[RASMB] Hb tetramer

Thu Feb 5 07:22:21 PST 2009

Just one further thought on the 'anomaly' of a value of 1.08 for the  
frictional ratio.

If you go back to that massive survey of globular protein properties  
given by Squire & Himmel you will find that their empirical equations  
linking s to M values etc imply an average f/f0 of 1.26 ± 0.06.

So a value of 1.08 is indeed a statistical outlier. But that does not  
in itself make it impossible.

Arthur

On Feb 4, 2009, at 19:50, 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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************************************************************************ 
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Arthur J Rowe
Professor of Biomolecular Technology / Director NCMH Business Centre
School of Biosciences
University of Nottingham
Sutton Bonington
Leics LE12 5RD

TEL:  0115 9516156
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