Lawrence J. Kaplan Williams College Williamstown. Massachusetts 01267
In a recent article in this Journal.. S~litteerber (1) re. ported the use of hemoglobin as a system for study in the introductory physical chemistry laboratory. We have also been employing hemoglobin as a system for laboratory study in a biochemistry course. Our rationale and approach to these experiments is essentially that of Splittgerber, in that students are more enthusiastic about a series of coherent experiments rather than individual exercises having no obvious connection. However, our series of experiments takes advantage of the fact that the tetrameric hemoglobin can be separated into its a and (3 subunits and that the subunits can be recombined to form regenerated hemoglobin. This generates a "family" of proteins for study and a number of interesting comparisons can be made between the native hemoglobin. its subunits. and reeenerated hemoelobiu. Most of the experiments can be completed in a 4-hr laboratory period but some reauire the student to return to the laboratory for brief periods of time.
-
Isolation of Hemoglobin The freshly collected whole blood (using acid citrate dextrose or henarin auticoaeulant) is treated essentiallv as described by kossi-Fanellikt al. (21, but the method outlined bv Snlitteerber (1) . . mav be used. The dialvsis of the final solution is carried out against water and the concentration determined with the Drabkin reagent (3) using either a of 7.2 a t 540 nm for cystandard curve or the El,,'" anomethemoglobin. The concentration of this solution is approximately 2-3% and also may be determined by direct = 8.63 a t spectrophotometric analysis employing El%' ,. 541 nm, assuming the protein in the oxyhemoglohin form.
. .
-
-
A Study of Hemoglobin and its Subunits Discontinuous Gel Electrophoresis Since the introduction of diac gel electrophoresis by Omstein (8)and Davis ( 9 ) , the technique has become a standard laboratory procedure for assessing homogeneity of protein samples. The disc gel technique involves the electrophoretic separation of proteins in a system which is discontinuous with respect to gel pore size, p H , and voltage. In the so-called stacking gel, conditions are such that the proteins are sandwiched between the chloride ion and the glycinate ion and "stack" into thin zones according to their electrophoretic mobility. As the proteins migrate into the running gel, the stacking phenomenon no longer occurs and they migrate according to their mobility a t high p H and small gel pore size. For more details, see the review of this technique which recently appeared in this Journal (10). The acrylamide (especially purified for electrophoresis) and other reagents are available from Eastman Kodak and the apparatus can be readily constructed from Plexiglas. The same apparatus and reagents can be used for isoelectric focusing and electrophoresis in SDS (see below). The hemoglobin system provides excellent samples to analyze by this procedure, and since a number of samples can be run a t one time the students can monitor a number of previous experiments. For example, the isolated hemoglobin can he analyzed and compared to commercially available hemoglobin and to the regenerated hemoglobin. The plasma originally removed from the erythrocytes provides a dramatic demonstration of the resolving power since many of the ~ l a s m amoteins are se~arated.Other nossible samples include: samples taken of the incubation mixture of hemoelobin and D-hvdroxvmercuribenzoate(5)and the individual fractio;~ cckecteh from the ion-exchange separation of the chains. In this connection. it is interestine to note the hemoglobin has a very different mobility fromalbumin which has a similar molecular weight but a different charge to mass ratio. This point can be made (quite dramatically) with the separated chains. While the @chainhas a mobility greater than native hemoglobin, the a chain does not migrate as rapidly as hemoglobin. The consequences of this are further investigated with isoelectric focusing.
