c a l c u l a t e d from the w e l l known r e l a t i o n of Laurant and K i l l a n d e r (53)i K a v = - Vn Vt - V0 f o r Ve values from s e v e r a l p r e p a r a t i o n s . The value i of K a v obtained was used to estimate roughly the molecular weight of DNase I from a p l o t of K a v vs M..W. The molecular weight of DNase I was found to be 40,000 d a l t o n s . This i s somewhat h i g h e r than the value / \ I V I \ Fraction N o . F i g . 7. Chromatography of a DNase I p r e p a r a t i o n on Sephadex G-100 column e q u i l i b r a t e d w i t h 0.01 M T r i s - H C l b u f f e r , pH 7.5, c o n t a i n i n g 5 mM C a + + . Dotted l i n e r e p r e s e n t s a b o s r p t i o n a t 280 nm, s o l i d l i n e r e p r e s e n t s u n i t s of DNase I a c t i v i t y , d o t t e d and dashed l i n e r e p r e s e n t s c o n d u c t i v i t y o f e l u a n t . 41 obtained by a n a l y t i c a l g e l f i l t r a t i o n and suggests t h a t i o n i c i n t e r a c t i o n s w i t h the g e l played no p a r t i n the s e p a r a t i o n . RECHROMATOGRAPHY ON DEAE 32 The p a r t l y p u r i f i e d m a t e r i a l was f u r t h e r p u r i -f i e d by rechromatography on DEAE c e l l u l o s e i n phos-phate b u f f e r . A s m a l l amount of enzyme came through the column on a p p l i c a t i o n of the sample. Reduction of the amount of p r o t e i n a p p l i e d d i d not e l i m i n a t e t h i s f r a c t i o n , suggesting t h a t i t was not due to ove r l o a d i n g of the column. Ca t l e y et a l (40) have shown t h a t the carbohydrate moiety of bovine DNase IA i s attached to the enzyme through an amide bond i n -v o l v i n g t h e p -COOH of a s p a r t i c a c i d . I t was p o s s i b l e t h a t the enzyme which does not absorb to the DEAE c e l l u l o s e i s DNase which has l o s t i t s sugar group. Such a molecule would c o n t a i n an e x t r a negative charge due to the -COOH group. This assumes t h a t the amide i s removed w i t h , o r subsequent t o , the removal of the sugar groups. When a l l the unbound enzyme had been washed from the column a g r a d i e n t of 0.02 M to 0.10 M phosphate was a p p l i e d . A s i n g l e peak of p r o t e i n was e l u t e d . A l l the remaining DNase I a c t i v i t y was 42 found In the l e a d i n g edge of t h i s peak as i s shown i n F i g u r e 8. I t was e s s e n t i a l to add calcium i o n to the enzyme s o l u t i o n immediately a f t e r e l u t i o n from the column i n order to s t a b i l i z e the p r e p a r a t i o n * . Over $0% of the e l u t e d enzyme a c t i v i t y disappeared when the pr e p a r a t i o n was s t o r e d f o r 12 hours i n 0.02 M phosphate b u f f e r c o n t a i n i n g 10 mM magnesium i o n . A summary of the p u r i f i c a t i o n of DNase I i s presented i n Table I I . Table *II.t-> T y p i c a l r e s u l t s f o r a three step p r e p a r a t i o n of DNase I from r a t i n t e s t i n a l mucosa. The crude enzyme p r e p a r a t i o n was chromatographed on DEAE c e l l u l o s e , concentrated and a p p l i e d to Sephadex G-100 and rechromatographed on DEAE c e l l u l o s e . F r a c t i o n T o t a l U n i t s of A c t i v i t y T o t a l P r o t e i n S p e c i f i c A c t i v i t y (unlts/mg) Crude DEAE 32 G 100 DEAE 32 462 341 240 60 1524 mg 136 mg 20 mg 1.1 mg 0.3 2.5 12.0 60.0 The enzyme was p u r i f i e d 200 f o l d r e l a t i v e to the crude p r e p a r a t i o n . The y i e l d was \$%. LO Fract ion N o . P i g . 8. Chromatography of a DNase I p r e p a r a t i o n on a DEAE c e l l u l o s e column e q u i l i b r a t e d w i t h 0,02 M phos-phate b u f f e r , pH 6.8, c o n t a i n i n g 10 mM Mg . E l u t i o n with a l i n e a r phosphate g r a d i e n t 0.02 M to 0.10 M a l s o c o n t a i n i n g 10 mM Mg + +. Dotted l i n e r e p r e s e n t s absorp-t i o n a t 280 nm, s o l i d l i n e r e p r e s e n t s u n i t s o f DNase I a c t i v i t y , d o t t e d and dashed l i n e r e p r e s e n t s c o n d u c t i v i t y of e l u t i o n g r a d i e n t . 44 PROPERTIES OF DNase I Some of the c h a r a c t e r i s t i c s of the p u r i f i e d and p a r t l y p u r i f i e d enzyme p r e p a r a t i o n s were i n v e s t i g a t e d . The enzyme e x h i b i t e d maximal a c t i v i t y a t pH 6.8 ( F i g . 