[RASMB] Hb tetramer

[mitrana at mail.utexas.edu]

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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