Separation and Preparation of Hemoglobin Chains Oxyhemoglobin is converted to carbon monoxyhemoglobin by gently bubbling CO through the solution. Then the tetrameric hemoglobin molecule is dissociated into the a and @ chains by reaction with p-hydroxymercuribenzoate (PMB) at p H 5.5 (4, 5). The use of this reagent converts the aSHand BSH chains (containing free sulfhydryls) to aPMR and OPMH chains (with blocked sulfhvdrvl). These chains are then separated on a carboxyme~hyl~~ephadex Carboxyl Terminal Sequence ion-exchange column (6). The ion-exchange resin is availThe a and @ chains provide an interesting model system able from Pharmacia Fine Chemicals and when 50-100 mg for the determination of the carboxyl terminal amino acid of hemoglobin are employed, the 0.7 X 15-cm disposable sequence. The actual sequence of the chains columns from Bio-Rad are convenient. Care must be taken a-chain -seryl-lysyl-tyrosyl-arginine with these procedures but reasonably good separations can be achieved with a stepwise p H gradient (7) employing 0-chain .histidyl-Iysyl-tyrosyl-histidine phosphate buffers of p H 6.8 and 7.5. complements the specificity of carboxypeptidase A and B (Sigma Chemical Co.); that is, an incubation of the @ chain Recombinailon of Subunits to Form Regenerated with carhoxypeptidase A (specific for aromatic amino Hemoglobin acids) will liberate the histidine and tyrosine, but an incuAliquots containing equal molar amounts of the a P M H bation with the a chain will not be productive if carboxyand pPMRchains are incubated with a 50-fold molar excess peptidase A is used (11). However, carhoxypeptidase B of cysteine in a 0.05 M phosphate buffer at p H 7.3. After (specific for lysyl and arginyl) will cleave the a-chain. If the reaction is monitored as a function of time by paper chrostanding for 1 hr, the solutions are dialyzed against the matography emploviny a phenol:water:ammonia solvent. phosphate buffer and the resulting a" and BSH subunits the a c i ~ ~sequence al are mixed together. The regenerated hemoglobin has many can he deduced from the relative intenproperties identical to the original hemoglobin (4,5). sity d r h e s p o t i obtained hy developing with ninhydrin. 64 1 Journal of Chemical Education
Isoelectric Focusing on Polyacrylamlde
The introduction of isoelectric focusing has permitted the determination of the isoelectric points without the time consuming (and expensive) technique of moving houndary electrophoresis. This steady-state electrophoretic technique involves the separation of proteins in a p H gradient. The proteins migrate until they are in a medium of p H equal to their respective isoelectric points; they focus a t this point since with no net charge they have no electroohoretic mobilitv (12). The D H eradient is obtained bv &nploying polyeiectrolytes c&edkmpholine (LKB ~r;duktor). Isoelectric focusinn on ~olvacrvlamideallows one to analyze a number of samples at once,-and this technique is also valuable for evaluating the purity of samples (13). Both the original hemoglohin and the separated a and 6 chains can he analyzed by this technique in the p H range 5-8. The exneriment works well if the samnle is incornorated into the'acrylamide solution before poiymerization and then electrofocused a t 350 V for 4 hr. Isoelectric ooints ohtained are in good agreement with those reported by Winterhalter and Colosimo (6) of 7.2 for hemoglohin, 7.5 for the a chain, and 6.1 for the 6 chain. Notice that the pI of the a chain is hieher than that of hemodohin which is consistent with its decreased mohility during disc electrophoresis. On the other hand. the 0 chain has a much lower D I and this accounts for i&highkr mohility upon electrophoresis.
-
Ultracentrifugation .
Employing a Spinco model L ultracentrifuge which has been adapted for analytical determinations, a sedimentation velocity experiment is performed (14). A series of pictures is taken through the schlieren optical system to monitor the movement of the protein houndary. From these pictures the students calculate the sedimentation coefficient and then the molecular weieht from the Svedhere eauation. The diffusion coefficient isbhtained from the litkature for the a~orooriateconditions. The sedimentation exoeriment can b e conducted on either hemoglobin or the individual chains ( 5 ) . Sodium Dodecyl Sulfate Electrophoresis
