Electron Microscopic Investigations on Soluble and Insoluble Bovine Fibrin

BIOCHIMICA ET BIOPHYSICA ACTA

BBA

445

35582

ELECTRON MICROSCOPIC INVESTIGATIONS ON SOLUBLE AND INSOLUBLE BOVINE FIBRIN*

R. GOLLWITZER, H. E. KARGES, H. HORMANN

AND

K. KUHN

Max Planck-! nstitut fiir Eiweiss- und Lederforschung, M iinchen (Germany) (Received December r8th, rg6g)

SUMMARY

Clots of soluble bovine fibrin were dissolved in various agents and reprecipitated by dialysis against phosphate buffer (pH 6.g). Cross-striated fibrils were obtained when the solvent I M KBr at pH 5·3 was used. Fibrils without cross-striation precipitated from 2% acetic acid, and more or less unstructured material resulted from 6 M guanidinium chloride or 2 M KCNS. The optical rotation of these fibrin solutions was established. The agents mentioned, besides 2 M KCNS, had a swelling effect in fibrin which was covalently cross-linked by activated Factor XIII. Electron micrographs of stabilized fibrin treated with I M KBr revealed subfibrils exhibiting a width equivalent to that of two fibrin monomers. Accordingly, the formation of covalent cross-links is limited to the subfibrils. A three-dimensional covalent network must be rejected. After the removal of KBr cross-striated fibrin fibrils reformed. When enzymatically cross-linked fibrin was treated with 2% acetic acid, 6 M guanidinium chloride or 2M KCNS, cross-striation could not be regained even after the removal of these solutes. ATP precipitated soluble fibrin in globular form. After the removal of ATP fibrin was able to reconvert to cross-striated fibrils.

INTRODUCTION

Thrombin acts upon fibrinogen to produce a fibrous clot called fibrin. This process involves a series of stages. First the Fibrinopeptides A and B are split off from the amino terminus of four of the six fibrinogen peptide chains1 - 4 . The resulting fibrin in comparison to fibrinogen exhibits a modified surface charge distribution. It aggregates to linear polymers which soon line up as fibrils. Primarily these fibrils are soluble in weak acids 5 or in solutions of lyotropic salts or urea 6 •7 . Under the influence of calcium and the fibrin stabilizing factor (Factor XIII) activated by thrombin 8 •9 , the fibrils gradually become insoluble in the above-named agents as a result of cross* A preliminary report of these findings appeared in H. E. Protides of the Biological Fluids, Pergamon Press, rg6g, p. I 55·

