Proc. Nat. Acad. Sci. USA Vol. 72, No. 7, pp. 2577-2581, July 1975

Biochemistry

Amino-acid sequence of activation cleavage site in plasminogen: Homology with "pro" part of prothrombin (zymogen activation/urokinase specificity/plasminogen structure)

LARS SOTTRUP-JENSEN*, MARIA ZAJDEL*, HENDRIK CLAEYSt, TORBEN E. PETERSEN*, AND STAFFAN MAGNUSSON* t *Department of Molecular Biology, University of Aarhus, DK-8000 Arhus C, Denmark; and tDepartment of Internal Medicine, Academic Hospital, B-3000

Leuven, Belgium

Communicated by K. M. Brtnkhous, April 21,1975

ABSTRACT A 38-residue fragment is isolated from carboxymethylated plasminogen. Residues 29-38 have the same sequence as the amino-terminal end of the light chain of plasmin. The sequence 1-28 is therefore the sequence of the carboxyl-terminal end of the heavy chain and contains the specific sequence at which urokinase (EC 3.4.99.26) and other plasminogen-activating serine proteases split. Two of the five carboxymethyl-cysteine residues in the isolated fragment are situated close to the cleavage site and the fragment is not itself a substrate for plasminogen-activators. Residues 1-11 show extensive sequence homology with residues 137147 and 242-252 in rot rombin, which are located in corresponding regions of the two internally homologous 83-residue structures in the non-thrombin part of the molecule, indicating that such structures may be a common feature of the non-protease part of the larger serine protease zymogens. The determination of the complete primary structure of prothrombin (1, 2) has led to the realization that the activation catalyzed by Factor Xa (prothrombinase, EC 3.4.21.6) involves the cleavage of only two peptide bonds, namely Arg274-Thr and Arg23-Ile. Prothrombin also contains a single thrombin-sensitive bond (Argl'6-Ser). The two FactorXa-sensitive bonds are preceded by the identical tetrapeptide sequences -Ile271-Glu-Gly-Arg- and Ile320-Glu-Gly-Arg, whereas the corresponding residues at the thrombin-sensitive site are different, namely -Vall'3-I1e-Pro-Arg. These findings indicated that a specific oligopeptide sequence immediately preceding the bond to be cleaved is required to define a particular protein as being a substrate for one of those highly specific trypsin-like serine proteases, such as Factors Xa, IXa, XIa, XIIa of the blood coagulation system, urokinase (EC 3.4.99.26) and others of the fibrinolytic system, kallikrein, and the proteolytic components of the complement system, which are involved in the activation and control of many extracellularly regulated processes. To test this possibility with respect to Factor Xa specificity we recently synthesized the compound Tos-LIle-LGlu-Gly-LArgp-nitroanilide and found it to be a good substrate for Factor Xa, but not for thrombin (EC 3.4.21.5), plasmin (EC 3.4.21.7), urokinase or streptokinase-plasminogen complex (T. E. Petersen, L. Sottrup-Jensen, and S. Magnusson, in preparation). The rate of Factor-Xa-catalyzed cleavage of this synthetic compound could not by itself explain the rapid rate of activation of prothrombin. One possible explanation for this is that the two -Ile-Glu-Gly-Arg- sequences may be Abbreviations: Tos, p-toluene sulfonyl; CTA, Committee on Thrombolytic Agents; Dns, 5-dimethylaminonaphthalene-l-sulfonyl; DEAE, diethylaminoethyl; CM, carboxymethyl. $ To whom reprint requests may be sent. 2577

