[RASMB] Hb tetramer

Tom Laue Tom.Laue at unh.edu
Thu Feb 5 05:19:00 PST 2009


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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Department of Biochemistry and Molecular Biology
University of New Hampshire
Durham, NH 03824-3544
Phone: 603-862-2459
FAX:   603-862-0031
E-mail: Tom.Laue at unh.edu
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