VIII Nov. 16, 1937
Dear Dr. Pauling:
This idea of writing a weekly letter may make the letters . discuss work weakly
[sic]. Taylor says that I spend so much time writing you that I do not have time
to get any work done. You have not fired me yet, but that may be because that you
are too busy.
The apparatus that I got with Webb Tuesday at Corona is in pretty good shape for developing
the dialyzing apparatus, requiring only a motor with a good reduction gear and the
glass parts, but Webb has not yet set it up completely. I was going to give him
the motor from my rotor-stirrer but I have used it a good deal this week, and may
want to go on with it. Webb has gone today for more blood although he has a stock
of two liters from our last visits that is not used. Ithink [sic] that I told you
a little last week about our plans to get the isoelectric point (rather important
to determine) and simultaneously get some data on the effect of salts on the pH and
solubility of globulins. I think that we have worked out a pretty good method that
will not be too difficult to put into practice. We will take a suspension of dialyzed
(isoelectric [handwritten annotation: "net charge zero"] and isoionic [handwritten
annotation: "equal amounts H+·OH- bound"]) hemocyanin, and titrate with various salts like NaCl and NaNCS and determine
simultaneously pH and amount of protein that has gone into solution. With hemoglobin,
NaCl affects the pH differently on opposite sides of the isoelectric point, decreasing
it on the alkaline side, increasing it on the acid side (Cohn, paper in the JACS Mar.
1937). This is probably a normal [handwritten annotation: "salt"] effect, and although
large, is in the direction of expected effects of ionic strength, or of appreciable
absorption of both Na+ and Cl-, not greatly different [handwritten annotation: "in amounts"]. With KNCS the pH
increases on addition to HB+ solution on both sides of the isoelectric points, pointing to absorption of NCS- far greater than of K+, the effect partially-offset by taking on of protons from the solution. Of course
with hemocyanin there is the added effect (sofar as I know as yet unexplained) of
salt in bringing the protein [edit: "globulin"] into solution, but a treatment of
the type you gave to Carpenter's experiments on gelatin may be significant. The
order of ions in the lyotropic series in bringing globulins (or any colloid) into
solution is the same order that prevails in salting out, which indicates strongly
that the effects are due mainly to the same forces.
Taylor has been working very hard the last few weeks, and has three complete determinations
of the absolute moment of hemoglobin (whole blood) worked out, involving the ordinary
(but very precise) magnetic measurements as well as both oxygen and carbon monoxide
capacities. I got blood for him twice last week and will have to go twice this
week. I determined some time- ago that there is absolutely no change of Δw of Hb
on laking corpuscles with saponin, but you may recall in our work that the moments
of ether-laked Hb solutions C and D may have been lower than moments of whole bloods
A and B. Taylor and I are laking with toluene, and find it satisfactory on 24-48 hours
standing in the refrigerator. The method used by Mirsky based on work of Henderson
(1921) (vol. missing from biol. libr.) involved diluting the washed corpuscles with
an equal volume of water (which in itself brings about considerable laking) and then
allowing to stand with toluene overnight. Taylor could find no intact cells and
very few 'ghosts' microscopically in [edit: "a solution from"] some corpuscles [handwritten
annotation: "without water"] that I had shaken with toluene, allowed to stand 48 hours,
and then centrifuged. This improvement allows us to obtain the more concentrated solutions,
and is free from objections inherent in peroxides or peroxide-formation in the presence
of ether. Of course we shall check the ether-laking but for future work probably
use the toluene.
Taylor is interested in getting the best value of the absolute moment of Hb, and finds
considerable difference (up to 5%) between concentrations calculated on the basis
of carbon monoxide capacity and oxygen capacity. (This would be predicted from
the work of Barkan.) The moment calculated from magnetic weighings and the former
capacity is accidentally very close to the value that we used (within several hundredths
of a Bohr magneton) so we will not have to make a serious correction in published
moments, since they should be referred to this standard rather than to one based on
oxygen. We shall have to think hard, when all of the data are ready, to explain
the probable source of some of the apparent differences.
Taylor is, by the way, waiting to hear from you about what to start next, as he is
near the end of this work.
I conceived the idea of fortifying Dodson's work on the magnetic titrations of HbO2 with Na2S2O4 with similar work based on the following reaction:
2 Hb + 2 NO-2 + S2O=4 = 2 HbNO + 2 SO=3, adding standard nitrite to Hb in the presence of excess S2O=4. In two carefully made titrations I ran out of reducing agent before I reached
the end-point, but the curves had a very nice linear early portion. After adding
an excess of nitrite I could get the final Δw by adding another portion of hyposulfite
(I prefer this name now to hydrosulfite). The only difficulty is that, with identical
initial volumes of Hb+ reduced, etc., the slopes if the linear portions differed appreciably. The slope
for the second titration was less, which may indicate that the nitrite-had decomposed
somewhat, although that does not seem too likely to me. The chief objections have
to our earlier work with Dodson are that the hyposulfite decomposed during the course
of the reaction, which would lead one to expect a curvature of the opposite nature
to the one expected, and that transformation of oxyhemoglobin to ferrihemoglobin occured
spontaneously to a large enough extent to cast doubts on the validity of the work.
Palmer's uncle-in-law, Baumberger at Stanford, told me once of the great success he
had once reducing oxyhemoglobin at a constant rate with bacteria (yeast, I believe
works too). If I could get a culture that would eat fast enough that the reaction
was over before multiplication of the beasts became serious, this would be a good
possibility. I believe that in the presence of organisms ferrihemoglobin would be
reduced as fast as it formed. I shall write him for advice. We sent him a slug of
sodium azide recently for his work, and I have met him several times.
I have a paper half written (and typed that far by GraceMary) which I want to submit
to you for approval on the acid-base functions of the heme of hemoglobin and myoglobin.
As long as I had all this material worked up for seminars, I thought that it would
be advisable
to see how it would look written up, but I do not intend to waste a
great deal of time on the matter because there is no experimental work of mine in
it. It consists merely of a thorough correlation of all of the work I can lay hands
on, interpreted from a structural viewpoint. There has been a lot of uncorrelated
work done in this field, and a fair amount of wrong mathematical treatment, but of
course, a fair amount of correct handling of isolated parts of the problem have appeared.
I shall leave the matter up to you.
I gave up my projected trip to Davis and Deep Springs because my passengers backed
out. I found that they were having only the morning session at Davis and thought that
the place was too far away for a trip for so short a meeting.
Mrs. Pauling said that you are staying at the Telluride House. I hope that you don't
get tellurium poisoning there. From what she says both lecture and book are progressing
wonderfully, for which I am very glad. However, do not forget to take a little time
off from work for relaxation once in a while.
[handwritten annotation: "Yours, Charles Coryell"]