only parts of two larger binding sites for Factor Xa in the prothrombin molecule (2). Since prothrombin contains the two so-called kringle structures, having 31 sequence identities in two structures of 83 residues each (positions 62-144 and 167-249) (1, 2), this explanation could not be excluded. To further elucidate this question we decided to investigate the primary structure of plasminogen, another large zymogen which is activated by limited proteolysis catalyzed by urokinase, by streptokinase-plasminogen complex (3, 4) or by a host-specific cell factor produced by virus-transformed malignant mammalian cells (5-7). The activation involves the conversion of the single-chain plasminogen to plasmin consisting of a heavy chain disulfide-bridged to a light chain. The only (8) or the last (9, 10) step in the activation appears to be the cleavage of that peptide bond which connects the C-terminal -Arg of the heavy chain to the N-terminal Val- of the light chain (3). The amino-acid sequence of the N-terminal region of the light chain is already known (11). On the basis of this sequence and since no small activation peptide has yet been demonstrated from the heavy chain to light chain overlap region, one could reasonably expect to find a chymotryptic fragment from reduced f14C]carboxymethylated plasminogen which would contain the activation cleavage site and have either of the two C-terminal sequences -Arg-Val-Val-Gly-Gly-CMCys-Val-Ala-HisPro-His or -Arg-Val-Val-Gly-Gly-CMCys-Val-Ala-His-ProHis-Ser-Trp-Pro-Trp. Such a fragment would stain specifically for arginine, histidine, and perhaps tryptophan, and it would also be radioactive. This paper reports the isolation and amino-acid sequence of such a fragment, and also notes the presence in plasminogen of structures that show extensive sequence homology with the two internally homologous kringle structures in the "pro" part of prothrombin. MATERIALS AND METHODS Human plasminogen was prepared by affinity chromatography according to ref. 12 as modified in ref. 13. It was homogeneous on sodium dodecyl sulfate disc gel electrophoresis and had a specific activity of 23.9 Committee on Thrombolytic Agents (CTA) U/mg when assayed without 6-aminohexanoic acid and 32.2 CTA U/mg in the presence of 1 mM 6-aminohexanoic acid. Chymotrypsin, three times crystallized (Worthington); trypsin, two times crystallized (Sigma) and thermolysin (Sigma) were obtained commercially. Urokinase (L0ven, Copenhagen) and (Abbott) had specific activities of 15750 CTA U/mg and 100000 CTA U/ml, respectively. Streptokinase (Kabi, Stockholm)-plasminogen com-

2578

Biochemistry: Sottrup-Jensen et al.

Proc. Nat. Acad. Sci. USA 72 (1975)

plex (1:1 molar ratio) was obtained by mixing solutions of the two materials. Pancreatic trypsin inhibitor (Kunitz) (Trasylol, Baeyer) had a declared specific activity of 10,000 kallikrein inactivator units (KIU)/ml (approximately 1.4 mg). One milligram of soybean trypsin inhibitor (Worthington) was claimed to inactivate 2 mg of trypsin. Analytical grade chemicals were used. 2-'4C-labeled iodoacetic acid (Amersham, U.K.) had a specific radioactivity of 51 Ci/mol. Sephadex G-25 (Pharmacia) and DEAE-cellulose DE-52 (Whatman) were used for columns. Preparative isolation of peptides by high-voltage electrophoresis was performed on Whatman no. 3 MM or no. 1 paper at 3 kV in buffers of pH 6.5 and pH 2.1 (14). Peptides were stained with cadmiumninhydrin (15). Specific color tests for peptides containing tryptophan (16), histidine and/or tyrosine (17), or arginine (18) were also used. CM-Cys-containing peptides were detected by autoradiography. Amino-acid compositions were estimated after hydrolysates (6 M HC1, 1100, 20 hr) had been subjected to electrophoresis at pH 2.1, 5 kV, 17 min on Whatman no. 1 paper and stained with collidine-ninhydrin (19). Quantitative amino-acid analyses were performed essentially according to ref. 20 using Locarte and Beckman 121 M analyzers with two-column systems. Amino-acid sequences were determined by the Dns-Edman method (21). Dns-amino-acid derivatives were identified by polyamide thin-layer chromatography (22). Peptide mobilities were related to charge and size (23).

0 C n

._

Chymotryptic Digest of Reduced, Carboxymethylated Plasminogen. Plasminogen (6 ,umol) was dissolved in 25 ml of 0.3 M Tris.HCl, 3 mM EDTA, pH 8.6, containing 4.2 mg of pancreatic trypsin inhibitor. Guanidine.HCl was added (final concentration 6 M) and 30 min later 240 Mmol of dithiothreitol. After reduction for 60 min, 600 ,mol of sodium iodoacetate (containing 50 ,uCi of iodo[2-14C]acetic acid) was added. Carboxymethylation was allowed to proceed for 30 min. After adjusting to pH 2.2 with acetic acid and desalting on Sephadex G-25 (4 X 120 cm) in 10% acetic acid the freeze-dried material from the void-volume peak was suspended in 500 ml of 0.15 M NH4HCO3 and digested (370, 1.5 hr) with 4.5 mg of chymotrypsin (pretreated with 400 ,ug of soybean trypsin inhibitor). All suspended material dissolved in 30 min. Diphenylcarbamylchloride (5.9 mg in 3 ml of methanol) was added to stop digestion. The mixture was freeze-dried, then dissolved in 40 ml of water, and adjusted to pH 8.3 with ammonia. A small amount of insoluble material was separated by centrifugation. The supernatant was applied to a DEAE-cellulose column, and the peptides were separated in a system of ammonium bicarbonate buffers (Fig. 1). The effluent was monitored by measuring the transmission at 280 nm and also by making three sets of peptide maps. One set was first stained with Cd2+-ninhydrin and then for tryptophan. The second set was stained for his-

,.

