304 BIOCHIMICA ET BIOPHYSICA ACTA

BB.\ e57OO

SOME PROPERTIES OF NITRO( ;EN-FIXING BREIS P R E P A R E D FROM

SOYBEAN ROOT NODULES

F. J. 13El{GEl{SEN

Division of Plan! lndusl~iV, CSIR() , Canberra (.4 l¢stralia)

(Received May 3rd, 1900)

SUMMARY

The preparation of nitrogen-fixing breis from soybean root nodules is described. Breis prepared in air and then rapidly placed in anaerobic conditions fixed only one- third as much nitrogen as breis prepared from nodules which had been equilibrated in argon for IO nrin. Breis prepared and stored under argon lost only one-third of their activity after I h. Nitrogen fixation required O,~ in the gas phase but with ]5o, _, in excess of 2 ')o, increased fixation was accompanied by an increased rate of decay of the system.

The addition of an adenosine 5'-triphosphate-generating system stimulated fixation to some extent. The addition of Io- a M 2,4-dinitrophenol was inhibit()rv and this inhibition was reversed by the addition of an adenosine 5'-triphosphate-generating system. These effects were small, suggesting that, if required for fixation, adenosine 5'-triphosphate from endogenous sources was adequate. Amounts of H,, evolxed by the breis were correlated with the amounts of nitrogen fixed. The newly fixed nitr~gen accumulated in the breis as soluble ammonia-N.

INTROI)UCTION

Although nitrogen fixation has been studied in cell-free extracts of several hee- living bacteria, the process in the legume-root nodule system has, until recently-, been studied only in intact nodules detached trom the host plant. Unsuccessful attelnpts to obtain nitrogen fixation using crushed, or homogenized soybean nodules or extracts of nodules supplied with factors necessary for fixation in the bacterial systems, have been reported from several laboratories 1.

Nitrogen fixation in breis of soybean root nodules was the subject of a prelimi- nary report by BERGERSEN 2. These active preparations were made by crushing the nodules in a stainless steel press, straining the resultant fluid through organdie and passing the entire preparation into an incubation flask under a stream of argon. This method had experimental limitations because a complete brei preparation was required for each flask and difficulty was experienced in obtaining reproducible preparations.

hr the present work the method has been modified to permit the preparation of larger amounts of brei and its subdivision into aliquots for different treatments, together with a method for withdrawing samples from incubation flasks during the

B i o c h i m . I~iop/~v.;. . l c / a , I3o (1900) 3 o 4 - 3 1 '-

NITROGEN-FIXING BREIS FROM SOYBEAN NODULES 305

course of an experiment. These methods have consistently produced breis with greater activity than before and they have been used to stud), nitrogen fixation in these preparations in more detail.

MATERIALS AND METHODS

Nodules Nodules aged 28-35 days were detached from soybean plants (var. Shelby in-

oculated with strain CC7~I of Rhizobil¢m japonicum) which had been grown in a glass- house as previously described a. They were washed in distilled water until free from growth medium and 5o-Ioo g were used for preparing a brei. Harvesting the nodules required 3o-45 min and they were chilled to ice temperature until used.

Apparatus The apparatus is diagrammatically illustrated in Fig. I. The press was con-

structed in three parts from stainless steel and when assembled was made gas-tight by the use of neoprene gaskets and O-rings. (Working drawings of the press will be supplied by the author on request.)

The whole system was connected to a cylinder of argon (welding grade dry argon supplied by Commonwealth Industrial Gases Ltd., Sydney, N.S.W., Australia) which

S

#' ,.

_ , = i t * " - - - D

_:_j C

B cc~ c M A

Fig. I. Diagram of the appara tus used for the prepara t ion of nodule breis. (a) The press assembly : A, B, C, D, E and F, stopcocks and valve for control of argon flow; H, perforated stainless-steel disc; G, discs of stainless-steel mesh (o.2 m m pore size) and nylon mesh (o.i m m pore size); L, piston with neoprene O-rings ; M, ice jacket ; K, receiving flask ; J, reservoir for buffered medium. The outlet from the receiving flask is closed with a water seal. (b) The reaction vessel: A, gas ent ry por t and stopcock; B and C, reagent sidearms with entry ports, one of which (B) is closed with a soft rubber cap; D, gas sampling a t t achment wi th 3-way stopcock; S, gas sample tube.

