ANALYTICAL BIOCHEMISTRY 35, 235-243 (1970)

An Improved Method for Quantitative Determination of Human Fetal Hemoglobin

W. A. SCHROEDER, T. H. J. HUISMAN, J. ROGER SHELTON, AND JERRY B. WILSON

Division of Chemistry and Chemical Engineering, Calijomiu Znstitute of Technology, Pasadenu, California 91109, and Laboratory of Protein

Chemistry, Medical College of Georgia and Veteralw Administration Hospital, Augusta, Georgia 30902

Received September 16, 1969

Some type of alkali denaturation procedure is the most commonly used means to determine the approximate percentage of hemoglobin F (Hb-F) in a human red-cell hemolyzate. The reliability of these and other meth- ods has been and continues to be the subject of controversy (1). Although Chernoff (1) minimizes the value of chromatographic procedures for the determination of Hb-F, any chromatographic procedure has the advantage that the Hb-F is separated from other components and is not determined by differential properties in a mixture. Our study of Hb-F in various hematological conditions (24, and unpublished) has led us to a renewed appreciation that the chromatographic determination of intermediate amounts of Hb-F in hemolyzates is subject to error because of minor components related to hemoglobin A (Hb-A) in the zone of Hb-F. Be- cause a more exact determination of Hb-F was necessary in these experi- ments, a new method was devised.

PRINCIPLE OF TH-E METHOD

The chromatographic separation on DEAE-Sephadex (5) of the Hb components in a freshly prepared hemolyzate of red cells from a normal adult is depicted in Figure la and of those in a normal newborn infant in Figure lb. In addition to the major component Hb-A0 in the normal adult, the minor components Hb-A, and Hb-A, are present. Hb-A, nor- mally amounts to 5 to 8% and is composed mainly of a hemoglobin that is identical to Hb-A0 in amino acid composition and has also been termed Hb-AI, (6). The apparent quantity of Hb-A, is increased by mishandling of blood or solutions of hemoglobin; products that are formed from Hb-A, by storage or warming but are unrelated to Hb-A, nevertheless chroma- tograph in its position (7). On the other hand, Hb-F,, the major Hb com-

235

236 SCHROEDER ET AL.

^_ A2 v3’ 3.1%

I I -7.0

!. .

PH -8.0

Q ---_ _ 7.5

g 0.5

5 .a t

A2 <O.I%

8.0

7.5

7.0

Effluent, ml

FIG. 1. Separation of hemoglobin components on a 0.9 X 45 cm column of DEAE- Sephadex with a gradient of Tris-HCl buffers: (a) normal adult; (b) newborn infant; (c) adult with increased W-F.

ponent of newborn hemoglobin, elutes in virtually the same position as Hb-A1 of the adult. A minor component Hb-F, that is also termed FI and is related to F, (8) is apparent when the percentage of F is high. The small amount of Hb-F, in Hb-A, of the adult or of Hb-A, in Hb-F, of the new- born infant is of no particular consequence in their quantitative chroma- tographic determination. In hematological conditions with elevated fetal hemoglobin, the zone in the position of A, and F. increases and the pres- ence of F, may become apparent (Fig. lc) : the quantity then is no longer a correct indicator of F, or of A, because both are present. The sum of F0 + A, can, of course, readily be calculated. If the percentage of F0 in F. + A, can be determined, the percentage of F, in the hemolyzate will also be known.

The most significant difference between Hb-A, and Hb-F, is the pres- ence or absence of isoleucine: the ay subunit of Hb-F, has 4 residues of isoleucine and the & subunit of Hb-A, has none. On the other hand, the my and the a/? subunits have 35 and 36 residues of leucine and 15 and 15 residues of phenylalanine, respectively. Consequently, a determination of the content of isoleucine in relation to leucine and phenylalanine is a

DETERMINATION OF #FETAL HEMOGLOBIN 237

measure of Hb-F,, in F,, + A, if A, is assumed to have the same amino acid composition as Hb-A,. The new procedure for determination of Hb-F, therefore, involves the chromatographic isolation of F, + A, and the determination of its isoleucine, leucine, and phenylalanine content. If Hb-F, is apparent on the chromatogram, it is combined with F, + A, prior to analysis.

