MARDER at agri.huji.ac.il (Jonathan B. Marder) writes:
>lenny at zeus.datasrv.co.il (Lenny Garfinkel) wrote:
>>This group appears to be dead, but I guess I can't lose by posting. I
>>have a fermentation supernatant (does not matter what organism) which I
>>suspect contains proteases. When I add a pure protein to the sup and
>>incubate for anywhere from 0.5 hr to overnight, I see an apparent
>>INCREASE in molecular weight on SDS-PAGE in the presence of
>>mercaptoethanol (yup, it runs slower). What sort of protein
>>modifications could cause this? Could clipping of a few residues result
>>in less SDS binding?
>>>Yes, especially if you chop off a bit of positively charged sequence
>========
Actually, and I hate to sound like a contrarian, but it's very likely the
slower migration is caused by a cleavage of hydrophobic residues.
Logic being, cleave hydrophobic residues and there is less SDS binding,
resulting in less net charge.
This is near and dear to me because this exact phenomenology happens to the
beta subunit of Chloroplast Coupling Factor I (CFI). After proteolysis of
a hydrophobic section of the tail, the now smaller mol. wt. core fragment
migrates slower on SDS gels. Very reproducible, and very maddening if your
goal is to separate the "usually faster running" beta subunit from the
"usually slower running" alpha subunit -- cleave the tail of the beta
subunit and the two pile up on the SDS gel (meaning separation is
difficult/impossible by this method.)
On native gels, sure, I'd buy your argument, but I think the negative
charge of SDS bound to protein completely swamps out the ionic constituents
of the protein. Indeed, this is likely why SDS is such a good method for
separating dispartite proteins based solely on mol. wt.
A more interesting question is the amount of SDS that actually binds to
protein. I tried some separations with my current protein (Rubisco) at
high concentrations and discovered (much to my chagrin) that I was "running
out of SDS" even though the initial conc. was 1%. At the protein
concentrations I was working at, lack of sufficient SDS meant the two
subunits didn't necessarily separate. Adding more SDS resolved the
problem. I can't remember how many SDSs/protein there were, but it was in
the hundreds per protomer.
--
Eric Larson | University of Illinois at Urbana-Champaign
USDA/Agronomy | 190 PABL; 1201 W. Gregory; Urbana, IL 61801
elarson at ux1.cso.uiuc.edu | Voice 217.244.3079 Fax 217.244.4419
Fidonet: 1:233/4.1 | My opinions are my own, but correct :-)
