Partial localization of the 5S RNA binding site on 23S RNA

BIOCHIMIE, 1972, 54, 41-45.

Partial localization of the 5S RNA binding site on 23S RNA. P. N. GRAY and R. MONIER. Centre de B i o c h i m i e et de Biologie Moldcnlaire, CNRS, Marseille. (27/1/1972). Summary. - - After splitting of 23S RNA in two fragments, according to the technique of ALLET and SPAHR [9], the larger fragment, which contains the 3'-end of the molecule, is still able to participate in the formation of a stable complex with 5S RNA in the presence of four proteins isolated from the 50S subunit. The smaller fragment has no significant binding activity. This observation suggests that the 23S RNA site(s) which participate in the cooperative binding of 5S RNA and the four proteins involve nucleotides belonging to the section of the molecule from which the 18S fragment originates.

INTRODUCTION. The study of the specific i n t e r a c t i o n s of isolated p r o t e i n s w i t h large RNA molecules can offer some i n s i g h t into the r e c o g n i t i o n a n d i n t e r a c t i o n of these molecules in a n a t u r a l assembly system such as the ribosome. The complexity of such a system is a p p a r e n t w h e n one considers that the bacterial ribosome c o n t a i n s 54-55 p r o t e i n s a n d three different RNA molecules [1, 2]. These not only form a very complex s t r u c t u r a l u n i t but a m u l t i f u n c t i o n a l one as well. Since the 7(~S bacterial ribosome can be separated into two subunits, 30S a n d 50S, this complexity can be r e d u c e d to a large extent. W h i l e the r e l a t i o n s h i p s b e t w e e n the RNA a n d p r o t e i n s of the 30S particle are being studied by a variety of t e c h n i q u e s [3-6], there is little evidence c o n c e r n i n g the r e c o g n i t i o n a n d i n t e r a c t i o n of the 33-34 p r o t e i n s a n d the two molecules of RNA, 5S a n d 2'3'S, w h i c h make up the 50S s u b u n i t [7-12].

50S s u b u n i t [14]. I n this report we a p p r o a c h the localization of p r o t e i n a n d 5S RNA b i n d i n g sites on the 23S RNA molecule. Our results d e m o n s t r a t e that the specificity for 5S RNA b i n d i n g to 23S RNA resides i n a 3'-terminal fragment of the 23S RNA. This b i n d i n g is specific, e q u i m o l a r a n d can be b r o u g h t about b y either a c r u d e m i x t u r e of 10 ribosomal p r o t e i n s or by the four purifi'ed p r o t e i n s we have s h o w n to be essential for f o r m i n g a 5S23S RNA complex [15]. EXPERIMENTAL. Ribosomes a n d ribosomal s u b u n i t s w e r e prepared from E s c h e r i c h i a colt RNase 1-10 [16] as previously described [17]. Ribosomal s u b u n i t s were stored as pellets at ~ 9 0 ° C .

T h e r e have been some recent reports concern i n g s t r u c t u r a l i n t e r a c t i o n s w i t h i n the 50,S s u b u n i t a n d these studies have relied on p a r t i a l degradation or u n f o l d i n g of the entire 50S subunit. Proteolytic enzymes have been used by CHANG a n d FLAKS [8] to study the i n a c t i v a t i o n of certain ribosomal functions. ALLET a n d SPAHR [9] have studied the protein-RNA complexes r e m a i n i n g after partial degradation of the 50S s u b u n i t w i t h p a n c r e a t i c nuclease. A t h i r d method receiving m u c h attention is the p a r t i a l u n f o l d i n g and s u b s e q u e n t release of some p r o t e i n s from the 50S particle by high salt c o n c e n t r a t i o n s [10, 12, 13].