9); i t was more than 10 times as a c t i v e a t pH 6.8 as a t pH 5 . 0 . The d i v a l e n t metal i o n requirements o f the enzyme were i n v e s t i g a t e d . D i v a l e n t metal i o n s were e s s e n t i a l f o r a c t i v i t y , the o r d e r o f e f f e c t i v e n e s s i n a c t i v a t i n g enzyme a c t i v i t y . b e i n g Mn + +7 M g + + 7 C a + + . An equlmolar mixture o f M g + + and C a + + was almost as e f f e c t i v e an a c t i v a t o r as Mn + +. F i g u r e 10 i l l u s t r a t e s the r e s u l t s o b t a i n e d w i t h the 200 f o l d p u r i f i e d DNase I. As may be seen from the diagram, C a + + Is a p a r t i c u l a r l y weak a c t i v a t o r o f the enzyme. Enzyme a c t i v i t y was i n h i b i t e d i n the presence o f K + o r N a + (47) o r haemoglobin (48). Samples o f com-m e r c i a l DNase I were run as c o n t r o l s i n these e x p e r i -ments and found to be i n h i b i t e d to a comparable ex-t e n t as shotm i n F i g u r e 1 1 . Thus, the r e s u l t s w i t h r a t i n t e s t i n a l mucosa enzyme c o n f i r m those o b t a i n e d by o t h e r i n v e s t i g a t o r s (47, 48) u s i n g bovine \ p a n c r e a t i c DNase I. 5 6 7 8 .. pH Units _ F i g . 9. The e f f e c t of pH on the a c t i v i t y o f 10 f o l d p u r i f i e d DNase I. The pH range was covered by a c e t a t e , T r l s and M.E.S. b u f f e r s , a l l 0.1 M. Concentration x 1 0 0 0 F i g . 10. The e f f e c t of metal i o n s on the a c t i v i t y o f 100 f o l d p u r i f i e d DNase I . A l l measurements o f a c t i v i t y were c a r r i e d out by the a c i d s o l u b l e o l i g o n u c l e o t i d e assay i n 0 . 1 M M.E.S. b u f f e r , pH 6.8. 4 7 P i g . 11. The e f f e c t of N a , K and haemoglobin on r a t DNase I . The open c i r c l e s represent ac-t i v i t y of the r a t enzyme, the t r i a n g l e s repre-sent a c t i v i t y of the bovine p a n c r e a t i c enzyme. 48 The time course of the a c t i o n of DNase I on DNA was i n v e s t i g a t e d . The r e s u l t s are shown i n Figure 12. The curve obtained was sigmoidal i n form which i s not p r e d i c t e d by present knowledge on the mode of a c t i o n of DNase I . The curve d i s p l a y s an i n i t i a l l a g which i s an a r t i f a c t of the a c i d s o l u b l e o l i g o n u c l e o t i d e assay. When an endonuclease such as DNase I a t t a c k s a t random a l o n g s t r a n d of DNA i t i s expected t h a t the f i r s t few s c i s s i o n s of the DNA by the enzyme w i l l not produce o l i g o n u c l e o t i d e s short enough to be a c i d s o l u b l e . This means t h a t the a c t i v i t y of the enzyme i n d i c a t e d by the assay w i l l be lower than the a c t u a l a c t i v i t y . With l o n g e r i n c u b a t i o n time the curve p l a t e a u s ; t h i s i s probably due to a u t o r e t a r d a t i o n . A u t o r e t a r -d a t i o n of the DNase I r e a c t i o n has been f r e q u e n t l y observed w i t h the bovine enzyme. PRODUCTS OF HYDROLYSIS - 5' OR 3'? C a l f thymus DNA was d i g e s t e d w i t h 200 f o l d p u r i f i e d DNase I and the d i g e s t chromatographed by the method of Tomlinson and Tener (41). The mixture Time min P i g . 12. Time course o f the a c t i o n o f DNase I on DNA, Assay tubes c o n t a i n e d 0.3 mg o f DNA d i s s o l v e d i n 2.9 ml of M.E.S. b u f f e r , pH 6.8, c o n t a i n i n g 10 mM Mn + +. 50 of o l i g o n u c l e o t i d e s was r e s o l v e d i n t o peaks c o r r e s -ponding to 1, 2, or 3-degrees of p o l y m e r i z a t i o n . P a r t of the mononucleotide m a t e r i a l was d e s a l t e d by io n exchange on DEAE c e l l u l o s e a f t e r the method of Tomlinson and Tener (41). This m a t e r i a l was d i g e s t e d by the method of Dixon (42) w i t h 5' n u c l e o t i d a s e from C r o t a l u s Adamanteus venom and analyzed f o r phosphate by the procedure d e s c r i b e d p r e v i o u s l y (see M a t e r i a l s and Methods). The r e s u l t s obtained are shown below i n Table I I I . Table I I I . N u c l e o t i d e s were produced by the a c t i o n of DNase I on DNA and separated by i o n exchange chro-matography on DEAE c e l l u l o s e i n 7 M urea. The n u c l e o t i d e s were p u r i f i e d , d i g e s t e