0.8

30 4

0.7

20 E c

RESULTS

40 1

j 0.6 0 8

F

0.5 I

m

50

I

z

0

60

0.4 'o

70

0.13

._

C

CA #A

nA

4p

OSR 80 90

A-

--AL-

~

~

~

~

~

~

~

~

-

--

-

0.2

- -

---/---W----

----

10

20

30

--

40

50

60

70

80

0

'r C

0.1 00

90

Fraction no.

20 r

0.8

30

0.7 2

40

0.6 °

CN 50

*"60

0.5 I z 0.4 'o

70

0.3 *~

80

0.2

£

0.1

c0

f 0

I

co

C

0

E c

Cu-W

0

90

100 90

100

110

120

130 140 Fraction no.

150

160

170

180

FIG. 1. Chromatographic separation of chymotryptic peptides from plasminogen. A column of DEAE-cellulose (1.3 X 75 cm) was equilibrated and developed with NH4HCO3 buffers of pH 8.3. The buffer concentration was increased as follows: equilibration: 20 mM; elution: 40 ml of 20 mM; linear gradient of 200 ml of 20 mM and 200 ml of 150 mM; linear gradient of 300 ml of 150 mM and 300 ml of 600 mM; 150 ml of 600 mM. Broken line: Buffer concentration. Solid line: Percent transmission at 280 nm. Peptide C was eluted in fractions 97-104, indicated by horizontal bar.

Proc. Nat. Acad. Sci. USA 72 (1975)

Biochemistry: Sottrup-jensen et al.

2579

Table 1. Amino-acid compositions, yields, and electrophoretic mobilities of chymotryptic fragment and peptides from subdigests C

CT1

CT1TL1

4.5 3.5 1.2 3.1 4.9

2.5 3.2 0.9 3.0

2.0 1.9

CT1TL2

CT1TL3

CT2

CT3

CTL1

0.9 1.2

0.8

0.9

1.9 2.2

CM Cys Asx Ser Glx Pro Gly Ala Val Tyr Phe Lys His Arg

3.0 1.9 1.3

2.0

Yield %

10

78

67

61

61

70

84

0*31

0.67

0.95

neutral

0.26

-0.40

-0.20

-3

-5

-4

0

-1

+1

+1

pH

6

3.0 4.4 0.9

3.2 1.0 2.0 2.1 0.9

1.0

1.0,

3.8

2.l1

pH mob/Ser

1.04

2.0 1.8 1.2

1.1

1.0v

2. 0

0.9 1.9 1.0* 2.5

1.0

1.0 0.8

CTL4

0 .9 1.4

CTL5

CTL6

1.7 1.4

0.8

1.0* 1.3

1.3

1.3 1.0

1.5

1.8 1.4

1.7 3.4 2.0*

2. 0*

CTL7

1.1

1.6

1.0

1.0*

2.0* 0.9

1.0

0.9

1.0* 2.0

1.0w 1.3

1.0

0.8 1.4

2.7

1.0*

1.0

17

29

2.1

1.9

1.0

.

mob/Asp charge5

1.2

CTL3

1.0

0.8 1.0 1.0

CTL2

0.88

54

60

0.97 neutral

-4 0.38

0 0.88

29 0.40

-2

-0.46 neutral

+2

0

60

69

0.51

-0.52

-1

+1

1.38

C, CT1, etc. are codes for the peptide identification; see legend to Fig. 2. * The amino acid used as base for calculating relative composition. t Yield expressed as % of amount digested. § The electrophoretic mobility relative to aspartic acid at pH 6.5 was used to determine the charge (23); the mobility relative to serine is indicated for peptides further purified at pH 2.1. After each of the first four cycles of Edman degradation of peptide CTL1 a sample of the remaining peptide was subjected to electrophoresis at pH 6.5. The mobilities found were used to locate the amide on residue number 4.