Biochim. Biophys..4cla, 13o (1966) 304 312

306 F.J. BERGERSEN

by mass spectrometer analysis, was shown to contain o.o25 % N 2, 0.oo 9 % O2, o.o12 % H 2 and 99.954 % argon.

Preparation of the brei The press was cooled to 0-4 ° and the buffered sucrose medium (O.I M KH2PO 4

(pH 7.0)-o.3 M sucrose-I mM Mg 2+) in the reservoir was equilibrated with argon while the nodules were being harvested. The nodules were placed in the press and the piston inserted and screwed down until it just cleared the nodules. Argon was then passed through the system for 15 rain at a rate of approx. 50o ml/min. Stopcocks C and Y were then closed, stopcock B opened and the piston raised in the press in order to draw in 15 ml of medium. Stopcock B and valve D were then closed and the piston returned to its former position before opening stopcock F and tightening the press to crush the nodules. The piston was then raised, taking care not to suck water from the seal through F into K. Argon was then passed to displace the brei into the receiving flask and the press was again tightened and the process repeated to produce about 30 ml of brei. Argon was passed through the receiving flask for a few minutes before closing the stopcocks E and F and removing the flask from the press.

Experime~ztal The flask of brei (K) and the reaction vessels (Fig. I) were continually flushed

with argon during the distribution of the brei and until the reaction vessels were closed with the gas sampling attachments 4 prior to filling with the experimental gas mixtures. A Io-ml glass syringe with stainless-steel canula was used to place 5 ml of brei beneath the surface of I ml of buffered medium in each vessel. Air was excluded from the canula by filling it with medium before use.

The gas sampling attachments were connected to a manifold which was in turn connected to a vacuum pump, mercury manometer and containers of gas mixtures containing ~SN,,. The reaction vessels were immediately evacuated and flushed three times with argon before filling with appropriate gas mixtures. In most experiments four or five vessels were used and these could be all set up in this way in the space of about 1. 5 min. Reagents, if any, were then mixed before incubating the vessels with shaking in a Warburg water bath at 23 °.

Samples (I ml) of the contents of the reaction vessels were withdrawn through the rubber caps at intervals during incubation by means of glass hypodermic syringes, capacity 2 ml, with 25 gauge needles. Again the dead space of the needles was filled with medium to exclude air. No significant contamination of the atmosphere in the vessels was detected following the taking of up to five samples.

Reagents The ATP-generating system consisted of (for each vessel) 5/xmoles ATP (sodium

salt), 5o ~moles creatine phosphate (sodium salt hydrate), and o.5 mg creatine phosphokinase. All of these were supplied by the Sigma Chemical Company, St. Louis, Mo., U.S.A., and were made up in water and neutralized before use.

2,4-Dinitrophenol was made up in water, neutralized and used to give a final concentration of io 4 M.

BiocLim. Biophys..4cta, 13o (~960) 3o4-3t2

NITROGEN-FIXING BREIS FROM SOYBEAN NODULES 307

Analytical Experience has shown that a minimum of 7 ° % of the fixed N can be recovered

in the soluble fraction of nodules, therefore in most experiments nitrogen fixation was measured by determining the atoms % 15N excess in the soluble non-protein N of each sample. This was done by centrifuging the i ml sample plus 0.5 ml buffer at 22ooo × g at 4 ° and making the supernatant to 3 N with HC1. The precipitated protein was removed by centrifugation and the supernatant was digested and analysed for N by the Kjeldahl method 5. Duplicate samples of the Kjeldahl distillates, each containing approx, o I m g N, were analysed for I~N using an Atlas M 86 mass spectro- meter as previously describedG, 7

In the experiments in which the distribution of the fixed N was determined, four identical reaction vessels were used and the entire contents were analysed from each, after varying periods of incubation The soluble and particulate fractions were separated centrifugally at 4 ° as before from 5 ml of the flask contents and the re- maining I ml was used to determine the total ~SN incorporation. The washings from the particulate fractions were added to the soluble fraction which was then made to IO ml; 2 ml of this was used to determine the total soluble lSN incorporation and the remainder was used for measurement of the ammonia-15N as described previously 7.

Brei protein (rag per reaction vessel) was calculated from the Kjeldahl N of the tungstic acid precipitate from 0.5 ml samples of brei. This method was used because the heterogeneous nature of the breis precluded the use of more direct colourimetric methods.