d w i t h 5' nuc-l e o t i d a s e and the d i g e s t assayed f o r phosphate. Digest N u c l e o t i d e 5'AMP 5' Water FOjf Number Nucleotidase JJ moles/ml 1 + + - 1.0 2 - + + - 0.87 3 + - - + 0.11 The 5' n u c l e o t i d a s e l i b e r a t e d phosphate from the mono-n u c l e o t i d e f r a c t i o n , demonstrating t h a t t h i s f r a c t i o n 51 contains phosphate i n the 5' p o s i t i o n . The amounts of mononucleotides and 5' AMP used, as measured by absorbance a t 260 nm, were roughly e q u a l . This r e s u l t , taken together w i t h the observation t h a t the amounts of phosphate l i b e r a t e d from each f r a c t i o n by 5' nu-c l e o t i d a s e were almost eq u a l , suggests t h a t most, o r a l l , of the mononucleotides were present as 5 ' mono-e s t e r s . U n f o r t u n a t e l y , t h i s was not t e s t e d by diges-t i o n w i t h 3 ' n u c l e o t i d a s e . CHARACTERIZATION OF THE ENZYME Based on the c r i t e r i a o r i g i n a l l y l i s t e d by L a s k o w s k i ( 1 8 ) , the enzyme p u r i f i e d from r a t i n t e s t i n a l mucosa may be c h a r a c t e r i z e d as a DNase I type enzyme. I t has a n e u t r a l pH optimum and r e q u i r e s d i v a l e n t metal ions f o r a c t i v i t y . Laskowski proposed f o u r major c r i t e r i a f o r the c l a s s i f i c a t i o n of n u c l e a s e s i 1) S p e c i f i c i t y toward the sugar moiety. Incubation of p a r t l y p u r i f i e d DNase I w i t h RNA under standard c o n d i t i o n s produced no incr e a s e i n A 2 3 Q . 2) E n d o n u c l e o l y t l c vs E x o n u c l e o l y t l c Mode of A c t i o n This was not t e s t e d d i r e c t l y but a l a g i n the a c t i o n of DNase on DNA was observed. This suggestJan e n d o n u c l e o l y t l c mode of 52 a c t i o n s i n c e an exonuclease would produce a c i d s o l u b l e mononucleotides immediately. 3) S p e c i f i c i t y of Cleavage to Form 5' o r 3' Monoesters T h i s was I n v e s t i g a t e d and the enzyme was found to produce mainly 5' monoesters. However, the v-•• p o s s i b i l i t y t h a t some 3' m a t e r i a l was produced was not e l i m i n a t e d . 4) Base S p e c i f i c i t y The base s p e c i f i c i t y o f the r a t enzyme was not i n v e s t i g a t e d as the base s p e c i f i c i t y o f DNase I i s thought to change throughout the r e a c t i o n . Numerous i n v e s t i g a t o r s have attempted to demon-s t r a t e a base s p e c i f i c i t y o f DNase I but none have been s u c c e s s f u l . More r e c e n t l y , Laskowski ( 2 ) has suggested t h a t these c r i t e r i a f o r DNase I s h o u l d be r e p l a c e d by a s i n g l e c r i t e r i o n , namely r e a c t i o n w i t h a p r o t e i n i n -h i b i t o r o f DNase I. The v a r i o u s f r a c t i o n s from the f i r s t chromato-g r a p h i c s t e p on DEAE c e l l u l o s e were t e s t e d f o r the presence o f a DNase I i n h i b i t o r as p r e v i o u s l y d e s c r i b e d . No evidence f o r the presence of a DNase I i n h i b i t o r was found. Attempts to prepare the i n h i b i t o r from r a t s p l e e n were u n s u c c e s s f u l . 53 AMMONIUM SULFATE FRACTIONATION The r e s u l t s o f ammonium s u l f a t e f r a c t i o n a t i o n o f the crude enzyme p r e p a r a t i o n are shown l n F i g u r e 13. Most of the enzyme p r e c i p i t a t e d between 30% to 50% s a t u r a t i o n o f ammonium s u l f a t e . T h i s gave a two f o l d p u r i f i c a t i o n and a 75% y i e l d . T h i s step was not used as p a r t o f the p u r i f i c a t i o n procedure. The r e s u l t s o b t a i n e d d i f f e r e d s i g n i f i c a n t l y from those o f Lee and Zbarsky (50). They found t h a t most o f the DNase I a c t i v i t y p r e c i p i t a t e d between 15# to 20% s a t u r a t i o n o f ammonium s u l f a t e . T h i s gave a 6k% y i e l d and a 20 f o l d p u r i f i c a t i o n . They a l s o found t h a t 85% of the p r o t e i n i n the crude enzyme e x t r a c t was p r e c i p i t a t e d a t 10$ s a t u r a t i o n o f ammonium s u l f a t e . Such r e s u l t s a re unprecedented f o r e x t r a c t s p repared from whole mammalian c e l l s . MOLECULAR WEIGHT The m o l e c u l a r weight o f the r a t enzyme was e s t i -mated by g e l