[RASMB] Hb tetramer

Thu Feb 5 06:38:05 PST 2009

Hi Tom,

what is independent of the viscosity is the mass M, since it's determined 
by the ratio of s and D, and therefore viscosity terms cancel out and one 
will get the correct M with an incorrect viscosity value.

However, for the frictional ratio this is not true, since it is governed by 
the ratio of M/s, and there's the viscosity term again.  Therefore, for 
estimating the hydrodynamic quantity f/f0 correctly, we'll need the right 
viscosity value.

Peter

At 08:19 AM 2/5/2009, you wrote:
>Hi Mitra-
>The viscosity would not cause the f/fo to be lower than expected since 
>both s and D will be affected. I believe Walter's comment on there being a 
>small amount of self association is the most likely cause.
>Best wishes,
>Tom
>
>mitrana at mail.utexas.edu wrote:
>>The Hb that I work with DOES self-associate upon deoxygenation but I 
>>don't expect much oligomers at the concentration that I am working - 
>>0.5uM Hb tetramer. It is possible that we have a problem with the v-bar 
>>but a paper from our lab (Demoll et al, Analytical Biochemistry 
>>363(2):196 2007) suggests that the additivity of Hb + heme may work. 
>>However the presence of IHP which is bound very tightly by the Hb is a 
>>problem. I account for the weight but not the v-bar of IHP. I think I'll 
>>go ahead and measure the IHP v-bar myself - haven't found it in the 
>>literature yet.
>>
>>More likely, there's a problem with our viscosity measurement - density 
>>isn't a problem since we have an Anton Paar. I am re-measuring the 
>>viscosity of our solution - 50mM Tris, 100mM NaCl, 2 mM IHP, 11 mM 
>>Na-dithionite, pH 7.25. In case anyone is wondering what Na-dithionite is 
>>doing in the buffer - it is to get rid of trace oxygen after our Hb 
>>sample + buffer is deoxygenated and to ensure that the Hb stays 
>>deoxygenated (with the Fe reduced) during the AUC run. And No! i have not 
>>found a way yet to measure the viscosity without somehow exposing the 
>>sample to oxygen - so there's that problem to consider as well.
>>
>>Current viscosity value that I am using to correct the s-value is 1.084 
>>cP but I think this is rather on the high side. Values calculated using 
>>SEDNTERP using only the Tris, NaCl, and the HCl gives a viscosity of ~ 
>>1.03 at 20 degC. I find it a little hard to believe that addition of 2 mM 
>>IHP and 11 mM Na-dithionite raises it to 1.08 but what do I know! Guess 
>>I'll find out.
>>
>>Anyways, thank you all for the comments. Off to the work space for me.
>>
>>
>>
>>Quoting Walter Stafford <stafford at bbri.org>:
>>
>>>Mitra,
>>>   It is also possible to get a slightly low f/fo (sometimes less than
>>>1.0) if there is a small amount of unaccounted for self-association
>>>going on.
>>>Walter
>>>
>>>John Philo wrote:
>>>>Mitra,
>>>>My recollection is that IHP does cause a slight compaction of the
>>>>structure, but I agree with Arthur that 1.08 sounds a bit low. I'm
>>>>wondering whether that might be because you are not using an
>>>>accurate vbar value. I wasn't doing AUC back in the dark ages when
>>>>I worked on hemoglobin, but I vaguely recall that the additivity
>>>>rule for vbar may not work well for heme + globin. I think you will 
>>>>find the experimental vbar in hemoglobin AUC papers by Stuart J.
>>>>Edelstein (? '60s-'70s).
>>>>John
>>>>
>>>>------------------------------------------------------------------------
>>>>*From:* rasmb-bounces at rasmb.bbri.org
>>>>[mailto:rasmb-bounces at rasmb.bbri.org] *On Behalf Of *Arthur Rowe
>>>>*Sent:* Wednesday, February 04, 2009 6:45 AM
>>>>*To:* mitrana at mail.utexas.edu; rasmb at server1.bbri.org
>>>>*Subject:* Re: [RASMB] Hb tetramer
>>>>
>>>>   Hi Mitra
>>>>
>>>>   An f/f0 value of 1.08 (±?) is low-ish, but within the ball park
>>>>   range for 'globular' proteins. I don't imagine that 2 mM Inositol
>>>>   would affect the (properly and fully corrected) s value.
>>>>
>>>>   Regards
>>>>
>>>>   Arthur
>>>>
>>>>
>>>>   Hello All
>>>>   Two questions, related ones -
>>>>
>>>>   1. Is a S20,w value of 5.01 too high for a DEOXY Hb tetramer of
>>>>   67180     Da MW? This gives a f/f0 of around 1.08. Just a reminder that
>>>>   deoxy Hb     tetramer does not dissociate to a dimer much (dissociation
>>>>   constant ~     10^-11 M).
>>>>
>>>>   2. can such an anomaly occur due to Hb interactions with the
>>>>   components of the buffer - in this case 2 mM of Inositol
>>>>hexakisphosphate (IHP)?
>>>>
>>>>   Glad to hear any comments and/or references to papers.
>>>>
>>>>   Mitra
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>>>>
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>>
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>--
>Department of Biochemistry and Molecular Biology
>University of New Hampshire
>Durham, NH 03824-3544
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