The initial gas mixtures and gas samples taken from the reaction vessels at the end of the incubations were routinely analysed as a control on air leakage and for measurements of respiration, H 2 evolution and 15• concentration in the N 2. These gas samples were analysed in the mass spectrometer and the partial pressures of the components calculated from the peak heights of the mass spectrum multiplied by the calibration for each component.

In all experiments the N2 of the gas phase contained in excess of 85 atoms °o o/ 15 N in 15N. The 15N content of the brei components was expressed in terms of ioo /o

the gas phase. The /~g 15N excess values therefore represented the nitrogen fixed in the experiment.

Gas mixtures These were prepared from good quality commercial Oe and the A described

above and contained 2o % ~SN 2 prepared from ~SNH4NO~ (Bio-Rad Laboratories, Richmond, California, U.S.A.) by oxidation of I~NHa with CuO at 6oo °. The mixtures were stored under pressure from a 3o-cm head of water in 2-1 containers and were admitted to the reaction vessels to a final pressure of 7oo mm Hg.

RESULTS

Breis of soybean nodules prepared in the manner described were seen in the light microscope to contain large numbers of intact bacteroids which were both individually free or aggregated in groups. There were no recognizable plant particles and no intact host cells. Electron microscope examination showed that membranous fragments were associated with the surfaces of the individual bacteroids and that

Biochim. Biophys. Acta, 13o (1966) 3o4 3t2

3 0 8 F . J . I3ERGEI¢SEN

the groups of bacteroids were embedded in membranous fragments and other small particles.

Every brei prepared with these methods actively fixed N,,. The amounts fixed varied with experimental treatment but with a gas phase containing about 6 % O.,, preparations fixed 6o-8o mffg N per mg brei protein. A few were more active, fixing up to z4o mffg N per mg. The time course of N,, fixation was variable, sometimes exhibiting a slight lag followed by a period in which fixation was linear with time and sometimes proceeding in a linear fashion without a lag before declining after about 3o min. The time for which fixation continued varied with Po2. In contrast to the time course of nitrogen fixation, respiration remained linear with time for up to I h. Table I shows the time course of 3 identical reaction mixtures and gives an indication of the practical limits of accuracy of the methods.

The data of Fig. 2a show the necessity for maintaining anaerobic conditions during preparation of the breis. In this experiment breis were prepared after o, 2 and IO rain of A flushing of the nodules before crushing, after which the breis were within 3o sec placed under argon atmospheres. About 0o % of the activity was lost when the nodules were crushed in air and ahnost 3o'!o was lost when ()nix" 2 rain A flushing was used.

T.\I~I~E 1

I':\'A].I'ATION OF TH]'2 MILTIIOI}

l ) a t a fo r t h r e e r e a c t i o n vesse l s g i x e n i{lel l t ical t r e a t m e n t , l : l a s k s c o n t a i n e d 4 iul 1)rci, I m l I}ui lered m c ( l i u l n an{1 in the gas p h a s e ] 7 % n~N2 (92 a t o m s %,), 4.3 % {}2 a n d 78 % .\. T i l t l)rei c o n t a i n e d (}5.3 m g p r o t e i n a n d 3 .18 m g so lub l e n o n p r o t e i n N.

7'h~m l&acli( .z . l/o~ns % 15A" r.rccss mHg ~\" f ixed ( . t in ) vcssH ]5 \ excess ( f ig ) per ~n 2 brci /)r,)h'itl

15 A o .o45 ].41 21.o l~ 1).o20 {}.{}2 14 . [ C o-o3{} <).()5 t 4-5

3 o A o. I {1 l 3 .23 49 .q l} o .o92 2.(} 3 44 .9 {i o . o g q 3- ] 3 48 .o

45 .k {1. 145 t.()o 7o.5 l~ o. ,.t4 4.57 7o.o (." /}. 132 4 . IQ I} t, I

~o~ 0 10 20 40 60

b / 0 10 20 40 60

Time (rain)

Fig . 2. (a) The effects of f lushing tile nodules in tile press with , \ before crushing. O O , no flushing; • - • , 2 rain f lushing; @ -:~, t o rain flushing. In a l l cases the brei was rapidly placed under an argon a t m o s p h e r e w i th in 3o sec. (;as phase conta ined 6.9 cJo 0 2 a n d 19 % 1aN 2. (b) The effects of s tor ing breis in ice under A. • • , brei immediate ly , af ter preparat ion; O O , after s torage f o r 30 r a i n ; ( D - - @ , after s torage for I ]l. (~raS phase conta ined 5.5 o,,,,o O.,. a n d -3' O.o lSN.,-_