f i l t r a t i o n on Sephadex G-100. E a r l y attempts to e s t a b l i s h the m o l e c u l a r weight o f the enzyme u s i n g samples c o n t a i n i n g s a l t gave w i d e l y d i f -f e r i n g m o l e c u l a r weight e s t i m a t e s r a n g i n g up to 40,00 d a l t o n s . In the f i n a l experiments, two, d e s a l t e d , .2Saturation of Ammonium Sulfate ... F i g . i 3 . F r a c t i o n a t i o n o f crude enzyme prepa-r a t i o n by ammonium s u l f a t e . H o r i z o n t a l bars r e p r e s e n t p r o t e i n i n supernatant, unshaded boxes r e p r e s e n t p r o t e i n i n p r e c i p i t a t e , curved l i n e s r e p r e s e n t enzyme a c t i v i t y . 55 p a r t l y p u r i f i e d samples of DNase I gave i d e n t i c a l e l u t i o n p r o f i l e s . The peak of a c t i v i t y e l u t e d i n the same tube i n each run. These r e s u l t s were f u r t h e r checked w i t h a sample of commercial DNase I which a l s o e l u t e d i n e x a c t l y the same p o s i t i o n as the r a t enzyme, Although the p r i n c i p l e s of g e l f i l t r a t i o n have been known s i n c e 1956 when Lathe and Ruthven (51) separated p o l y s a c c h a r i d e s and p r o t e i n s on s t a r c h g e l s , no r i g o r o u s t h e o r e t i c a l treatment e x i s t s . I t i s g e n e r a l l y agreed t h a t the t o t a l volume V t of a g e l column i s given byt V t = v 0 +V1 + V m where V 0 i s the volume out s i d e the g e l g r a i n s , i s the volume i n s i d e the g e l g r a i n s and V m i s the volume of the g e l m a t r i x . For molecules able to penetrate p a r t of the i n n e r volumei V e - V c + K d . V l where V Q i s the e l u t i o n volume and K d i s a constant. Porath (52) assumed th a t the a c c e s s i b l e volume I n s i d e the g e l p a r t i c l e was cone shaped and p r e d i c t e d t h a t i ^/ K d < v/M.W.-where M.W. i s the molecular weight. 56 I t i s u s u a l i n p r e p a r i n g c a l i b r a t i o n curves f o r molecular weight determinations on Sephadex to p l o t . K a v vs Log M.W. K a v was d e f i n e d by Laurant and K i l l a n d e r (53) &s» K a v »Ze - V o Vt - v 0 and I s an empircal r e l a t i o n s h i p . In the present work, the data obtained have been analyzed by both of the above methods. A n a l y s i s of the data by the method of Porath (52) i n d i c a t e s a molecular weight of 29,700 d a l t o n s , whereas a n a l y s i s by the method of Laurant and K i l l a n d e r (53) i n d i c a t e s a molecular weight of 30.500 d a l t o n s . Lieberman et a l (54) c a l c u l a t e d t h a t the molecular weight of DNase I from the s m a l l i n t e s t i n e of r a t was between 32,00 and 35.000 d a l t o n s . Commercial DNase IA has a molecular weight of 30,072 daltons (11)j t h i s value corresponds w i t h t h a t obtained f o r the DNase I from r a t i n t e s t i n a l mucosa. 57 GEL ELECTROPHORESIS A DNase I p r e p a r a t i o n , which had been p a r t l y p u r i f i e d on DEAE 32, was chromatographed on Sephadex G-100 as p r e v i o u s l y d e s c r i b e d . Samples o f DNase I were taken from the l e a d i n g edge, c e n t r e , and t r a i l i n g edge o f the enzyme peak t o g e t h e r w i t h a sample of the enzyme p r e p a r a t i o n a p p l i e d to the G-100 column and a n a l y z e d by g e l e l e c t r o p h o r e s i s . F i g u r e 14 shows the o p t i c a l d e n s i t y t r a c e o b t a i n e d f o r the v a r i o u s f r a c -t i o n s . I t was expected t h a t f r a c t i o n s taken from the l e a d i n g edge of„ the enzyme peak would be comprised mainly o f p r o t e i n s p o s s e s s i n g m o l e c u l a r weights g r e a t e r than t h a t o f DNase I . S e p a r a t i o n by polyac(rylamlde g e l e l e c t r o p h o r e s i s i s dependent on charge and molecu-l a r weight. Thus, s i n c e the p r o t e i n sample c o n s t i t u t e d a s i n g l e f r a c t i o n from an a n i o n exchange column,it might be expected to c o n t a i n p r o t e i n s o f s i m i l a r charge. F r a c t i o n a t i o n o f t h i s sample by g e l e l e c t r o p h o r e s i s , t h e r e f o r e , would be p r i m a r i l y dependent on m o l e c u l a r weight d i f f e r e n c e s . I f t h i s assumption i s c o r r e c t , the p r