tidine and tyrosine, the third for arginine. All three sets were examined by autoradiography for CM-cysteine and the autoradiographic spots were also used to check reproducibility between sets. Each set of maps was obtained by applying 0.75% of the material from each consecutive pair of tubes as contiguous 1 cm lines over the width of the paper. After electrophoresis at pH 6.5, 3 kV, 40 min, the area containing neutral peptides was cut out and stitched to a new paper and the peptides were separated by electrophoresis at pH 2.1, 3 kV, 40 min. On the basis of the information from the maps the peptides were collected in 28 pools, each of which was purified by paper eleqtrophoresis. The maps showed close to 150 well-defined spots, only four of which contained Arg, His, Trp, and CM-Cys. During preparative isolation they segregated to different peptides, none of which contained all three of the residues Arg, His, and CM-Cys. Another three spots contained Arg, His, and CM-Cys, but not Trp. Two of these spots were rather weak. The third spot was strong in all three "stains" and corresponded to peptide C (Table 1) isolated from pool 18 by preparative paper electrophoresis at pH 6.5 and pH 2.1. Peptide C was found to contain the desired heavy chain to light chain overlap. The complete sequence of peptide C was deduced by combining data from amino-acid analysis (Table 1) and sequencing (Fig. 2) of peptide C and of the peptides isolated from tryptic and thermolytic subdigests of C. The peptides CTL.3 to CTL5 are thermolytic split variants and were obtained in very low yield (30-45 nmol), and the amino-acid analyses of those three peptides were performed on only 100-200 pmol and are inaccurate. However, the sequence data obtained from these peptides were unambiguous, and

the balance of evidence is therefore sufficient to prove the given in Fig. 2. Attempted Degradation by Urokinase and by Streptokinase-Plasminogen Complex. Peptide C (10-15 nmol) was incubated for 1 hr at 370 with either of 20 CTA-U of urokinase (Abbott), 1050 CTA-U of urokinase (L0ven), or 0.11 nmol of streptokinase-plasminogen complex in a total volume of 20 ul of 0.15 M NH4HCO3. All three incubation mixtures were then subjected to high-voltage electrophoresis at pH 6.5, and the paper was stained with Cd2+-ninhydrin. Corresponding amounts of the respective activators were examined as blanks. In all three cases no peptide other than the original peptide C with a mobility of 0.31 relative to Asp was detected. sequence

DISCUSSION Since the sequence of residues 29-38 of peptide C is identical to that of the first 10 residues of the light chain of plasmin as determined by Robbins et al. (11), it is concluded that peptide C is the desired heavy chain to light chain overlap fragment and that residues 1-28 constitute the C-terminal end of the heavy chain. The Arge-Val bond is then the bond which is cleaved by the plasminogen activators during conversion of plasminogen to plasmin. This agrees with the C-terminal Arg found by Robbins et al. (3) and with the data of Wiman and Wallen (9) which indicated that no small peptide is released from this region of plasminogen during activation. The negative results of the attempted degradation of peptide C by urokinase and by streptokinaseplasminogen complex prove that the carboxymethylated

Biochemistry: Sottrup-Jensen et al.

2580

2

1

3

5

4

Proc. Nat. Acad. Sci. USA 72 (1975) 8

7

6

10

9

12

11

15

14

13

17

16

18

19

ASP-TYR-CYS-ASN-VAL-PRO-GLU-CYS-ALA-ALA-PRO-SER-PHE-ASP-CYS-GLY-LYS-PRO-GLUI

,

,

,

,

,

,

,

,

,

,

c I

CT1 , I CT1TL2

I,

CTlTLl !

,V

,

,

,

,

.

I

I

CTjTL2 ' ,V IF , , CTL2 CTL3

,

,

I,,

I

CTL1

CTL5

20

21

22

23

24

25

26

27

28 29

30

32

31

33

34

35

36

37

38

-VAL-GLN-PRO-LYS-LYS-CYS-PRO-GLY-ARG-VAL-VAL-GLY-GLY-CYS-VAL-ALA-HIS-PRO-HIS C cont.

if CT1 cont.

I

,

, I

r

CT2

,1

1

f

CT3

I

I

I

I

I

I

I

CT1TL3 cont. i

I

I

,F

f

I

ii

CTL4

p

CTL6

CTL7

CTL5 cont.

