Toxkron, Vol. 26, No . 2, PP. 218-221, 1988.

00"1-0101/88 53 .00+ .00Printed in Grat Britain

© 1988 Perpmon Journals Ltd .

ISOELECTRIC FOCUSING OF PROTAC®, THE PROTEIN CACTIVATOR FROM COPPERHEAD (AGKISTRODONCONTORTRI~ VENOM: A NOTE ON EXPERIMENTAL

PROBLEMS

J . MEIER, CH. ADLER and K. STOCKERPentapharm Ltd, 4002 Basle, Switzerland

(Acceptedfor publication 2 September 1987)

J. MB1ER, Clt . ADLER and K. $TOCKER. Isoelectric focusing of Protac~, the protein C activatorfrom copperhead (Agkistrodon contortrix) venom: a note on experimental problems . Toxicon 26,218-221, 1988 . - The isoelectric point of Protac® was recently estimated to be at pH 3 .However, further investigations using different experimental procedures revealed that Protacm,due to its particular binding properties, is able to folal complexes with carrier ampholytes . Thus,the actual isoelectric point of Protean was found to be in the basic region .

THE PROTEIN C activator Protac® from Agktstrodon contortrix venom is a glycoproteinwith a molecular weight of 37,000 that converts the zymogen protein C into a productwith the enzymatic properties of activated proteinC. Using thin layer polyacrylamide gelson polyester films (Servalyt®-Precotes®, SERVA GmbH, Mannheim, F.R.G.)(RADOLA, 1980) and a system of carver ampholytes establishing a pH gradient, theiscelectric point of Protac® was estimated to be pH 3 (STOCKER et al ., 1987a). In testsystems containing contact activators ofthe intrinsic blood coagulation pathway, a strongaffinity of Protac® for the electronegative surfaces of kaolin and silica particles wasobserved (STOCKER. et al ., 1987b) . This phenomenon seems to contradict an isoelectricpoint in the acidic pH range.

In immune electrophoresis using a barbital buffer, pH 8 .6, Protac® showed only aweakmobility to the anode. Moreover, Protac® bound with a high affinity to immobilizedheparin as well as to carboxymethylated, sulfophenylated or sulfoethylated polymers .This affinity to polyanions also indicates the basic character of this protein. ExxER andVAASJOKI (1987), based on their observations that the protein C activator fromA. contortrix venom fails to bind to the chromatofocusing medium PBE94 (Pharmacia,Uppsala, Sweden), suggest a basic iscelectric point. The obvious contradiction of ourisoelectric point determination and the cationic behaviour of Protac® led us to aninvestigation of this phenomenon .

Iscelectric focusing became a widely used procedure for iscelectric point estimationsince several ready-for-use systems are commercially available. However, those

21s

Short Communications

3

ao00 o~

ôxaô â â

00

â .a g ââ é ~ ~ .Neri O~ O~ a

IaoN

I~o

I~o . ~ ô

..

âh

Éq

äW W

ao

~aa

8 8ô ô

â~8 0

n

à à à àn

a

à àa aà~

â~i3 syn

ô~1~ w

O O O O� ~

I I I I I I "~ Ôen en v~e+i

en en ~O

ca

220

Short Communications

procedures using carrier ampholytes have several drawbacks, such as instability of the pHgradient, uneven conductivity and buffering capacity, extremely low and unknown ionicstrength and limited loading capacity due to isoelectric precipitation . Furthermore, it isknown that, occasionally, proteins may associate with carrier ampholytes, acquiring acombined iscelectric point different from that of free proteins . This may even causemultiple minor bands, since the ampholytes themselves occur as discrete components(SCOPES, 1982) . The binding capacity for such complexes is strongest in the pH 3 area ofthe gel (GIANAZZA and RIGHETTI, 1978) . Although most data available refer topolyanions, it has been shown that binding can occur also with basic proteins (FRATER,1970) . Whether conformational changes, blocking of charged groups or a combination ofsuch events with addition of further charges are provoked by the protein - ampholyteinteraction remains unknown. However, binding is affected by the type of charge, itsdensity and spatial orientation of the protein (GIANAZZA and RIGHETTI, 1978) .

Table 1 shows the different experimental tests we performed to determine the iscelectricpoint of Protac® . In the case of the Servalyt®-Precotes® gels used, an extended pre-runto a final voltage of 1,700 V before application of the sample in the neutral pH range ofthe gel led to a shift into the basic pH region (the manufacturer recommends a pre-run upto 500 V before sample application) . This observation is in agreement with data obtainedby focusing an acidic protein from wool (FRATER, 1970) . An experimental series using theAmpholine~ system (LKB, Bromma, Sweden) revealed that the self-prepared gels also ledto a separation of Protac® between pH 6 and 9, if the system contained 8 M urea . Urea inthis concentration is able to dissociate complexes with carrier ampholytes (GIANAZZA andRIGHETTI, 1978) .The Immobiline® system (LKB, Bromma, Sweden), a method using immobilized pH

gradients (BJELLQVIST et al., 1982), was also used for isoelectric point determinations .Protac® was always found at the cathodic end, when gels with a pH range of 3 - 5 or 3 - 6were used. In gels with a pH range of 6- 10, Protac® was separated in three major bandsat pH 7.8, 8.0 and 8.8 respectively . A similar pattern was found in experiments carried outby Ek (LKB; personal communication) .As concluded from these findings and in contrast to our previous report, the isoelectric

point of Protac® is located in the basic region . However, the question as to whether theformation of more than one band from a preparation, which in SDS - PAGE,immunoelectrophoresis and analytical ultracentrifugation, appeared homogeneous, hasto be attributed to a true inhomogeneity or to an artifact, remains unknown.

Acknowledgements -We thank DrM. HÄUSERMANN (LKB Switzerland) and Dr K . EK (LKB Bromma) for theirexcellent cooperation . Miss A. ENTZ (Pentapharm Ltd) kindly prepared the manuscript .

REFERENCES

BJELLQVIST, W., EK, K ., RIGHETTI, P. G, GIANAZZA, E ., GOERE, A., WESTERMEIER, R . Snd POSCEL, W. (1982)Isoelectric focusing in immobilized pH gradients : principle, methodology and some applications . J. Biochem.Biophys. Methods 6, 317.

EXNER, T. and VAASJOKI, R. (1987) Partial characterization of a protein C activator from Agkistrodoncontortrix contortrix venom . Thromb. Haemostas. (in press) .

FRATER, R . (1970) Artifacts in isoelectric focusing . J. Chromat. 30, 469.GIANAZZA, E. and RIGHETTI, P. G. (1978) Binding of polyanions to carrier ampholytes in isoelectric focusing .Biochim. biophys. Acts 340, 357.

RADOLA, B. J . (1980) Ultrathin layer isoelectric focusing in SO-100 iJm polyacrylamide gels on silanized glassplates or polyester films. Electrophoresis 1, 43 .

SCOPES, R . (1982) Protein Pur~cation : Principies and Practice. New York : Springer .

Short Communications

221

STOCKER, K., F~sctiea, H., Matt, J ., BAOOLI, M. and Svrrvnsew, L . (1987x) Characterisation of the protein Cactivator Prolog from the venom of the southern copperhead (Agkistrndon contortrlx) anahe. Toxirnn 2S,239 .

S~o~x, K., Ftsc~a, H. and Matt, J . (1987b) Practical application of the protein C activator Protacm fromAgkistrodon rnntortrix venom . Folio haentat. (in press).