o t e i n s i n the f r a c t i o n s from the l e a d i n g edge o f the peak sh o u l d produce bqnds c l o s e to the 58 o r i g i n o f the g e l . The p r o t e i n s p e c i e s l n the f r a c -t i o n s from the middle o f the enzyme peak sh o u l d mi-g r a t e towards the c e n t r e r e g i o n o f the g e l w h i l e those i n the f r a c t i o n s from the t r a i l i n g edge sh o u l d move to the end o f the g e l . T h i s was observed as may be seen by i n s p e c t i o n o f F i g u r e 14. Since the t o t a l a c t i v i t y o f each f r a c t i o n and the t o t a l p r o t e i n a p p l i e d to each g e l were known, i t should be p o s s i b l e to i d e n t i f y one band as the enzyme band. Assuming t h a t each band r e p r e s e n t s one p r o t e i n and t h a t the a r e a under each band of the o p t i c a l d e n s i t y t r a c e s i s p r o p o r t i o n a l to the amount o f pro-t e i n p r e s e n t , then A e°£ E and A e/E = K where A i s the area of any p r o t e i n band, A e i s the a r e a o f the enzyme p r o t e i n band, E i s the amount o f enzyme p r e s e n t and K i s a c o n s t a n t . The r a t i o A n/E i s c a l c u l a t e d f o r n - l to n = the t o t a l number of p r o t e i n bands on the g e l f o r each g e l of the s e r i e s . One band sh o u l d d i s -p l a y a constant r a t i o o f band a r e a to enzyme a c t i v i t y f o r a l l the g e l s ; t h i s band i s expected to be the enzyme band. Tables IV and V below show the r e s u l t s o b t a i n e d . 59 Table IV. A c t i v i t y determined by the a c i d s o l u b l e o l i g o n u c l e o t i d e assay and o p t i c a l d e n s i t y a t 280 nm of the samples of p u r i f i e d en-zyme e l u t e d from Sephadex G-100 which were subjected to g e l e l e c t r o p h o r e s i s . F r a c t i o n # A280 A260 (assay) 9 1.28 0.01 11 0.47 0.31 12 0.48 0.76 14 0.23 0 .03 Table V. R e l a t i v e i n t e n s i t y of each p r o t e i n band as determined by area under o p t i c a l d e n s i t y t r a c e s f o r each of s e v e r a l bands from g e l s corresponding to f r a c t i o n s 11 and 12. Band # 5 6 7 8 Areas 787 863 535 172 R e l a t i v e 1.0 i . l 0.68 0.25 I n t e n s i t y 3.4 An/2 3.1 2.1 0.7 Area .' , 180 432 504 86 R e l a t i v e 1 2.32 2.71 0.46 I n t e n s i t y 1.32 3.1 3.6 0.6 F i g . 14. O p t i c a l d e n s i t y t r a c e s of polyacrylamide g e l s . Gels were run a t 150 V f o r 2 hours, s t a i n e d w i t h Coomaise Blue and de s t a l n e d e l e c t r o p h o r e t i c a l l y . 61 These r e s u l t s suggest t h a t e i t h e r band 6 or 8 i s the enzyme band. I t was not p o s s i b l e from these data to e s t a b l i s h which i s the enzyme band. Indeed, the data obtained c a s t doubt on the assumption t h a t each band represents a s i n g l e p r o t e i n s p e c i e s . In-sp e c t i o n of Figure 14B shows a s p l i t peak which i s not r e s o l v e d i n Figure 14C. An attempt to determine the exact l o c a t i o n of the enzyme was made by assa y i n g DNase I a c t i v i t y i n s i t u In the g e l s . DNA was added to acrylamlde g e l s and samples c o n t a i n i n g DNase I were subjected to e l e c t r o p h o r e s i s . A f t e r e l e c t r o p h o r e s i s these g e l s were incubated a t 37° C f o r 24 hours and developed i n 0.5 N HC1. A broad d i f f u s e zone of c l e a r i n g was observed c o v e m i n g the l a s t t h i r d of the g e l . Other attempts to l o c a l i z e DNase I a c t i v i t y have a l s o f a i l e d . F l a n a g a n (60) embedded a polyachrylamlde g e l i n a DNA-agar g e l prepared by the method p r e v i -o u s l y d e s c r i b e d . The system was incubated f o r 24 hours a t 37° C and developed i n 0.5 N HC1. No DNase a c t i v i t y was detected. The experiment was repeated w i t h the polyacrylamide g e l s s p l i t lengthwise and then embedded i n the DNA-agar g e l , but nov enzyme a c t i v i t y c o u l d be detected. 62 Figures 15A and 15B show o p t i c a l d e n s i t y scans of ten f o l d p u r i f i e d Dnase I (15A) and 300 f o l d p u r i -f i e d DNase I (15B). The 300 f o l d p u r i f i e d sample shows only one major peak corresponding to e i t h e r peak 5 or peak 6 on Figure 14A. On the b a s i s of these data i t seems probable t h a t band 6 represents the DNase I enzyme. 