PEETERS,

Proc. r6th Call. on

Biochirn. Biophys. Acta, 207 (1970) 445-455

R. GOLLWITZER et al. linking by covalent bonds10 . Cross-linking takes place via a transamidase reaction involving side chains of glutamine and lysine residues of adjacent fibrin molecules9·11-13. Only the a- and y-peptide chains of fibrin participate in the formation of intermolecular bonds 14 - 16. Electron micrographs of fibrin show cross-striated fibrils 17 - 19 . One heavy and one thin dark band alternate within an identity period of 240 A (ref. 20). To date, cross-striated fibrils have only been obtained during the coagulation process. They could not be reconstituted from solutions of fibrin in diluted acids or in lyotropic agents. Clearly, cross-striation is due to a special arrangement of the fibrin molecules within the fibre. Various hypotheses 20 - 23 are concerned with this superstructure. The theories have to consider that the individual molecules, according to HALL AND SLA YTER 20 , consist of three globular units connected linearly to one another by thin rods. The diameter of the two terminal nodules is about 6o A, that of the central nodule only so A. For the present investigations fibrin was treated with various agents which dissolve fibrin and which dissociate enzymatically cross-linked fibrin into substructures. Subsequently, the dissolved or swollen material was tested for its capability to reconstitute fibrils with or without electron microscopic cross-striation. Differences in the behaviour of soluble and of cross-linked fibrin should indicate a stabilizing effect of the intermolecular cross-links on the tertiary structure. In addition, electron microscopic investigation of cross-linked fibrin treated with the swelling agents gave information concerning the spatial arrangement of the fibrin molecules and the intermolecular cross-links within the fibril. MATERIALS AND METHODS Bovine fibrinogen was purified according to the method of BLOMBACK AND BLOMBACK24 from a crude specimen of coagulability 80-84% received from Behringwerke, Marburg. The purified material was dissolved in 0-3 M NaCl and stored frozen. Coagulability 97-98%. In order to remove Factor XIII fibrinogen was precipitated once or twice according to LoEWY et al. 25 with (NH 4 ) 2S0 4 followed by dialysis against 0.3 M NaCl for 2 days. Coagulation with thrombin in the presence ofCaC12 and cysteine (see procedure for insoluble fibrin) produced fibrin which, after 2 h of syneresis, was soluble in 2% acetic acid or in 30% urea. The amount of fibrin present in each experiment was determined from the fibrinogen content of the stock solution and from the coagulability which was evaluated by spectroscopic methods, according to HoRMANN AND GoLLWITZER 26 . For fibrinogen dissolved in various solvents the values for Lis (280 m/1/320 m11) indicated in Table I were determined by means of methods published earlier 26 . Absorbance measurements were made on the spectral photometer Beckman DU. Factor XIII was obtained from 20 1 of fresh bovine blood stabilized by addition of 500 ml 0.3 M trisodium citrate (pH 7-4) per 5 1. Subsequently the procedure of LOEWY et al. 25 was followed. Fraction 5 was used for further investigations. r mg of protein nitrogen equaled the activity necessary to transform rooo mg of fibrin to insoluble material (see below) within 2 h in the absence of cysteine. Stock solutions containing approx. 2 mg protein per ml were made in 0.3 M NaCl and stored frozen. In order to prepare fibrin for electron microscopic experiments r ml stock Biochim. Biophys. Acta, 207 (1970) 445-455

ELECTRON MICROSCOPY OF FIBRil\

447

solution of fibrinogen (concentration 0-4-0.8%) was diluted with 5-3 ml of 0.3 M NaCl and IO ml of o.oi6 M phosphate buffer (pH 6.35) containing 0.075 M NaCl. If soluble fibrin was desired, a watery solution of IS units (N.I.H.) of thrombin was added. For the preparation of cross-linked fibrin the same amount of thrombin dissolved in o.I ml of o.I M CaCl 2 (pH 6.5-7.0) was used and the clotting mixture was further supplemented by I ml stock solution of Factor XIII and I ml of o.or M cysteine hydrochloride (pH 6.5-7.0). 2 h later the gel was transferred to a nylon towelling for syneresis and washed 3 times with 0.15 M NaCl. Insolubility was tested after 24-h incubation of clotted fibrin with ro ml of 30% urea or 2% acetic acid. TABLE J EXTINCTION VALUES OF FIBRDIOGEK IN VARIOUS SOLVENTS

-----

--

- - - - -

Solvent 0.3 M NaCI o.os M phosphate buffer (pH 6.9) 2% acetic acid 1M NaEr (pH 5-3) 30% urea 6 M guanidiniurn chloride (pH 5-45) 2 M KCNS (pH 5 .8)

15.04 15-49 15.16 15.29 15-45 15.60 15.94

All dissolutions, incubations and precipitations of soluble and of cross-linked fibrin were carried out by dialysis at 6° using Visking tubings. Volume inside, IO ml; outside, approx. 2 l. Dialysis lasted 2-3 days with stirring and solvent exchange 3 times daily. Composition of the phosphate buffer (pH 6.g): 44.6 g KCl, ro.g g Na 2HP04 ·2 H 2 0, 5.28 g KH 2 P0 4 made up to 2l. For electron microscopic observation the Elmiscop I from Siemens, Berlin, with double condensor and 70-tJ-m objective aperture was used. High tension 8o kV, electron microscopic magnification 40 ooo: r. Copper nets (Veco Zeefplatenfabriek NV; Eerbeck, Netherlands) coated with a collodium film were used as sample carrier. The coated net was drawn with forceps in a direction parallel to its plane through the fibrin gel (fibrils or swollen fibrin). Larger pieces of gel were removed with some other forceps. The net was then laid for 5-ro min on the surface of a large drop of 2% phosphotungstic acid of pH 4 (set with I M NaOH). Excess acid was thoroughly rinsed out with water. Optical rotation was measured in temperature-controlled cuvettes at 20° with the photoelectric polarimeter o.005° manufactured by Zeiss at 405 mtt. Readings were taken at intervals of IO-I5 min until constant values were obtained. Nitrogen was determined by the micromethod of STRAUCH 27 or Kjeldahl. All reagents were A.R. with the exception of pure NaBr, Merck, Darmstadt, and guanidinium chloride, Schuchardt, Munich. Disodium salt of ATP crystallized A.R. was procured from Serva, Heidelberg, and thrombinum purum from Behringwerke, Marburg.