32P-5S RNA was p r e p a r e d as described by MONIRR and FEUNTEUN [18] a n d was the generous gift of Dr G. BELLEMARE. I n t a c t 23S RNA a n d 16S RNA were p r e p a r e d by phenol-SDS extraction of p u r i f m d ribosomal s u b u n i t s a n d isolated by sucrose g r a d i e n t s e d i m e n t a t i o n [19]. The large ¢ 1 8 S ~ , a n d small ¢ 13S >>, fragments of 2i3S RNA were o b t a i n e d by c o n t r o l l e d p a n c r e a t i c r i b o n u c l e a s e deg r a d a t i o n of 50S s u b u n i t s a c c o r d i n g to the method of ALLET a n d SPAHR [9]. The g r a d i e n t fractions c o n t a i n i n g the large a n d small f r a g m e n t particles were pooled, c o n c e n t r a t e d by Diaflo ultrafiltration (UM-10 m e m b r a n e ) a n d reisolated u n d e r the same conditions. The RNA from these purified large and small <> fragments were then extracted as described above for 23S RNA.

Rather t h a n a p p r o a c h the entire 50S particle we have attempted to study the i n t e r a c t i o n s of the isolated 23,S RNA and 5S RNA with isolated ribosomal proteins. In a previous study ~ve reported on the f o r m a t i o n of a specific stable complex contain i n g 23S, 5S RNA a n d only a few p r o t e i n s from the

Electrophoresis through a g a r o s e - a c r y l a m i d e composite gels was used to d e t e r m i n e size a n d h o m o g e n e i t y of the various RNAs used [20]. After electrophoresis the gels were stained w i t h met h y l e n e blue, destained w i t h w a t e r a n d s c a n n e d w i t h a Joyce-Loebl Chromoscan.

P. N. Gray and R. Monier.

42

T h e r i b o s o m a l p r o t e i n s w e r e p r e p a r e d by 2MLiE1 t r e a t m e n t of 50S subunits. F r a c t i o n I, w h i c h c o n t a i n s about 10 50S proteins, w a s obtained by D E A E cellulose c h r o m a t o g r a p h y as d e s c r i b e d by GRAY and M o i m n [14]. T h e purified proteins, L2, L6, L18, L25, w e r e p r e p a r e d f r o m f r a c t i o n I proteins by CM cellulose c h r o m a t o g r a p h y at 4°C [21]. T h e i d e n t i f i c a t i o n of the p r o t e i n s in f r a c t i o n I a n d of those n e c e s s a r y for c o m p l e x f o r m a t i o n is desc r i b e d e l s e w h e r e [15].

tion I p r o t e i n s or a m i x t u r e of the four isolated p r o t e i n s L2, L6; L18 and L25 [15]. Using the same p r o t e i n p r e p a r a t i o n s , the e x i s t e n c e of s i m i l a r complexes can be d e m o n s t r a t e d by sucrose g r a d i e n t s e d i m e n t a t i o n (fig. 2).

T h e p r e s e n c e or absence of the f o r m a t i o n of a 5S R N A - r i b o s o m a l RNA c o m p l e x w a s d e t e r m i n e d by sucrose g r a d i e n t c e n t r i f u g a t i o n of the d e s i r e d assay mixtures. E a c h assay m i x t u r e w a s c o m p o s e d of 5'0 to 75 ~g r i b o s o m a l RNA, 2 to 3 ~g a2P-5S RNA, a s a t u r a t i n g a m o u n t of specific proteins, 10-20 !~g, a n d buffer to m a k e an i n c u b a t i o n v o l u m e of 10'0 Ixl. T h e i n c u b a t i o n buffer w a s 10 mM Tris-HCl, p H 7.6; 20 mM MgC12 ; 300 mM KC1 ; 6~ mM 2-mercaptoethanol. All assays w e r e p r e p a r e d at 0 ° to 2°C, m i x e d , i n c u b a t e d at 30°C for 15 minutes, q u i c k l y c h i l l e d and a p p l i e d d i r e c t l y to the sucrose gradients. G r a d i e n t c e n t r i f u g a t i o n w a s done in 10 mM TrisHC1 p H 7.6, 10 mM MgC12, 30 mM KC1, 6 mM 2-merc a p t o e t h a n o l c o n t a i n i n g 5 p. cent to 20 p. cent Sigma u l t r a p u r e sucrose. A B e c k m a n Spinco SW50-L r o t o r c o n t a i n i n g 5 ml g r a d i e n t s w a s used. See figure legends for c e n t r i f u g a t i o n c o n d i t i o n s . The gradients were scanned and fractionated with an ISCO Model D f r a c t i o n a t o r at 254 m~. T w e n t y 0.25 ml f r a c t i o n s w e r e collected f r o m e a c h gradient, dissolved in Bray's s c i n t i l l a t i o n fluid and c o u n t e d in a P a c k a r d Tri~Carb s p e c t r o m e t e r [22]. RESULTS. T h e f r a g m e n t a t i o n of 23S RNA resulted m essent i a l l y the same t w o pieces as d e s c r i b e d by ALLET and SPAI~R [9]. T h e sizes and h o m o g e n e i t y relative to i n t a c t 23S a n d 16S RNA w e r e d e t e r m i n e d by gel e l e c t r o p h o r e s i s and are s h o w n in figure 1. T h e t w o f r a g m e n t s m i g r a t e as two RNA m o l e c u l e s c o n t a i n i n g a p p r o x i m a t e l y 3/5 and 2/5 of the orig i n a l 23S RNA. This size d e t e r m i n a t i o n agrees well w i t h the r e l a t i v e s e d i m e n t a t i o n coefficients of 18S and 13S. H o w e v e r , since the f r a g m e n t s o b t a i n e d are quite large it can be e x p e c t e d that t h e r e w i l l be some size h e t e r o g e n e i t y d e p e n d i n g on the p o s i t i o n of cleavage. This is obvious in the 18S p r e p a r a t i o n w h e r e t h e r e seems to be a range of sizes of f r a g m e n t s b e t w e e n 16S and 18S w i t h t h e ] 8 S p r e d o m i n a n t . It w a s p r e v i o u s l y r e p o r t e d that a stable c o m p l e x , w h i c h c o n t a i n e d both 23S and 5 S R N A and w h i c h w a s r e t a i n e d on Millipore filters, could be f o r m e d by u s i n g e i t h e r t h e frac-