63 Fig. 15. Optical density traces of polyacrylamide gels. Fig. 15A shows a sample of enzyme 10 fold purified by a single chromatography on DEAE cellu-lose. Fig. 15B shows a sample of enzyme purified 300 fold. 64 INTERACTIONS BETWE ? CALCIUM ION AND DNase I The i n t e r a c t j 3 between c a l c i u m i o n and DNase I are o f c o n s i d e r a b l i n t e r e s t . in the presence of 10"^ M c a l c i u m lor. he bovine enzyme i s s t a b i l i z e d a g a i n s t p r o t e o l y s S ( 4 ) . D e s p i t e the important r o l e p l a y e d by c a l c i u m i o n i n t i e s t a b i l i z a t i o n o f DNase I, t h i s c a t i o n i s n o t the most e f f e c t i v e a c t i v a t o r o f the enzyme. P r i c e e t a l (7) have suggested t h e r e f o r e t h a t the bovine enzyme possesses s e v e r a l b i n d i n g s i t e s f o r metal i o n s . B i n d i n g o f c a l c i u m i o n a t one s i t e causes a change i n the shape o f the enzyme molecule which r e -duces i t s s u s c e p t i b i l i t y to p r o t e o l y t i c a t t a c k . T h i s s i t e i s s p e c i f i c f o r c a l c i u m i o n . DNase I a l s o r e q u i r e s metal i o n s f o r c a t a l y t i c a c t i v i t y . However, s i n c e the enzyme i s not a p p r e c i a b l y a c t i v a t e d by the c a l c i u m i o n c o n c e n t r a t i o n which pro-t e c t s i t from p r o t e o l y s i s , a second b i n d i n g s i t e has been p o s t u l a t e d . Magnesium o r manganese i o n s a t con-c e n t r a t i o n s o f 5 x 10"^ M s t r o n g l y a c t i v a t e the enzyme, whereas a s i m i l a r c o n c e n t r a t i o n o f c a l c i u m i o n has l i t t l e a f f e c t on enzyme a c t i v i t y . 65 M g + + a t a c o n c e n t r a t i o n o f 1 mM i s a weak a c t i -v a t o r w h i l e 0.2 M C a + + i s i n e f f e c t i v e as an a c t i v a t o r of the enzyme.. In c o n t r a s t , a mixture o f these two i o n s , a t the c o n c e n t r a t i o n s s t a t e d , s t r o n g l y a c t i v a t e s the enzyme. In the presence o f 10"^ M Mg + +, a concen-t r a t i o n o f 5 x 10""^ M c a l c i u m i o n a c t i v a t e s , whereas i n the presence o f h i g h e r c o n c e n t r a t i o n s o f C a + + DNase I a c t i v i t y i s i n h i b i t e d , as i s shown i n F i g u r e 18. To study f u r t h e r the i n h i b i t i o n by C a + + , Lineweaver-Burk- p l o t s were d e r i v e d f o r a number o f c a l c i u m i o n c o n c e n t r a t i o n s . As shown i n F i g u r e 16, a l l the l i n e s i n t e r s e c t a t the same p o i n t on the 1/v axes, i n d i c a t i n g t h a t c a l c i u m i o n i s a c o m p e t i t i v e i n h i b i t o r . The dat a o b t a i n e d were a n a l y z e d by the c l a s s i c a l Lineweaver and Burk e q u a t i o n (55) as follows« 1 = K m f l + 1 ^ 1 + 1 Where V i s the r e a c t i o n v e l o c i t y , i s the M i c h a e l l s -Menten co n s t a n t , V m i s the maximal r e a c t i o n v e l o c i t y , I i s the c o n c e n t r a t i o n o f i n h i b i t o r and S i s the c o n c e n t r a t i o n o f magnesium i o n . F o r £l] = 0, the sl o p e o f the l i n e i n the double r e c i p r o c a l p l o t i s K m/V m. Thus, i t i s p o s s i b l e to estimate how s t r o n g l y c a l c i u m i o n bi n d s t o the DNase I-DNA system when i t F i g . 16. Lineweaver Burk p l o t showing the r e s u l t s obtained w i t h calcium ion a t d i f f e r e n t c o n c e n t r a t i o n s . _ K L X F i g . 1 ?. Plot of K i , the i n h i b i t o r constant f o r Ca* + acting as a competitive i n h i b i t o r , against the concentration of C a + + . F i g . 18, S t i m u l a t i o n of DNase I a c t i v i t y by C a + + i n the presence of 1.0 mM Mg + +. A c t i v i t y was de-termined by the a c i d s o l u b l e o l i g o n u c l e o t i d e assay. 