[RASMB] Hb tetramer

Allen Minton minton at helix.nih.gov
Thu Feb 5 08:57:36 PST 2009

I have been witnessing an extended discussion on 
possible reasons for Mithra's results, but they 
are all based upon the assumption that he was 
looking at an Hb tetramer.  There have been tens 
of publications reporting S20,w values for many 
different Hbs under a variety of conditions, and 
all of the reported values lie between 4.1 and 
4.5 S.  Mithra's value of 5S indicates the 
presence of a significant amount of higher MW 
material, so I doubt that the calculated value of f/fo has physical meaning.


At 08:19 AM 2/5/2009, Tom Laue 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,
>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>:
>>>    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.
>>>John Philo wrote:
>>>>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).
>>>>*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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>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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