Biochim. Biophys. Acta, 207 (1970) 445-455

R. GOLLWITZER et al. RESULTS Investigation of soluble fibrin Many of the agents suitable for dissolving fibrin have a denaturing effect. In order to demonstrate whether dissolved fibrin is native or whether it is more or less denatured, the solution was dialysed against phosphate buffer (pH 6.g) and the precipitate formed was observed in the electron microscope. The presence of crossstriated fibrils was conclusive evidence that no conformational change had taken place. Fibrils without cross-striation or structureless precipitates indicated denaturation of various degrees. The following dissolving agents were used: I M KBr (or N aBr), 2% acetic acid, 6 M guanidinium chloride or 2 M KCNS. I M KBr was first described by SHULMAN et al. 28 in I953 as a mild dissolving agent for fibrin. So far, the physicochemical experiments of DoNNELLY et al. 29 have shown that in this agent fibrin occurs in monomeric form at pH 5.3, whereas a higher pH causes aggregation. Fibrin dissolved slowly, when the clot was dialysed against a large excess of I M KBr. This procedure eliminated the pH changes observed during the dissolution of fibrin in I M KBr30 •31 and removed all low-molecular-weight participants of the coagulating system. Subsequ~nt dialysis against phosphate buffer (pH 6.g) precipitated fibrils which, under the electron microscope, revealed the same cross-striation

Fig. I. Cross-striated fibril obtained from solutions of fibrin in r M KBr (pH 5-3) by dialysis against 0.05 M phosphate (pH 6.g)-o.3 M KCI. Bar (in every figure) represents o. r I'·

with a period of 220 ± IO A as the original material (Fig. I). However, the fibrils were thinner (diameter 200-JOO A) than those produced by coagulation. Thicker fibrils were obtained by stepwise dialysis against phosphate buffer first kept at pH 4·5 and subsequently raised to pH 6.g. At pH 4·5 fibrin remained in solution and precipitated slowly, when dialysed against buffer pH 6.g. Apparently, no conformational change had taken place in I M KBr.

Fig. 2. Fibril without cross-striation obtained from solutions of fibrin in 2% acetic acid by the procedure indicated in Fig. I.

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ELECTRON MICROSCOPY OF FIBRIN

TABLE II SPECIFIC OPTICAL ROTATIO:t-; OF FIBRINOGEN A:t-;D FIBRIN IN VARIOUS

SOLVE~TS

MEASURED AT 405 IDfl

Solvent

0.3 MNaCl I M NaBr (pH 5.3)•

2%

acetic acid 23.33% urea 6 M guanidinium chloride 2M KCNS

Fibrinogen

Fibrin

--147·8' - I38·9° 0 - I83 . .')

insoluble

···I5!.00

- I55·00

-Lfil.7"

-245·7'' - IJ6.z"

- I33·9°

·- I39·2o -

I83·3o

• Compare ref. 32.

When fibrin was dissolved in 2% acetic acid and dialysed against phosphate buffer (pH 6.g), fibrils were obtained which did not reveal any cross-striation under the electron microscope (Fig. 2). Thus, limited damage had taken place in 2% acetic acid, not enough, however, to affect the ability to form fibrils. This capability was noticeably affected by 6 M guanidinium chloride or 2M KCNS. During dialysis against phosphate buffer fibrin dissolved in 6 M guanidinium chloride formed a precipitate which, under the electron microscope, only seldom revealed fibrillar structures. From 2 M KCNS solutions the same procedure precipitated an almost entirely unstructured fibrin. The electron microscopic observations concerning the influence of solvents on the conformation were supplemented by measurements of the optical rotation of the various solutions of fibrin at 405 mtt (Table II). The values of fibrin always agreed well with those of fibrinogen in the same solvent33 (see Table II). The specific optical rotation of material dissolved in 2% acetic acid or in 6 M guanidinium chloride differed considerably from fibrin dissolved in I M NaBr. Surprisingly the rotational value in 2M KCNS, which has a strong denaturing effect, was the same as that for I M NaBr in which dissolved fibrin retained its original conformation. Possibly in this case factors influencing the optical rotation which can be attributed to conformational changes are cancelled by solvent factors which are not dependent on conformation. The changes in the optical rotation during transfer from 2% acetic acid to 6 M guanidinium chloride were reversible. The specific rotation shown by fibrin in a solution TABLE fll SPECIFIC OPTICAL

