Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Enzyme inactivation by chemical treatment
Reexamination Certificate
1999-03-17
2001-05-22
Naff, David M. (Department: 1651)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Enzyme inactivation by chemical treatment
C435S219000, C435S023000, C514S277000
Reexamination Certificate
active
06235509
ABSTRACT:
DESCRIPTION
The present invention concerns a process for inhibiting serine proteases by derivatization with sulfonic acid derivatives or fluorophosphates, a reagent for avoiding unspecific derivatization of other proteins during the inhibition of serine proteases by sulfonic acid derivatives or fluorophosphates as well as a reagent kit for the specific inhibition of serine proteases.
The catalytically active binding pocket of serine proteases can be specifically labelled and hence inhibited by sulfonic acid derivatives, and in particular by fluorides or fluorophosphates thereof. This can, on the one hand, be used to determine the protease category (serine, cysteine, metallo, aspartyl protease) and, on the other hand, to protect proteins from proteolytic degradation (Markwardt in Pharmazie 26 (1971), 401-404, J. Kraut, Ann. Rev. Biochem. (1977), vol. 46, p. 331-358).
Although the selectivity of the reaction is very high, unspecific secondary reactions with other proteins can be occasionally detected e.g. when using the inhibitors AEBSF (4-(2-aminoethyl)-phenyl-sulfonyl fluoride), PMSF (phenylmethyl-sulfonyl fluoride), diisopropyl fluorophosphate (DFP) or amidinophenylmethylsulfonyl fluoride (APMSF). These depend above all on the pH conditions of the reaction, the protein concentration, the sulfonyl fluoride or fluorophosphate concentration and the type of accessible amino acids. Thus at high pH values, reactions can occur at the amino terminus, on lysines and on tyrosines of proteins. Such secondary reactions are of course especially undesired for proteins which are used therapeutically or diagnostically. Furthermore this can lead to falsifications of the measurement results (e.g. determination of the protein concentration by absorption at 280 nm). The secondary reactions can for example be detected by sequence analysis or simply by mass spectroscopic analyses.
Methods Enzymol. 182 (1990), p. 87, 88 and 180, 181 describes a method for detecting protease contaminations in protein preparations and mentions protease inhibitors together with information on concentration ranges in which such protease inhibitors are to be used. Processes or reagents for inhibiting an unspecific derivatization of proteins when using protease inhibitors are not disclosed.
In a further document of the prior art (Database Biol. Abs., 89: 451839) the inhibitory effect on serine proteases and in particular on trypsin and &agr;-chymotrypsin of 2-ethyl-6-methyl-3-oxypyridine antioxidants phosphorylated on the oxy group is described. An increase in the specificity of protease inhibitors is not disclosed.
Hence the object of the present invention was to provide a method for avoiding unspecific secondary reactions when serine proteases are inhibited by sulfonic acid derivatives and fluorophosphates.
This object is achieved by a process for inhibiting serine proteases by derivatization with sulfonic acid derivatives or fluorophosphates which is characterized in that, in order to avoid unspecific derivatization of other proteins, a compound of the general formula (I)
is added in which
X denotes C or N,
R
1
denotes H, methyl, ethyl or OH,
R
2
and R
3
each denote H, methyl, ethyl, OH or residues which together form a ring closure with a maximum of 6 C atoms in the ring and
R
4
denotes H, OH or an optionally substituted alkyl, alkenyl or alkinyl group with up to 4 C atoms provided that at least one of R
1
, R
2
, R
3
or R
4
represents an OH group and the compound has a pKa of at least 8.
It has surprisingly turned out that the addition of compounds of formula (I) enables unspecific derivatization of proteins other than serine proteases to be practically completely prevented. The requirements which the compounds of formula (I) have to fulfill for this are primarily the stated pKa value since compounds with a lower pKa value do not adequately prevent the unspecific secondary reactions. Furthermore the optional residues on the groups R
1
, R
2
, R
3
and R
4
and the possible ring closure by the residues R
2
and R
3
, should not allow the compound to reach a size which makes it ineffective due to steric hindrance. Attainment of the maximum size of the residues can be easily established by simple preliminary experiments. Within the scope of the present invention it is preferred to use a cresol in particular p-cresol or 3-hydroxypyridine as the compound of formula (I). Both compounds are extremely effective.
The process according to the invention is therefore preferably used to process fermentations, culture broths and cell lysates; i.e. only interfering serine proteases are irreversibly inhibited without an unspecific derivatization of the desired protein occurring. This is for example advantageous when producing proteins which are intended for therapeutic measures if the protein is obtained by genetic engineering and the cell lysate is treated with sulfonic acid derivatives or fluorophosphates to avoid loss of activity by serine proteases. The compound of the general formula (I) added in the process according to the invention has in addition a low molecular weight and can thus easily be subsequently separated during the purification e.g. by dialysis.
A further subject matter of the present invention is a reagent for avoiding unspecific derivatization of proteins when serine proteases are inhibited by sulfonic acid derivatives or fluorophosphates to which a compound of the general formula (I)
is added in which
X denotes C or N,
R
1
denotes H, methyl, ethyl or OH,
R
2
and R
3
each denote H, methyl, ethyl, OH or residues which together form a ring closure with a maximum of 6 C atoms in the ring and
R
4
denotes H, OH or an optionally substituted alkyl, alkenyl or alkinyl group with up to 4 C atoms provided that at least one of R
1
, R
2
, R
3
or R
4
represents an OH group and the compound has a pKa of at least 8.
In a particularly preferred embodiment the reagent according to the invention contains a cresol, in particular p-cresol, or 3-hydroxypyridine.
As already mentioned above, the reagent according to the invention can be used advantageously in the processing of fermentations and culture broths in which serine proteases are present and where it is intended to inhibit these by sulfonic acid derivatives in order to avoid losses of desired proteins.
A further subject matter of the present invention is a reagent kit which contains a) a sulfonic acid derivative or fluorophosphate and b) a compound of the general formula (I)
in which
X denotes C or N,
R
1
denotes H, methyl, ethyl or OH,
R
2
and R
3
each denote H, methyl, ethyl, OH or residues which together form a ring closure with a maximum of 6 C atoms in the ring and
R
4
denotes H, OH or an optionally substituted alkyl, alkenyl or alkinyl group with up to 4 C atoms provided that at least one of R
1
, R
2
, R
3
or R
4
represents an OH group and the compound has a pKa of at least 8.
Such a reagent kit can be used advantageously for the inhibition according to the invention of serine proteases by derivatization with sulfonic acid derivatives or fluorophosphates to avoid unspecific derivatization of other proteins. In particular interfering proteases can be irreversibly inhibited in culture broths by this means as already mentioned. It is therefore advantageous to provide a reagent kit according to the invention as a package unit in order to carry out the process according to the invention. In a particularly preferred embodiment the reagent kit according to the invention contains AEBSF, APMSF or PMSF as the sulfonic acid derivative or a fluorophosphate such as DFP and a cresol in particular or 3-hydroxy-pyridine as b). Both components of the reagent kit a) and b) must be present in separate vessels since they would otherwise already react together before carrying out the actual process and their effectiveness would suffer.
The process according to the invention, the reagent and the reagent kit enable for the first time the specific inactivation of serine proteases in protein mixtures without changing the other proteins that are present in this process.
Arent Fox Kintner & Plotkin & Kahn, PLLC
Meller Mike
Naff David M.
Roche Diagnostics GmbH
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