BIOCHIMIE, 1972, 54, n ° 1.

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FIG. 1. - - Aerylamide-agarose composite gel e]ectrophoresis of ribosomal RNA. RNA was prepared from purified 50 S and 30 S ribosomal subunits as well as from the two <> fragments resulting from pancreatic ribonuelease treatment of 50 S subunits. 75100 ~g of each RNA was applied to a composite 3 p. cent aerylamlde - 0.5 p. cent agarose gel. After eleetrophoresis and staining, gels were scanned with a Joyee-Loebl Chromosean to determine size and homogeneity of each RNA preparation. A, 23 S RNA. B, 16 S RNA. C, RNA from larger <> fragment. D, RNA from smaller < fragment. Migration was from right to left. No c o m p l e x is f o r m e d in the absence of proteins. Using either p r o t e i n p r e p a r a t i o n the m o l a r r a t i o of 5S RNA to 23,S RNA in the r e s u l t i n g complex w a s a p p r o x i m a t e l y 0.9. No a p p a r e n t alteration in the s e d i m e n t a t i o n rate of 23S RNA upon c o m p l e x f o r m a t i o n could be noted. Since the total a d d e d m o l e c u l a r w e i g h t of the 5S RNA and four p r o t e i n s is only 10 p. cent of the 23S RNA molecular w e i g h t no change in s e d i m e n t a t i o n w o u l d be e x p e c t e d due to the mass i n c r e a s e alone. On the

43

Partial localization of the 5S RNA binding site on 23S RNA. with with ~( 18 total

other hand, a major conformational alteration might occur which could change the sedimentat i o n r a t e . S u c h a c o n f o r m a t i o n a l c h a n g e is n o t evident, ho~,ever, from the experimental results.

t h e 23S R N A c o u l d b e f o r m e d j u s t as w e l l only four ribosomal proteins. Assuming the S ~) f r a g m e n t to b e a p p r o x i m a t e l y 3 / 5 of t h e 23S RNA, t h e m o l a r r a t i o o f 5S R N A t o ~<18S ~

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Fro. 2. - - Analysis for the f o r m a t i o n of a complex between 5 S RNA and 23 S ribosomal RNA. Assay m i x t u r e s w i t h a 5 S RNA to 23 S RNA m o l a r ratio of 1.2 ~vere applied to 5-20 p. cent sucrose gradients in 10 mM Tris-HC1, pH 7.6, 10 mM MgCI~, 30 mM KC1 and 6 mM 2-mercaptoethanol. Conditions of centrifugation were 49,000 rpm, I°C, 2.5 hours. S e d i m e n t a t i o n is from left to right A : 32p-5 S RNA plus 23 S RNA w i t h o u t ribosomal proteins. B, ~2P-5 S RNA plus 23 S RNA with fraction I proteins. (2, 132p-5 S RNA plus 23 S RNA with the four ribosomal proteins L2, L6, L18, L25.