ROTATIO~

OF FIBRIK Dl:RIKG

CO~SECUTIVE

TREATMEKTS WITH VARIOUS SOLVENTS

Solvent exchange by dialysis.

Consecutive treatment Solvent

I. 2%

acetic acid 6 M guanidinium chloride 3· o.os :VI phosphate (pH 6.9) 4· 2% acetic acid

2.

Time (h) -I9!.7° -250.2°

Ppt. -

I94·6°

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of 2% acetic acid became more negative during dialysis against 6 M guanidinium chloride. When fibrin was subsequently precipitated by dialysis against phosphate buffer (pH 6.9) and redissolved in 2% acetic acid, the specific rotation originally found in this solvent was regained (Table III).

Investigation of enzymatically cross-linked fibrin Coagulation of fibrinogen by thrombin in the presence of added excess Factor XIII and calcium yielded cross-linked fibrin which no longer could be dissolved in all the aforementioned solvents suitable for fibrin. However, most of these solvents caused a considerable swelling of the fibrous structure. Examination of the swollen material under the electron microscope revealed structures that varied according to the denaturing properties of the swelling agents used. The same held true when the capability to reconstitute fibrils was tested.

Fig. 3· Subfibrils of enzymatically cross-linked fibrin dissociated by longitudinal junction within the subfibrils.

I

lVI KBr (pH 5.3). Arrows:

Enzymatically cross-linked fibrin, treated with r M KBr, appeared divided into numerous subfibrils ordered lengthwise (Fig. 3). These had a uniform width of about 120 A, corresponding to twice the diameter of a fibrinogen molecule. In the middle of the subfibrils a thin white line occasionally became visible which may represent a junction between two polymer strands. A cross-striation of the subfibrils was no longer perceptible. If KBr was removed by dialysis against phosphate buffer (pH 6.g), the original compact fibrin fibrils reappeared, with cross-striation recognizable under the electron microscope (Fig. 4). Biochim. Biophys. Acta, 207 (1970) 445-455

451

ELECT RO:'\ MICROSCOPY OF F IBRI N

Fig. 4· Cross-striated fibrils ob t a ined fro m cross-linked fi brin prev iously swollen with (pH 5-3) b y dia lysis against 0.05 M ph osphate (p H 6.9) --0.3 M l
I

.iVI KBr

Enzymatically cross-linked fibrin , treated with 2% acetic acid, also showed a division into parallel subfi brils wh en observed under t he electron microscope34 • The subfibrils, however, appeared t o be damaged t o some ext ent (Fig. 5). Dialysis of the swollen mat erial against phosphate buffe r (pH 6.g) again induced formation of the compact fibrils, but these were no longer cross-striated. Obviously the 2% acetic acid had some damaging effect on the molecules in cross-linked fi brin similar to that upon soluble fibrin. Finally, 6 M guanidinium chloride transformed enzymatically cross-linked fibrin into a subst ance which, under the electron microscope, revealed striated structures showing no preferred direction of arrangement. Dialysis against phosphat e buffer restored poorly developed fibrils. In 2 M KCNS only limited swelling of cross-linked fibrin was observed . In one experiment, already clotted but not yet cross-linked fibrin was treated with Factor XIII in the presence of calcium and thrombin . The insoluble material obtained by this two-step procedure, when dialysed against r M NaBr and subsequently observed in the electron microscope, showed division into subfibrils of a diam-

Fi g. 5· Subfi brils of cross- linked fibrin d issociated b y

2%

acet ic ac id.