];iochim. BiopIl'c.~.. 1 or'a, ] 30 { t 90{}) 3o4--312

N I T R O G E N - F I X I N G BREIS FROM SOYBEAN NODULES 309

The breis were m o d e r a t e l y s table when kep t in ice under A as shown in Fig. 2b. Even af ter I h under these condi t ions a useful amoun t of ac t i v i t y remained.

The effects of p•2 Fig. 3 shows the resul ts from two exper iments in which two ranges of 2/502 w e r e

used. P re l imina ry exper iments had shown tha t only a t race of N 2 f ixat ion occurred o, and 2 o~ 02 , f ixat ion remained l inear wi th t ime for at wi thou t added O 2. At I ,o ,o

BO

4 0

)-2o z

7 0 E

c1

4O

0

160

120

O 10 2 0 4 0 6 0 0 10 2 0 4 0 6 0

T ime (rr, in)

Fig . 3- T h e effects of Po2 u p o n n i t r o g e n f i x a t i o n b v b re i s of nodu le s . D a t a f r o m t w o e x p e r i m e n t s . .... o.,; s o. O.,.(b) Q ,5~,6 0` O o ; • 0 , (a) O O, i °~ O2 ; ~ ) _ @ , 2 o / O2 ; ~ _ _ ~ , 4 o • 0 , , ..o ,, o / o _ . .

I o , r • % O.,; O O , 5 ,o O.z- T h e b re i in (b) h a d a b o u t t w i c e t h e a c t i v i t y of t h a t u sed in (a).

least I h. This has also been shown in other exper iments . The ra te of f ixat ion at o~ Highe r Po2 values gave grea ter f ixat ion b u t the curves 2 O/.o 0.~ was twice t ha t a t I ,,o.

indica te t ha t this was accompanied by grea ter decay of the system. A t 15 °o 02, f ixat ion ceased af ter IO min and only 6o mffg N per mg brei prote in was fixed, com- pa red with more than 18o mffg at IO ° o O., (Fig. 3b). Consequent ly 4-6 °o O., was used for most subsequent exper iments .

Tke effects of A T P The Po2 results suggested t ha t increased energy m a y have been avai lable to

the brei sys tem at higher 35o2 levels and a number of exper iments were per formed using the creat ine p h o s p h a t e - c r e a t i n e kinase sys tem of genera t ing ATP. Add i t iona l ATP prov ided in this w a y consis tent ly s t imu la t ed f ixat ion bu t seldom b y more than lO_15 .... ,o (Fig. 4)- The s t in lu la t ion was not grea ter a t 2 % 0 2 than at 6 .... /o 0.,. Un- coupling ox ida t ive phosphory la t ion wi th 2 ,4-dini t rophenol (IO -4 M) inh ib i ted f ixat ion by IO-I5 °o and this inhibi t ion was pa r t i a l ly reversed b y the addi t ion of the ATP- genera t ing system.

H~ evolutio~ Consistent increases in the pa r t i a l pressure of H 2 were measured in the seven

exper iments in which rel iable measurement s were made. This evolut ion of H 2 was small, being an increase from approx, o .o i % in the ini t ia l gas mix tu re to o.o6 o/ • o

after i tl incubat ion. When the amounts of N2 fixed per flask at the t ime of sampl ing the gas were p lo t t ed agains t the increases in pI~,, (Fig. 5) i t was found t ha t there was a posi t ive and h ighly significant corre la t ion (r = o.91 ) be tween the two quan t i - ties. The correla t ion held under the influence of increasing Po2 and also wi th t ime

Biochim. Biophys. Acta, 13o (1966) 304 312

3 1 0 F. J. BERGERSEN

during the course of a fixation experiment. The slope of the regression line suggests that 1 .5 / ,moles of H 2 were evolved from the brei while I / , m o l e of NH a was being produced from o .5 / ,mole of N2.