FIG. 2. Sequence of the chymotryptic plasminogen fragment containing the "heavy chain-light chain" overlap. C is the whole chymotryptic frgment. Two subdigests of C (180 nmol each) were made, a tryptic (enzyme:substrate, 1:100, 370, 1 hr, 0.15 M NH4HCO3, pH 7.8) giving peptides CT1 to CT3, a thermolytic (enzyme:substrate, 1:60, 45°, 3 hr, 50 mM N-ethylmorpholineacetic acid buffer, pH 7.8, with 1 mM CaCl2) giving peptides CTL1 to CTL7. Of the tryptic peptides, CT1 was further subdigested (100 nmol) with thermolysin (enzyme:substrate, 1:300, 1 hr, same temperature and buffer) giving peptides CTITLI to CT1TL3. Solid lines correspond to amino-acid compositions, short diagonal lines to sequence results. Peptide CT2 was sequenced twice. Vertical arrow indicates the activation cleavage site.

fragment is no longer a substrate for these enzymes. The reason may be either that the extra negative charge introduced on residue P4§ (Cys2) by the carboxymethylation blocks the binding of the activator or that this binding involves not only residues P1 to P4, or perhaps P1 to P6, but also additional residues situated in the immediate vicinity in the native tertiary structure. The presence of Pro in position P3 (Pro26) is interesting, because it excludes the possibility that the substrate-binding geometry for the plasminogenactivating serine proteases is the same as that observed for chymotrypsin with regard to the P3 site (24). A Gly in position P2 (Gly27) also occurs in Ac-Gly-LLys-OMe, reported to be a substrate for urokinase (25). Except for an Arg in P1 the only similarity between the sequence -Lys23Lys-Cys-ProGly-Arg- at the activation cleavage site described here and the sequence -Arg-Lys-Ser-Ser-Ile-Ile-Arg- in positions 62-68 (26), which is the only urokinase-sensitive bond in maleylated plasminogen (27), is the two basic residues LysLys in P5 and P6, and Arg-Lys in P6 and P7, respectively. The fact that urokinase did not split maleylated plasminogen at the activation site (27) can be explained by suggesting that two negative charges on the maleylated lysyls in P5 and P6 are not acceptable, whereas one negative in P6 matched by one positive in P7 might still be acceptable. The dissimilarity of sequence at the two urokinase sites in plasminogen raises the question whether the split at position 68 leading to the formation of plasminogen B is not normally catalyzed by plasminogen-activators but perhaps by a different serine protease. § Numbering is from C toward N-terminal, and is relative to the activation cleavage site.

Residues 1-11 of peptide C show extensive sequence homology with residues 137-147 and 242-252 in prothrombin (1, 2) (Fig. 3), which constitute corresponding parts of the two homologous kringle structures, one of which is located in the A-fragment, the other in the S-fragment part of prothrombin. The 31 identities in the two prothrombin kringle structures include the 6 Cys residues, which in both fragments are bridged 1-6, 2-4, 3-5. The homologous decapeptide sequences (Fig. 3) contain the two Cys residues number 5 and 6 from the two prothrombin kringles, indicating that residues 1-11 in peptide C constitute part of a kringle structure in plasminogen. At present partial sequence data on other fragments from the same chymotryptic digest of CMplasminogen indicate that at least an additional 14 frag137 138 139 140 141 142 143 144 145 146 147

A:

-Glu-Glu-Cys-Ser-Val-Pro-Val-Cys-Gly-Gln-Asp-

S:

-Glu-Tyr-Cys-Asn-Leu-Asn-Tyr-Cys-Glu-Glu-Pro-

242 243 244 245 246 247 248 249 250 251 252

1

2

3

4

5

6

7

8

9

10

11

-Asp-Tyr-Cys-Asn-Val-Pro-Glu-Cys-Ala-Ala-ProFIG. 3. Sequence homology between the heavy chain part of peptide C and the non-thrombin part of prothrombin. A: residues 137-147 are located in the A-fragment part (residues 1-156) of prothrombin. S: residues 242-252 in the S-fragment part (residues 157-274). A and S together constitute the non-thrombin part (residues 1-274) of prothrombin (582 residues) (1, 2). C: the first 10 residues of peptide C (Fig. 2). The underlined residues in C are identical with corresponding residues in either the A- or the S-

C:

fragment or both.

Biochemistry: Sottrup-jensen et al.