Biochim. Biophys. A cta, 20 7 {r970) 445-455

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eter identical with that of fibrin which had been clotted by thrombin in the presence of Factor XIII. Precipitation of dissolved or swollen fibrin by dialysis against ATP ATP, due to its polyanionic nature, is capable of precipitating proteins from acidic solutions in which they exhibit a positive charge. Under these conditions the aggregation of the protein molecules to structures of superior order is modified. Particularly, this has been investigated in the case of collagen solutions which during dialysis against ATP release the so-called 'long spacing segments' that differ from native-type fibrils 35 • The native structure of the individual molecule is retained during this process, but the arrangement of the monomers in the precipitate is altered 36 . The question was raised as to whether ATP might also induce the precipitation of modified structures from dissolved fibrin. When a solution of fibrin in I M KBr was dialysed against 0-4% ATP solution at pH 4.5, a precipitate resulted which appeared to be globular in the electron microscope. The fibrin was nevertheless native. It could be dissolved, although slowly, by I M KBr and converted to cross-striated fibrils by dialysis against phosphate buffer of pH 6.g (Fig. 6). Fibrin dissolved in 2% acetic acid, 6 M guanidinium chloride or 2 M KCNS precipitated also in globular form when dialysed against 0-4% ATP solution.

Fig. 6. Cross-striated fibril reconstituted from soluble fibrin previously precipitated in the presence of ATP. Fibrin was first dissolved in I M KBr (pH 5.3) and dialysed against 0.4% ATP (pH 4.5). The structureless precipitate was redissolved in I M KBr (pH 5.3) and transformed to fibrils by dialysis against o.o5 M phosphate (pH 6.g)-o.3 M KCI.

In contrast enzymatically cross-linked fibrin which had been swollen in I M KBr aggregated to fibrils during dialysis against 0-4% ATP solution (pH 4-5). These fibrils, however, did not reveal any cross-striation. The original cross-striation was reproduced if the subfibrils were dissociated once more by I M KBr and dialysed against phosphate buffer of pH 6.g (Fig. 7). This revealed that the molecules them-

Fig. 7· Cross-striated fibril reconstituted from cross-linked fibrin previously transformed to fibrils without cross-striation by treatment with ATP. Procedure analogous to that described in Fig. 6.

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ELECTRON MICROSCOPY OF FIBRIN

selves had not been damaged. Apparently, only the arrangement of the subfibrils in a superstructure exhibiting cross-striations was affected in the presence of ATP. Cross-linked fibrin, swollen in 2% acetic acid or 6 M guanidinium chloride, reaggregated during dialysis against ATP solutions to fibrils. These, surprisingly, were formed far more precisely than fibrils obtained by dialysis against phosphate buffer but exhibited no cross-striation. TABLE IV ELECTRON MICROSCOPIC APPEARAKCE OF SOLVENT-TREATED FIBRIN

Solvent

Solvent-swollen, cross-linked fibrin

Ppt. after removal of solvent (a) By phosphate (pH 6.9) Soluble fibrin Cross-linked fibrin (b) By ATP (pH 4-5) Soluble fibrin Cross-linked frbrin

r i\1] KBr (pH 5-3)

2% acetic

6 M guani-

2

Subfibrils, diameter

Subfibrils, thickness less defined

Shapeless films

Limited swelling

Crossstriated fibrils

Fibrils without crossstriation

Distorted structures Poorly developed fibrils

I20

A

l~lobuli •

acid

Globuli Fibrils' Fibrils Without cross-striation

dinium chloride

Globuli Fibrils

M KCNS

Globuli

'After removal of ATP capable of converting into cross-striated fibrils.

DISCUSSION

The electron microscopic results obtained after treatment of soluble as well as enzymatically cross-linked fibrin by several solvents are summarized in Table IV. The investigations concerning fibril formation from solutions of fibrin show that the solvents used affect fibrin to a varying extent. No denaturation occurred in I M KBr. For the first time cross-striated fibrils of fibrin could be reconstituted from this solvent. Limited denaturation, however, appeared to have taken place in 2% acetic acid, since fibrils were obtained which did not exhibit any cross-striation. More pronounced structural changes were effected with, for example, 6 M guanidinium chloride. However, these more severe effects showed themselves to be to a large extent reversible. This was evident from the optical rotational values. Fibrin dissolved in 6 M guanidinium chloride and subsequently transferred to 2% acetic acid exhibited the same optical rotation as fibrin dissolved in acetic acid. The above-named solvents, besides 2 M KCNS, have a swelling effect on enzymatically cross-linked fibrin. The influence on the native structure which accompanied the swelling action varied in a manner analogous to that observed for soluble fibrin. When swollen in I M KBr, distinct subfibrils of uniform thickness became visible. As soon as the swelling agent was removed they reaggregated to fibrils with Biochim. Hiophys. Acta, 207 (I970) 445-455