The distribution of fixed N in the breis The data of Table II clearly show that the fixed N accumulated in the soluble

fraction as NHa-N which, after 50 rain, contained almost 6 atoms % ~SN. There was a steady increase in the quantity of soluble NHs-N recovered with time of incubation. For this reason the fig aSN excess values were calculated for the amount present at each time interval and not for the mean value as was done with the other fractions.

.c 8 0 - a

o

F~ol

EC z

®

12 , , ,

0 j ~' 0.5 ~0 1.5

0 10 20 40 60 0 15 30 45 60 0 2~10-4 4~K)-4 6~10-4 7irne (min) 14 2 evolut ion

Fig. 4- (a) The effects of add ing an ATI ' -genera t ing sys t em ( @ - - @ ) and 2,4-dini t rophenol (Q • ) to a n i t rogen- f ix ing brei ( O - - O ). (b) The reversal of the inh ib i t ion produced by 2,4-dini t rophenol by the fu r the r add i t i on of the ATP-genera t ing sys tem. O - - O , brei alone; • • , brei plus din i t ropheno l ; @ - - @ , brei plus din i t rophenol phts ATP. ( ;as phases conta ined (a) 5.5°.0 0 . 2 - i - o 1 5 N . ~ / ,, , ; (b) 5.8°o O2-~6°/o 18N.,.

Fig. 5- The cor re la t ion be tween N.2 f ixat ion and i t 2 evolut ion. D a t a for seven exper iments . The do t t ed lines show the corre la t ion in ind iv idua l expe r imen t s ; O, @, the effects of Po.2; x , the corre la t ion a t in te rva l s dur ing an exper iment . H a evo lu t ion shown as the change in pH 2 (lower scale) in flasks of 7 ° ml vo lume wi th a t o t a l gas pressure of 700 ran1 Hg at 23°: The upper scale gives gnaoles H 2 evolved.

T A B L E II

T H E D I S T R I B U T I O N O F F I X E D N I N T H E H R E I G

Data for the fixed N of four ident ica l reac t ion vessels whose con ten t s were ana lysed af ter four per iods of incuba t ion . E x c e p t for the soluble NHa-N in which i n d i v i d u a l values were used, the fig 15N excess va lues were ca lcu la ted from the mean mg N va lues and the m e a n a toms % excess values. Each vessel con ta ined 86.2 mg brci p ro te in and the gas phase was 2o % 15N 2 (95 a toms %)---

o , o / 5.4 :o 0 .2-74.6 /o argon.

Incubat ion Bacteroid + parlicl~ lime (rain) L'rei non-protei~ N Soluble N H 3 N non-protein N

A toms °//o 15N excess mg ;\: A toms c!~ 15N excess A toms ~o ISN exc~ 15N excess (fig) 15N excess (itg) 15N excess (ltg)

5 0.0o9 0.41 o. 126 0.282 0.36 o.oo 4 o.o6 20 o.126 5.77 o.162 3.341 5.41 o.oI 4 o.21 3 ° o. 19o 8.69 o. 171 4.857 8. 31 o.o22 0.32 5 ° 0.295 13.45 0.250 5.967 14.92 0.030 0.54

Mean mg N 4.56 ~ 0.63 - - ~.5 o ~ 0.07 per vessel

t3iockim. Biophys. Acla, 13o (1966) 3o4-312

NITROGEN-FIXING BREIS FROM SOYBEAN NODULES 311

The relatively small amounts of fixed N associated with the bacteroids and sub- cellular particles is considered to be contamination from the soluble fraction as the particulate material was washed only once. The total soluble 15N excess values were very similar to both the brei non-protein N and soluble NHa-N values and were therefore omitted from Table II. The greater variability of the brei non-protein N (4.56 ± 0.63 compared with 1.5o ± 0.07 for the particles and 2.99 -1- 0.07 for the soluble non-protein N) was attributed to the fact that this was analysed in the residue remaining in the reaction vessels after removal of 5 ml for the analyses and recovery may not have been quantitative in some instances.

DISCUSSION

N 2 fixation in breis differs in several ways from fixation in intact detached nodules. In the intact system, N 2 fixation proceeds in a linear fashion with time for at least 1.5 h (ref. 6), whereas in the breis there was frequently a lag before the maximum rate was attained and this was quickly followed by a declining rate as the system decayed. Increasing Po2 up to 50 % 02 increased the rates of fixation in intact nodules 6 and further increases above 50 % decreased the rates of fixation. In the breis, increased flo2 above 2 % increased •2 fixation but also increased the decay of the system and rates of fixation could not be determined. The inhibition of fixation in the brei at 15 % 02 seemed to follow solely from a greatly shortened period of activity. Because of these effects it was not possible to do any work on the kinetics of N 2 fixation in the breis.