Proc. Nat. Acad. Sci. USA 72 (1975)

2581

ments are derived from various parts of different kringle structures. Four kringles of the same size as those found in

4. Summaria, L., Hsieh, B. & Robbins, K. C. (1967) J. Biol.

prothrombin would constitute about 330 residues and if "connecting stretches" of 20 residues for each kringle are assumed, a four-kringle structure would account for about 410 or more of the approximately 520 residues that constitute the heavy chain of plasminogen B. This is the first indication of a general structure common to these large serine protease zymogens where an acidic N-terminal region of 40-80 residues is followed in sequence by one or more kringle structures (80-120 residues) and the potential serine protease structure constitutes the C-terminal 220-260 residues. Although the function of the kringle structures is not known, their similarity to proteolytic enzyme inhibitors, particularly the pancreatic secretory trypsin inhibitor (2) suggests that they contain specific binding sites for structures with which the particular zymogen interacts.

5. Unkeless, J., Dano, K., Kellerman, G. M. & Reich, E. (1974) J.

We wish to thank J. Plough, L0ven, and G. Barlow, Abbott, for gifts of urokinase; H. Nilehn, Kabi, for a gift of streptokinase; H. Bennich for providing access to a Beckman 121 M amino-acid analyzer at the Biomedical Center, University of Uppsala; and Laila Br0ns, Lene Kristensen, Lone Christensen, and Margit Skriver for excellent technical assistance. L. S. received a fellowship from the Danish Science Research Council. Financial support was given by the Danish Science Research Council, the U.S. National Heart and Lung Institute (Grant no. HL16238 HEM), and the Novo Foundation. 1. Magnusson, S., Sottrup-Jensen, L., Petersen, T. E. & Claeys, H. (1975) in Prothrombin and Related Coagulation Factors, eds. Hemker, H. C. & Veltkamp, J. (Universitaire Pers, Leiden, Holland), pp. 25-46. 2. Magnusson, S., Petersen, T. E., Sottrup-Jensen, L. & Claeys, H. (1975) in Proteases and Biological Control, eds. Reich, E. et al. (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). Chap. 9., in press. 3. Robbins, K. C., Summaria, L., Hsieh, B. & Shah, R. J. (1967) 1.

Biol. Chem. 242, 2333-2342.

Chem. 242,4279-4283.

Biol. Chem. 249,4295-4305. 6. Quigley, J. P., Ossowski, L. & Reich, E. (1974) J. Biol. Chem.

249,4306-4311.

7. Christman, J. K. & Acs, G. (1974) Biochim. Biophys. Acta 340,

339-347.

8. Sodetz, J. M., Brockway, W. J., Mann, K. G. & Castellino, F. J. (1974) Biochem. Biophys. Res. Commun. 60,729-736. 9. Wiman, B. & Wallen, P. (1973) Eur. J. Biochem. 36,25-31. 10. Rickli, E. E. & Otavsky, W. J. (1973) Biochim. Biophys. Acta 295,381 384. 11. Robbins, K. C., Bernabe, P., Arzadon, L. & Summaria, L.

(1973) J. Biol. Chem. 248,7242-7246. 12. Deutsch, D. G. & Mertz, E. T. (1970) Science 170, 1095-1096. 13. Claeys, H. & Vermylen, J. (1974) Biochim. Biophys. Acta 342,

351-359.

14. Ambler, R. P. (1963) Biochem. J. 89,349-378. 15. Heilmann, J., Barrollier, J. & Watzke, E. (1957) Hoppe-Seyl-

er's Z. Physiol. Chem. 309, 219-220. 16. Smith, J. (1953) Nature 171, 43-45. 17. Pauly, H. (1904) Hoppe-Seyler's Z. Physiol. Chem. 42, 508-

517. 18. Yamada, S. & Itano, H. A. (1966) Biochim. Biophys. Acta 130, 538-540. 19. Levy, L. A. & Chung, D. (1953) Anal. Chem. 25,396-399. 20. Spackman, D. H., Stein, W. H. & Moore, S. (1958) Anal.

Chem. 30,1190-1206. 21. Gray, W. R. (1967) in Methods in Enzymology, ed. Hirs, C. H. W. (Academic Press, New York), Vol. 11, pp. 469-475. 22. Woods, K. R. & Wang, K.-T. (1967) Biochim. Biophys. Acta

133,369-370. 23. Offord, R. E. (1966) Nature 211, 591-593. 24. Segal, D. M., Cohen, G. H., Davies, D. R., Powers, J. C. & Wilcox, P. E. (1971), Cold Spring Harbor Symp. Quant. Biol.

36,85-90. 25. Walton, P. L. (1967) Biochim. Biophys. Acta 132, 104-114. 26. Wiman, B. (1973) Eur. J. Biochem. 39, 1-9. 27. Wiman, B. & Wallen, P. (1975). Eur. J. Biochem. 50, 489494.