[Protein-analysis] Re: serine protease inhibition by plastic extractable material

rolando via proteins%40net.bio.net (by rolando.perdomo At infomed.sld.cu)
Mon Nov 20 21:40:12 EST 2006


Thanks a lot for your support. I fact I am a beginner using ultrasonic
for cell disrupting. Now I am very enthusiastic to get more hints about
the issue. To get an idea: I am studying a serine protease which comes
from the hemocytes from the lobster (these cells have hardness similar
to lymphocytes). In the past I used a glass piston homogenizer.
However, currently I would prefer ultrasonic treatments due to is more
efficient and less handling would required. I am using a cycle of 30
sec at 20 Watt for a total of 2 min on ice (300 ul sample). This cycle
did not seem particularly aggressive to me in the first time. As I am a
novice ultrasonic user, I tested the cycle using a commercial
preparation of trypsin, and the activity was not affected by the cycle.
In addition, other enzyme naturally occurring in the hemocytes, the
phenoloxidase, was not affected neither. What called me the attention
was that even compared to glass piston homogenization the serine
protease activity was lower. That is why I thought that maybe
extractable materials from plastic could be the cause. In addition,
sometime I heard that this could occur. I am considering that maybe
this serine protease indeed can be less resistance to the heating
stress caused by the ultrasonic than commercial trypsin and
phenoloxidase. Perhaps I should reduce the cycles, for instance to 15
sec, and to work with glass reservoir. I was bought by the brochure of
the ultrasonic equipment, which emphasize that the microprobe can be
used with eppy tubes and ultrasonicate very low volume of samples.
Looking forward to your thoughts and comments.
DK ha escrito:

> In article <1163981995.186709.246890 At b28g2000cwb.googlegroups.com>, "rolando" <rolando.perdomo At infomed.sld.cu> wrote:
> >Hi  Every one,
> >
> >I found that during preparation of cellular extract to analyze serine
> >protease activity, if sample is sonicated in 1.5 ml eppendorf centrigue
> >tubes, the proteolytic activity is diminished. In other hand, if the
> >same cells are lysed by sonication using a glass reservoir no
> >inhibition appears. Does anyone know about relation of protease
> >inhibition and plastic-extractable material??? References????? Looking
> >forward to receive some light.
>
>
> Bad experiment. If you were to blame plastic-extractables, you should
> be able to get inhibition by sonicating buffer in the eppy and adding it
> to you protease.
>
> If both sonications are done on ice, the more likely explanation is
> that heat dissipation is too slow in plastic => protein heats up too
> much => activity is diminished due to partial denaturation. 
> 
> DK



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