Polyacrylamide gels equilibrated with sodium dodecyl sulfate and employing P-mercaptoethanol have been widely used to determine the molecular weight of proteins (15, 16). The sodium dodecvl sulfate hinds to the oroteins and the resulting negative iharge causes the subunits to dissociate due to electrostatic reoulsion. The 0-mereantoethanol reduces disulfide bonds within the prbtein anh between subunits. Hemorlobin suhiected to this orocedure dissociates into monomers and tbe molecular weight ohtained is about 16,000. As calibration standards. B-lactodobulin (mol wt 18,000) or myoglohin (mol wt 16,000), ovalbumin (mol wt 46,000) and bovine plasma albumin (mol wt 68,000) are sufficient to establish the relative mobility versus log mol wt graphs. This experiment is an excellent alternative for those who do not have an ultracentrifuge and also emphasizes the versatility of electrophoresis on polyacrylamide gels. Gel Filtration
Gel filtration is another technique that has become a standard biochemical procedure in recent years. The hasic principle involves a molecular filtration in which small pro-
teins enter the gel matrix and are retarded in their movement while larger proteins flow around the gel in the void volume and move more rapidly down a gel filtration column. T o illustrate these principles, Dewhurst (17) reported two experiments on relatively simple systems. One, which is applicable to this set of experiments, involves the determination of the hemoglohin binding capacity of serum haptoglobin. The procedure outlined by Dewhurst is followed employing the hemoglohin isolated by the student and the serum (or plasma) originally separated from the whole hlood. Besides these procedures we are presently performing a few experiments which are similar to those outlined by Splittgerber (I), some of which contain additional interesting features. Viscosity of Native and Denatured Hemoglobin
The intrinsic viscosity of hemoglohin is obtained as outlined by Splittgerber ( I ) and the data treated to obtain molecular parameters such as the radius of the molecule. In addition, the intrinsic viscosity is determined in 6 M guanidine hydrochloride and from the equation [q] = 0.716 the numher of residues, n, per chain is calculated according to the procedure of Tanford et al. (18). Assuming a mean residue molecular weight of 110, a molecular weight of approximately 15,000 is ohtained. Minimum Molecular Welght
The minimum molecular weight of hemoglobin can be determined by a chemical procedure and compared to the hiophysical procedures. The protein is digested with perchloric acid and hydrogen peroxide to liherate the iron. The iron is reduced with hydroxylamine and reacted with o-phenanthroline. The colored complex that forms is stable and measured spectrophotometrically a t 509 nm. A standard iron curve is prepared using ferrous ammonium sulfate. Molecular weights in the range of 15,000-17,000 are routinely obtained. This lahoratory program is presented to supplement those techniques discussed by Splittgerber (1) and to extend the application of the hemoglobin system into the biochemical laboratory. Certainly, many of the techniques outlined by Splittgerber could he used in the biochemical lahoratory hut with the techniques outlined here, one now has a truly wide range of experiments on hemoglobin applicable to the physical, biophysical, and biochemical laboratory. Individual copies of these experiments are available upon requesk. Literature Cited
,
~~.~ ~
~~~
,~~
15) Bucci, R.;Fnmtieelli. C.. ~ h j aE.. ~Wyman, ~ ~ J..~ Antonini. ~ , E.. and Kunsi~Fanelli. A . J Mol. Hinl.. 12, 188 (1965). 10.621 119711. 161 Winterhalter. K. H.,andCoL~rim~!.A..Rioehsmisrry, 171 Gauche., G. M.. J . C H E M . EDUC..46.729 (1969). 18) Omstein. L..Ann. N Y A m d Sci.. 121.921 (1964). 191 Davis. R.J., Ann. N Y. Arod. Sri., lZI.404 (19861. (101 Brewer, J. M..and Arhuurth. R. B.. J . C H E M . E D U C . 4 6 . 1 1 11969). 1111 1:uidutfi.G.. Ruzchem. Riuphys. A d a . 42. 177 11960). 1121 Svensron. H.. in "Pmtider B i d Fluids. Proc. 15th Coll~o.." . ( E d i t o r Peetern. H I . Elrevier, Amsterdam, p. 515, 1967. I1:Il W r i ~ l e yC..SCIP!ICP , Toula. 15. li 119681. . 1141 Crillith. 0. M..and Cmpper. L.,Anal. R i u c h ~ m . 31,218119691. 1151 Dunker, A. K.,and Kueckert. R. R., J. Riol. Chem.. 244.5076 (19691. 1161 \Yeher, K., and OsL,m,M.,J. H i d C h m , 2 4 4 , 4 1 6 11969). 1171 I3ewhuril. F.. J. CHEM. EDUC.46.864 (19691. 1181 T s n i w d , C.. Kawahars. K..a n d Lapanjo, S.,J. A m s r C h m S ~ o . . 89.729 119671
.
Volume 53, Number
1, January 1976 1 65