f r a g m e n t i n t h i s c o m p l e x a p p r o x i m a t e s 0.82. S i n c e the f r a g m e n t a t i o n results in several d i f f e r e n t sized p i e c e s , as s h o w n b y gel e l e c t r o p h o r e s i s , a n d t h e <( 1 8 S , f r a g m e n t c a n l o s e u p to 20 p. c e n t of t h e 3 ' - t e r m i n u s d u r i n g d e g r a d a t i o n [9], t h e o b s e r v e d molar ratio agrees reasonably well with the e x p e c t e d r a t i o , a s s u m i n g t h a t o n e 5S R N A is b o u n d p e r ¢ 18S >> f r a g m e n t . A g a i n , t h e s e d i m e n t a t i o n

W h e n t h e 23S R N A w a s f r a g m e n t e d i n t o a n <<18S >> f r a g m e n t , c o n t a i n i n g t h e 3 ' - t e r m i n u s of t h e 23S R N A m o l e c u l e a n d a <~ 13S >> f r a g m e n t c o n t a i n i n g t h e 5 ' - t e r m i n u s of t h e 23S R N A [9], t h e f o r m a t i o n of a 5S R N A c o n t a i n i n g c o m p l e x w~as p o s s i b l e o n l y w i t h t h e <<18S >> f r a g m e n t . T h e s e d i m e n t a t i o n p a t t e r n s a r e s h o w n i n f i g u r e s 3 a n d 4. T h e 5S RNA-<< 18 S >> f r a g m e n t c o m p l e x , like t h a t

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Fin. 3. - - Analysis for the f o r m a t i o n of a complex between 5 S RNA and <<18 S >> RNA f r o m t h e larger <> f r a g m e n t of t h e 50 S subunit. Assay m i x t u r e s containing a m i x t u r e of 5 S RNA and ¢ 18 S ~ f r a g m e n t in the m o l a r ratio of 0.94 in 100 ~1 were applied to 5-20 p. cent sucrose gradients. Conditions of ceutrifugation w e r e 49,000 r p m , 1°C, 3.0 hours. A, 32P-5"S RNA p l u s <( 18 S .~> f r a g m e n t w i t h o u t ribosomal proteins. B, z2p-5 S RNA plus <( 18 S > ) t r a g m e n t w i t h fraction I proteins. C, 32P-5 S RNA plus <( 18 S ~; f r a g m e n t with the four ribosomal proteins L2, L6, Li8, L25. S e d i m e n t a t i o n is from left to right.

BIOCHIMIE, 1972, 54, n ° 1.

P. N. Gray and R. Monier.

~4

On the contrary, no significant complex form a t i o n o c c u r s w h e n t h e (~ 13S >> f r a g m e n t d e r i v e d f r o m t h e 5 ' - e n d of t h e m o l e c u l e is u s e d . T h e s e observations suggest that the specific sites on the 23S R N A m o l e c u l e , w h i c h a r e r e s p o n s i b l e f o r t h e c o o p e r a t i v e b i n d i n g of 5S R N A a n d p r o t e i n s L2, L6, L18 a n d L25, a r e c a r r i e d b y t h e p o r t i o n of t h e 23S n u c l e o t i d e s e q u e n c e l o c a t e d b e t w e e n t h e reg i o n c l e a v e d b y RNlase a n d t h e 3 ' - e n d . T h i s p o r t i o n still e n c o m p a s s e s a b o u t 3./5 of t h e 23S m o l e cule, b u t i t s h o u l d b e p o s s i b l e to d e l i m i t t h e binding sites more accurately by similar experiments using shorter specific fragments.