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the original cross-striation. Treatment with 2% acetic acid or 6 M guanidinium chloride produced material which contained damaged subfibrils or had no distinct structures at all. It was reaggregated to fibrils without cross-striation by removing the solvent. The intermolecular cross-links which render fibrin insoluble have apparently no special stabilizing effect on the conformation of the individual fibrin molecules connected to each other. However, during deswelling they induce a reformation of fibrous structures. The individual subfibrils, which were dissociated by I M KBr and which contained native cross-linked fibrin molecules, no longer showed any cross-striation. Distinct cross-bands, however, reappeared if the subfibrils were again rearranged according to their original superstructure. As early as HALL 18 , it was recognized that fibrils with a diameter below 200 A do not exhibit cross-striation. Clearly the crossstriation is observed only in a unit of multiple subfibrils whose polar groups are in register. This statement could be of considerable significance for the explanation of the cross-striation (cf. ref. 37). Since the native structure of enzymatically cross-linked fibrin was not affected during swelling with I M KBr, the electron micrograph of the swollen substance makes possible more detailed statements concerning the arrangement of fibrin molecules within the subfibrils and thus, also, the arrangement of cross-links. The subfibrils have a diameter twice that of the terminal nodules of the fibrin molecule. They do not, however, reveal any grainy structures, in which the individual nodules might be recognizable. Apparently, the individual nodules arrange themselves during fibril formation very close to one another, possibly with a certain degree of deformation. This tight package of the fibrin monomers within the subfibrils is not affected by I M KBr. The electron microscopic results on fibrin subfibrils justify a discussion of the arrangement, in fibrin, of the intermolecular bonds formed by activated Factor XIII. The subfibrils appear to be independent units without regular connections to adjacent subfibrils. A three-dimensional covalent cross-linking must be rejected. The intermolecular bonds are limited to the unit of the subfibrils. There, they appear to be arranged in such a way that a predominantly longitudinal association of fibrin molecules is achieved, while the diameter of the subfibrils is restricted to that of two monomers. The dimerization of the polymer strands occurs as early as the polymerization of the fibrin monomers to fibrin fibrils and not during the later formation of covalent cross-links by activated Factor XIII. This is apparent from the fact that fibrin, which was stabilized by the addition of Factor XIII subsequent to clotting, can also be dissociated into subfibrils with a diameter of I20 A by dialysis against I M KBr. Therefore, already existent double strands are cross-linked by activated Factor XIII. Various authors 38 - 40 have previously postulated the arrangement of fibrin molecules into dimeric strands during fibril formation on the basis of physicochemical experi, ments. The extent to which the intermolecular cross-links stabilize the fibrous structure of fibrin becomes evident from comparative investigations on the influence of ATP on soluble and on cross-linked fibrin incubated with I M KBr. Globular precipitates were obtained from solutions of fibrin in the presence of ATP. This globular appearance was due to an altered arrangement of the individual molecules. No change of conforBiochim. Biophys. Acta, 207 (1970) 445-455

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ELECTRON MICROSCOPY OF FIBRIN

mation was observed. Probably, the change in aggregation was due to the influence of ATP on the electrostatic interaction of the fibrin monomers. In fibrin stabilized by activated Factor XIII, the possible aggregations of the monomers are limited by the intermolecular cross-links. Even in the presence of ATP, the subfibrils of swollen fibrin could only reaggregate to fibrils. The superstructure, however, was altered to some extent, as shown by the lack of cross-striation. The altered structure induced by ATP was converted to the original one by further incubation with I M KBr and subsequent dialysis against phosphate buffer (pH 6.g). ACKNOWLEDGEMENT

We are indebted to Miss A. Lichtenauer for technical assistance. REFERENCES I 2 3 4 5 6 7 8 9 IO II IZ I3 q 15 I6 17 I8 I9 zo 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

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