69 i s a c t i n g as a c o m p e t i t i v e i n h i b i t o r . The v a l u e s o f o b t a i n e d by s u b s t i t u t i o n i n the equ a t i o n above are t a b u l a t e d i n Table V I . Table VI. Values o b t a i n e d f o r K l t the i n h i b i t o r b i n d i n g constant, f o r d i f f e r e n t con-c e n t r a t i o n s o f C a + + i n the DNase I-DNA system. C o n c e n t r a t i o n s 5 lOmM 15 mM 20 mM of C a + + 5 x 10-3 2.8 x 10-3 2 z 10-3 6 x 10"^ 2.5 x 10-3 1.4 x 10-3 1.35 x 10~3 4.6 x 10-^ 2 x 10-3 1.9 x 10-3 .86 x 10~3 1.8 x 10-3 1.05 x 10-3 3.3 x I O - 3 1.97 x 10"3 1.32 x 10"3 5.3 x 10" The r e s u l t s i n d i c a t e t h a t the ext e n t o f b i n d i n g of c a l c i u m i o n i n c r e a s e s w i t h i n c r e a s i n g c o n c e n t r a t i o n s of t h i s metal i o n . The dependence o f on the concen-t r a t i o n o f c a l c i u m i o n i s i l l u s t r a t e d i n F i g u r e 17 where K± i s p l o t t e d a g a i n s t C a + + . The curved p l o t v may be e x p l a i n e d i n e i t h e r o f two ways. Calcium i o n may e x e r t i t s e f f e c t by b i n d i n g to e i t h e r DNase I o r DNA. 1) I f C a + + b i n d s t o the enzyme the c o m p e t i t i v e i n h i b i t i o n may be e x p l a i n e d as f o l l o w s J DNase I a c t i v i t y r e q u i r e s the b i n d i n g o f two metal i o n s a t d i f f e r e n t s i t e s . Optimal a c t i v i t y i s o b t a i n e d Values o b t a i n e d f o r K i by sub-s t i t u t i o n l n the Lineweaver Burk equ a t i o n Average v a l u e f o r K i 70 when one s i t e i s o c c u p i e d by c a l c i u m and the o t h e r by magnesium. I n c r e a s i n g the c o n c e n t r a t i o n of c a l c i u m i n c r e a s e s the number of enzyme molecules which have both s i t e s f i l l e d by c a l c i u m . T h i s b i - c a l c i u m complex i s o n l y s l i g h t l y a c t i v e c a t a l y t i c a l l y and so the t o t a l r e a c t i o n v e l o c i t y V i s decreased. Hovfever, s i n c e the b i - c a l c i u m complex possesses some c a t a l y t i c a c t i v i t y , the v e l o c i t y V i s g r e a t e r than the expected v e l o c i t y f o r the complex c o n t a i n i n g both magnesium and c a l c i u m a c t i n g a l o n e . Since the Llneweaver Burk eq u a t i o n was d e r i v e d f o r a dead end i n h i b i t o r , h y d r o l y s i s due to the b i c a l c i u m complex causes an i n c r e a s e i n the v a l u e of V over the p r e d i c t e d v a l u e and a decrease i n K A w i t h i n c r e a s i n g c o n c e n t r a t i o n o f metal i o n . 2) I f c a l c i u m i o n e x e r t s i t s e f f e c t s by b i n d i n g to DNA, then some r a t i o o f c a l c i u m i o n to magnesium i o n must be o p t i m a l f o r enzyme a c t i v i t y to account f o r the observed r e s u l t s . T h i s r a t i o i s about 2 t l (MgiCa). I f the c o n c e n t r a t i o n s of b o t h metal Ions are r a i s e d , the enzyme i s s t i l l o p t i m a l l y a c t i v a t e d , i f the r a t i o remains 2 i l . Comparison o f f i g u r e s 18 and 19 i l l u s t r a t e t h i s p o i n t . I n h i b i t i o n by c a l c i u m thus appears to be due to departure from t h i s ^3 i n t h e p r e s e n c e o f 5 mM C a + + . A c t i v i t y was d e -t e r m i n e d by t h e a c i d s o l u b l e o l i g o n u c l e o t i d e a s s a y . 72 optimum r a t i o . The s i g n i f i c a n c e o f the r a t i o i s n o t understood. The d a t a o b t a i n e d are not s u f f i c i e n t t o decide between these a l t e r n a t i v e s . However, P r i c e (8) has c a l c u l a t e d an average d i s s o c i a t i o n c o n s t a n t of 1.4 x 1Q~5 M f o r the two s t r o n g metal i o n b i n d i n g s i t e s o f the bovine enzyme. A number o f weaker s i t e s a l s o e x i s t . In the p r e s e n t study, the v a l u e s o b t a i n e d f o r ranged from 3 x 10-3 M to 5 x 10"^ M. T h i s weak b i n d i n g p r b v i d e s I n d i r e c t evidence t h a t c a l c i u m i o n b i n d s to the DNA r a t h e r than the enzyme. 73 CONCLUSIONS DNase I a c t i v i t y associated, w i t h a 105.000 x g supernatant o b t a i n e d from an homogenate o f r a t i n t e s -t i n a l mucosa t i s s u e was p u r i f i e d and c h a r a c t e r i z e d . The p r o p e r t i e s o f t h i s enzyme were compared w i t h those of the bovine p a n c r e a t i c DNase I, which has been i n -t e n s i v e l y c h a r a c t e r i z e d . 1) A 3 step column chromatography procedure was employed to o b t a i n an approximately 200 f o l d p u r i -f i c a t i o n o f the crude enzyme p r e p a r a t i o n . On s e v e r a l o c c a s i o n s a 400 f o l d p u r i f i c a t i o n was a c h i e v e d . 