r a t e of t h e c o m p l e x is n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t of t h e c o n t r o l (< 18'S >> f r a g m e n t . T h e r e is n o s i g n i f i c a n t b i n d i n g of 5S R N A t o t h e s m a l l e r > R N A f r a g m e n t ( f i g u r e 4, B). S i n c e a n e a r l y t w o f o l d e x c e s s of t h e (< 13,S >) f r a g ment was used in this particular assay, the o b s e r v e d b i n d i n g c o r r e s p o n d s to a b o u t 0.11 m o l e of 5S R N A p e r m o l e of <<13S >> f r a g m e n t . T h i s 1o~" l e v e l of b i n d i n g is c e r t a i n l y r e l a t e d to c o n t a m i n a t i o n of t h e ¢ 13S ~> p r e p a r a t i o n b y t r a c e s of t h e <<18S>) f r a g m e n t o r b y s m a l l e r R N A f r a g m e n t s , c o n t a i n i n g t h e 5S R N A r e c o g n i t i o n site.

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Fro. 4. - - Analysis for t h e f o r m a t i o n of a complex between 5 S RNA and <<13 S >> RNA f r a g m e n t f r o m the s m a l l e r <> f r a g m e n t of 50 S s n b u n i t s or w i t h 16 S RNA f r o m t h e 30 S r i b o s o m a l subunits. Conditions of c e n t r i f u g a t i o n were t h e same as in figure 3. The m o l a r ratio of 5 S RNA to <<13 S >> RNA was 0.6. The 5 S RNA to 16 S RNA m o l a r ratio was 0.7. S e d i m e n t a t i o n is f r o m left to right. A, 32p-5 S RNA plus <<13 S >) RNA w i t h o u t r i b o s o m a l proteins. B, ~2P-5 S RNA plus <<13 S >> RNA w i t h f r a c t i o n I proteins. C, 32p-5 S RNA plus 16 S RNA w i t h f r a c t i o n I proteins. T h e s p e c i f i c i t y f o r 5S R N A b i n d i n g to 23S R N A or a fragment thereof can be demonstrated by the t o t a l l a c ~ of b i n d i n g to 16S R N A i s o l a t e d f r o m t h e 30S r i b o s o m a l s u b u n i t (figure 4, C). DISCUSSION. It had been previously established that a comp l e x c o n t a i n i n g e q u a l m o l a r a m o u n t s of 23S R N A a n d 5 S R N A c a n b e f o r m e d i n t h e p r e s e n c e of a f e w 50S r i b o s o m a l p r o t e i n s . T h e f o r m a t i o n of s u c h a c o m p l e x a p p e a r s to i n v o l v e c o o p e r a t i v e i n t e r a c t i o n s b e t w e e n t h e 5S R N A a n d t h e p r o t e i n s [14]. In the present communication we demonstrate t h a t t h e i n t e g r i t y of t h e 2,3'S R N A m o l e c u l e is n o t r e q u i r e d f o r t h e c o n s t i t u t i o n of a c o m p l e x i n v o l v i n g 5S R N A a n d t h e f o u r 50S p r o t e i n s L2, L6, L18 a n d L25. A c o m p l e x , w h i c h c a n b e i d e n t i f i e d b y s u c r o s e g r a d i e n t s e d i m e n t a t i o n , is s p e c i f i c a l l y f o r m e d w h e n 23S R N A is r e p l a c e d b y t h e <<18S >> fragment derived from its 3'-end by RNase degrad a t i o n a c c o r d i n g t o ALLET a n d SPAHR [9].

BIOCHIMIE, 1972, 54, n ° 1.

Ac&no,wledgements. This C.E.A., tifique Damon

work was supported i n p a r t by grants f r o m the t h e D~l~gation G6n~rale h la Recherche Scienet Technique, a n d b y g r a n t DRF-628 f r o m the R u n y o n F u n d for Cancer Research. RfiSUM~.

Apr6s clivage du RNA 23S e n deux f r a g m e n t s selon la t e c h n i q u e d'ALLET et SPAHR [9], le f r a g m e n t le plus long (18S), qui r e n f e r m e l'extr6mit6 3' de la mol6cule, est encore capable de participer h la f o r m a t i o n d ' u n complexe stable avec le RNA 5S en p r d s e n c e de q u a t r e prot~ines isol~es de la sous-unit~ 50S. Le f r a g m e n t le plus court (13S) n ' a pas d'activit~ dans ce~te association. Cette o b s e r v a t i o n sugg6re que le ou les sites du RNA 23S qui p a r t i e i p e n t h l'association cooperative avec le RNA 5S et les q u a t r e prot6ines i m p l i q n e n t des nucl~otides a p p a r t e n a n t h la p o r t i o n de la molecule qui d o n n e naissance au f r a g m e n t 18S. ZUSAMMENFASSUNG.

