dilute NaOH causes hydrolysis

Achim Recktenwald, PhD achim at ibex.ca
Tue Nov 26 09:04:39 EST 1996


Reno T. Nguyen wrote:
> 
> 
> I've been extracting proteins from various matrices (e.g., algae and
> sediments) using 0.5 N NaOH and then estimating total protein by a
> colorimetric assay.  This method seems to work well, as long as the
> standards are treated the same way. NaOH has been used by other
> researchers for extraction of sedimentary protein.
> 
> I was hoping to analyze the MW distribution of proteins extracted as
> described above. After buffering to a pH of 6.8, I used one of Millipore's
> ultrafiltration devices to concentrate the proteins; diluted the salts
> using buffer; ran SDS-PAGE.  None of the protein standards (e.g., BSA)
> were observed in the correct MW range.  However, I did notice a smear in
> the low MW range suggesting protein hydrolysis.
> 
> I'm aware that 2 to 4 N NaOH (at 100 deg C for 4 to 8 h) will normally
> hydrolyze proteins completely. Would a NaOH concentration as low as 0.5 N
> hydrolyze proteins to some extent, even if the samples are extracted at
> room temperature instead of 100 deg C?


0.5M NaOH is used for in-place-cleaning of chromatographic columns under
GLP/GMP conditions. The use of 2 to 4M NaoH at elevated temperatures is
used to get a COMPLETE hydrolysis of the protein. 
So, yes, you have to expect some limited hydrolysis wit 0.5M NaOH.

Achim



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