The high levels of fixation obtained with IO % 02 in the gas phase are in contrast with results from the intact system in which the state of the leghaemoglobin is indica- tive of effective 02 concentrations several orders of magnitude lower than this s. At this partial pressure of 02 (IO %) the bacteroids in the breis must be expending a major part of their reducing power in aerobic respiration. These results are in conflict with the hypothesis developed from work with the intact system, in which the diversion of bacteroid reducing power from the reduction of N2 was regarded as resulting from penetration of 02 into the region in which fixation was occurring*, 9.

The evolution of H 2 from intact nodules was much greater in relation to N 2 fixation than H e evolution from breis. Intact nodules evolved up to 25/*moles of H 2 for every/*mole of N 2 fixed and there was a tendency for evolution to increase more than fixation in response to increased Po2 (ref. 4). In the breis, however, there was a constant relationship of 3/.moles H2 evolved for each /.mole of N2 fixed. This is comparable with the level of the 2H 2 exchange reaction in intact nodules, in which 2-3/.moles of H~H were formed for every/.mole of N 2 fixed 4.

The relatively slight effects obtained when an ATP generating system was added to the breis and when oxidative phosphorylation was uncoupled with dinitrophenol, indicated that endogenous sources of energy were adequate for nitrogen fixation. These results also showed that the stimulatory effects of O 2 are not due in any great measure to the provision of respiratory energy from host plant mitoehondria in the breis since the stimulation by added ATP was no greater at 2 % O 2 than it was at 4% although fixation increased 3-fold over this range of Oa concentration. It is, however, possible that increased availability of energy within the bacteroids due to

Biochim. Biophys..:tcta, 13o (1966) 3o4 3 ~-

3 1 2 F. J. BERGEI,~SEN

increased respiration may stimulate fixation and this effect may not be obtained bv adding exogenous ATP.

The failure of the breis to synthesize amino compounds from the fixed NHa-N contrasts with the rapid removal of fixed NHa-N into a-amino-N in the intact svstemL This failure may be related to the rise in soluble NHa-N (12 4/*g) which occurred during the course of incubation of the breis ('Fable II). Only 15/*g of this rise was due to accumulated fixed NHa-N. These results suggest that extensive deamination was occurring in the breis and under these conditions the newly fixed N remained as XH a.

The moderate stability of the nitrogen fixing activity at low temperature under argon will permit further work to be done and it is hoped that the study of individual components of the system will be possible.

ACKNO\VLEDGEMENTS

Mrs. A. MARCINA was responsible for skilled analytical assistance and the staff of the workshops of the Division of Plant Industry, especially Mr. G. LEI~ION and Mr. P. BYRNE, were largely responsible for the development of the press. Helpful discussions with manv colleagues in the Division have also provided a major eoln- ponent in this work.

R E I"F~ R E N( lCS

I C. ('. I)I,:LXVICHI'., 5"ciel/cc, 151 (19{~()) 1565. 2 12. J. ]{I-RGERSEN, ]~iOC]lilll. I~ioph)'s..Iota, xJ.5 ([90O) -'47. 3 F. J. BI'ZRC;ERSI~N, J . (;e~t. Microbiol., 19 (r958) 312. 4 F. J. 131~RG~,;RSFx, A z t s l r a l i a l ~ J . l]ioA Sci., iO (1903) (~09. 5 K. |)M~C;H AND It . \:. TR XCEV, Moder~ 3let]lods qf Plalzt . l Jzal3,sis, Spr inge r , Ber l in , t950, p. 479. 0 F. J. Bt~ROERSEN, J . (;e~. ~llicrobio/., "-9 (1902) I I 3. 7 F. J. I3ERCFRSFN, Atfsh'ali~t~t J. 1~iot. Nci., ~8 (1965) 1. 5 1:. j . 13I~RGERSEN, Nalttrc, J94 (i902) lO59. ~) i t. J. 13ERG1~RSI~;N, Baclcriol. t&v., 24 U90°) 24t~.

I~i~chim. t~'io F'zvs. A eta, T 30 ([900) 3o4 -3 t 2