Re: proteolysis at arg

Dr. Ming F. Tam (MBR405@ccvax.sinica.edu.tw)
Thu, 16 Jul 1998 15:07:00 +0800

In our hands, the Arg-C from Boehringer Mannheim behaves just like
trypsin--cut at both Arg and Lys. We have contacted BM and they claimed
that we have used too much enzyme for the cleavage (1:10), hence the
non-specificity in cutting. We changed the enzyme/substrate ratio to 1:50.
It was working better, but the cleavage at Lys was not eliminated.

Ming

>>Dear Philip,
>>
>>One possibility is to use the endoproteinase Arg-C (sold by Boehringer
>>Mannheim
>>among others), a protease that should specifically cleave C-terminal of Arg
>>residues. I have however not used that enzyme myself and I vagely remember
>>hearing or reading that the enzyme activity was not as robust as one
>>could hope.
>>
>>Maybe some ABRfer may wnat to comment on this. Another possibility is to
>>denature the protein, reduce/alkylate the Cys and to derivatize the Lys with
>>maleic acid anhydride (the two steps can be performed together), and then to
>>digest the protein with trypsin. Since the Lys are now derivatized,
>>trypsin will
>>
>>only cleave after arginines.
>>
>>Hope that it helps
>>
>>Axel Ducret
>>
>>Philip Majerus wrote:
>>
>>> I have need to cleave a large protein at R in order to identify a
>>> particular carboxymethy cys residue. Greg Grant suggested that I poll the
>>> group for advice as to a suitable protease and protocol to do such if there
>>> is one available. I have about 500 pmol of tritiated protein that is mr
>>> 140000. Thanks for your help Phil Majerus
>>>
>>> Phil Majerus
>>
>>--
>>Axel Ducret, Ph.D.
>>Senior Research Biologist
>>Merck-Frosst Canada Inc.
>>Dept. Biochemistry and Molecular Biology
>>P.O. Box 1005
>>Pointe-Claire-Dorval PQ H9R 4P8
>>Canada
>>
>>tel. + (514) 428-3428
>>fax + (514) 428-4900
>
>
>

*********************************
Dr. Ming F. Tam
mbr405@ccvax.sinica.edu.tw
Institute of Molecular Biology
Academia Sinica
Nankang, Taipei 11529
Taiwan, ROC
*********************************