[RASMB] Hb tetramer

Tom Laue Tom.Laue at unh.edu
Thu Feb 5 09:56:48 PST 2009


You're right- sorry! Tom

Peter Schuck wrote:
> 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
>>>>>   _______________________________________________
>>>>>   RASMB mailing list
>>>>>   RASMB at rasmb.bbri.org
>>>>>   http://rasmb.bbri.org/mailman/listinfo/rasmb
>>>>>
>>>>>
>>>>>
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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
>> www.bitc.unh.edu
>> www.camis.unh.edu
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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
www.bitc.unh.edu
www.camis.unh.edu




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