Nach Spalten der 23S-RNA in zwei Bruchstiicke nach d e r Technik yon ALLET u n d SPAHn [9] ist das l~ingere Bruchstiick (18S), 'welches das 3'-Ende des Mo-

Partial localization of the 5S RNA binding site on 23S RNA. lekiils enth~ilt, noch dazu fiihig an der Bildung eines stabilen Komplexes m i t der 5S-RNA in der Gegenwart yon vier aus der 50S-Untereinheit isolierten P r o t e i n e n t e i l z u n e h m e n . Das kiirzeste Bruehsttick (13S) h a t in dieser Assoziation keine Aktivit~it. Diese Beobachtung liisst vermuten, dass das oder die Zentren der 23SRNA, welehe an der kooperativen Assoziation mit der 5S-RNA und den vier P r o t e i n e n teilnehemen, aus Nnkleotiden bestehen, die in dem Teil des Molekiils, welcher das 18S-Bruchstiick erzeugt, e n t h a l t e n sin& REFERENCES. 1. Kaltsehmidt, E. ~ W i t t m a n n , H. G. (1970) Proc. Natl. Acad. Sci., U.S., 67, 1276. 2. Nomura, M. Mizushima, S., Ozaki, M., Traub, P. .~ Lo'wry, C. V. (1969) Cold Spring Harbor Symp. Quant. Biol., 34, 49. 3. Schaup, H. W., Green, M. ~ Kurland, C. G. (1970) Molec. Gen. Genetics, 109, 193. 4. Brimacombe, R., Morgan, J., Oakley, D. G. ~ Cox, R. A. (1971) Nature N~w Biol., 231, 209. 5. Chang, F. N. a Flaks, J. G. (1970) Proc. Natl. Acad. Sci. U.S., 67, 1321. 6. Sypherd, P. S. (1971) J. Mol. Biol., 56, 311. 7. Stoffler, G., Daya, L., Rak, K. H. ~ Garrett, R. A. (1971) J. Mol. Biol., 62, 411.

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8. Chang, F. N. • F l a k s , J. (1971) J. Mol. Biol., 61, 387. 9. Allet, B. & Spahr, P. F. (1971) Eur. J. Biochem., 19, 250. 10. Homann, H. E. a Nierhaus, K. H. (1971) Eur. J. Biochem., 20, 249. 11. Forget, B. G. a Reynier, M. (1970) Biochem. Biophys. Res., Commun., 39, 114. 12. Gormly, J. R., Yang, C. H. & Horowitz, J. (1971) Biochim. Biophys. Acta, 247, 80. 13. Reynier, M. ~ Monier, R. (1968) Bull. Soe. Chim. Biol., 50, 1583. 14. Gray, P. N. ,~ Monier, R. (1971) FEBS Letters, 18, 145. 15. Gray, P. N., Garrett, R. A., Stoffler, G., Monier, R. V~Tittmann, H. G., s u b m i t t e d for publication. 16. Gesteland, R. F. (1966) Y. Mol. Biol., 16, 67. 17. Kikuchi, A. ~ Monier, R. (1970) FEBS Letters, 11, 157. 18. Monier, R. ~ Feunteun, J. (1971) Methods in Enzymologv Vol. 20, part C, pp. 494. Eds Moldave K. Grossman, L. (Academic Press, Ne~v York). 19. Marcot Qtteiroz, J. ~ Monier, R. (1965) Y. Mol. Biol., 14, 490. 20. Dahlberg, A. E., Dingman, C. W. & Peacock, A. C. (1969) J. Mol. Biol., 41, 139. 21. Hindennach, I., Stoffler, G. ~ W i t t m a n n , H. G. (1971) Eur. J. Biochem., 23, 7. 22. Bray, G. A. (1960) Analyt. Biochem., 1, 279.