2) Ammonium s u l f a t e f r a c t i o n a t i o n e f f e c t e d o n l y a 2 f o l d p u r i f i c a t i o n o f the crude enzyme pre -p a r a t i o n . T h i s method, t h e r e f o r e , was n o t Inc l u d e d - i n the r o u t i n e p u r i f i c a t i o n procedure. The d i s c r e -pancy between t h i s r e s u l t and the f i n d i n g s o f Lee and Zbarsky (50) may be due to d i f f e r e n c e s i n te c h -n i c a l d e t a i l , f o r example, c o n t r o l o f pH. 3) The l a b i l i t y o f the i n t e s t i n a l mucosa DNase I i n c r e a s e d d r a m a t i c a l l y d u r i n g the p u r i f i c a -t i o n procedure. While magnesium i o n s were e f f e c t i v e i n s t a b i l i z i n g the crude enzyme e x t r a c t , o n l y c a l c i u m i o n s s t a b i l i z e d the most p u r i f i e d enzyme. Protease a c t i v i t y was d e t e c t e d i n the enzyme p r e p a r a t i o n s 74 by the c a s e i n d i g e s t i o n method of K u n i t z (61). The s p e c i f i c a c t i v i t y . o f protease i n c r e a s e d on p u r i f i c a -t i o n of the DNase I , i n d i c a t i n g t h a t the protease was being c o - p u r i f l e d . 4) Calcium ions a c t i v a t e d as w e l l as s t a b i l i z e d DNase I a c t i v i t y . I t was of i n t e r e s t , t h e r e f o r e , to study the b i n d i n g of metal ions i n the DNase I-DNA system. This was done i n d i r e c t l y by examining the e f f e c t s on DNase I a c t i v i t y of a C a + + p l u s M g + + a c t i v a t i n g system i n which the co n c e n t r a t i o n of each metal i o n was v a r i e d independently of the other. I t was found that calcium i o n behaved as a competitive i n h i b i t o r of DNase I a c t i v i t y . The i n h i b i t o r con-s t a n t , K^, was estimated to be 5 z IO--* M. This value d i f f e r s s i g n i f i c a n t l y from the K^ value ( d i s s o c i a t i o n constant f o r calcium b i n d i n g to DNase I) determined by g e l f i l t r a t i o n and suggests t h a t the metal ions are b i n d i n g to the DNA. 5) On the b a s i s of i t s ,pH optimum, i n a c t i v a t i o n by iodoacetate, s e n s i t i v i t y to i n h i b i t i o n by N a + or haemoglobin and molecular s i z e as estimated by g e l f i l t r a t i o n on Sephadex G-100 the DNase I enzyme from r a t I n t e s t i n a l mucosa was i d e n t i c a l w i t h the bovine p a n c r e a t i c DNase I . I t i s p o s s i b l e , t h e r e f o r e , t h a t 75 these enzymes are homologous o r d i f f e r o n l y In a s m a l l number of amino a c i d r e s i d u e s . 6) I f h i g h l y p u r i f i e d DNase I from r a t i n t e s t i n a l mucosa were a v a i l a b l e a t r y p t i c " f i n g e r p r i n t " c o u l d be prepared and compared d i r e c t l y w i t h a s i m i l a r " f i n g e r -p r i n t " o f the bovine p a n c r e a t i c DNase I.to determine whether the enzymes are Indeed homologous. Any d i f -f e r i n g p e p t i d e s c o u l d be r e a d i l y i d e n t i f i e d s i n c e the sequence of p a n c r e a t i c DNase I Is known (11). I f d i f -f e r e n t p e p t i d e s d i d e x i s t , these c o u l d be a n a l y s e d . Thus a complete sequence of DNase I from r a t i n t e s -t i n a l mucosa c o u l d be o b t a i n e d w i t h a minimum of ex-p e r i m e n t a l e f f o r t . U n f o r t u n a t e l y , the p a r t i a l l y p u r i f i e d enzyme de-s c r i b e d here i s not homogeneous. Gel e l e c t r o p h o r e s i s of a p r e p a r a t i o n which had been p u r i f i e d 300 f o l d demon-s t r a t e d the presence o f one dominant p r o t e i n band t o -g e t h e r w i t h s e v e r a l minor bands. P r e p a r a t i v e g e l e l e c t r o p h o r e s i s of t h i s p a r t i a l l y p u r i f i e d enzyme p r e p a r a t i o n f o l l o w e d by g e l f i l t r a t i o n on Sephadex G-100 might y i e l d a DNase I p r e p a r a t i o n o f r a t i n t e s -t i n a l mucosa s u i t a b l e f o r sequencing s t u d i e s . i 76 BIBLIOGRAPHY 1. German, G., a n d O k a d a , S., 23_, 621-623 (1957) B i o c h i m . B i o p h y s . A c t a . 2. L a s k o w s k i , M., S r . , A d v a n c e s i n E n z y m o l o g y 29, 169 f f (1967) 3. L i n d b e r g , U. B i o c h e m . 6, 335-342 (1967). 4. 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