METHODS

Analytical Chromatography of Hemoglobin

Approximately 40 mg of hemoglobin is chromatographed on a 0.9 X 45 cm column of DEAE-Sephadex (A-50, capacity 3.5 + 0.5 meq/gm, par- ticle size 40-120@, Pharmacia Fine Chemicals, Inc.) with a gradient of Tris-HCl buffers (5). The percentage of each component is calculated from the absorbancy readings.

Isolation of F, + A,

The appropriate pooled fractions1 of the zone in the position of F, + A, are adjusted to pH 6.5-6.6 with 0.1 N HCl and diluted with an equal volume of distilled water. One or two drops of a 2% KCN solution are added. An aliquot of the F,, + A, solution (containing 0.5 to 5 mg of hemoglobin) is passed through a column (0.5 X 2 cm) of CM-Sephadex (C-50, capacity 4.5 i 0.5 meq/gm, particle size 40-120~, Pharmacia Fine Chemicals), which has been equilibrated with a developer 0.05 M in Tris with maleic acid added to give pH 6.5. All hemoglobin concen- trates at the top of the column and is then eluted completely with a few milliliters of 0.05 M Tris-HCl developer, pH 8.2. The Tris developers contain 100 mg KCN in 1000 ml.

Amino Acid Analysis

After the eluate of the CM-Sephadex column has been dialyzed over- night at 4” against distilled water, the approximate concentration is de- termined, and the sample is blown dry and hydrolyzed in 6 N HCl for 24 hr at 110” (9). After evaporation of the acid, the sample is dissolved in 1 ml of pH 2.2 buffer and centrifuged. Depending upon the sensitivity of the amino acid analyzer, an appropriate aliquot portion is taken for analysis.

A long column analysis is sufficient because analytical data for iso- leucine, leucine, and phenylalanine only are necessary. The conditions of chromatography may be modified to reduce the time of analysis. The 0.9 X 60 cm column is equilibrated with pH 3.25 buffer. After the sample

’ I f Hb-Fl is apparent on the chromatogram, it should be included.

238 SCHBOEDER E-J.’ AL.

has been applied, the space above the column (about 5 ml) is filled with pH 3.25 buffer. However, the lines to the top of the column are filled with pH 4.00 buffer (which is prepared by adjusting the pH of the pH 4.25 buffer), and elution is made with pH 4.00 buffer. Change to pH 4.25 buffer is set to occur after leucine has emerged. When the flow rate of developer is 68 ml/hr and the temperature is 56”, adequate resolution can be maintained on spherical resins, and the chromatogram is complete in two hours.

Analyses were made both in Pasadena and Augusta. The modified development was used in Pasadena and the regular development with pH 3.25 and pH 4.25 buffers at a flow rate of 68 ml/hr in Augusta. The spher- ical resins in the two analyzers have somewhat different properties: in Pasadena, the baseline is not reached between isoleucine and leucine even with regular development whereas it is reached in Augusta, but the tyrosine-phenylalanine separation is excellent in Pasadena and marginal in Augusta. High-sensitivity cuvets (10) were used in Pasadena and long- path cells (11) in Augusta.

Calculation After the pmolar amounts of isoleucine, leucine, and phenylalanine have

been calculated, the percentage of F, in F. + A, is as follows:

To F. in F. + A1 = pmole Ile X 100

2 ’ (

rmole Leu 35.5 +

rmole Phe 15 >

Estimation of Hb-F by Alkali Denaturattin

The method of Betke, Marti, and Schlicht (12) was used.

RESULTS AND DISCUSSION

All procedures for the determination of Hb-F, including the present one which will be termed the File method, have a basic problem: control experiments with accurately known amounts of Hb-F cannot be made. To a considerable extent, an estimate of accuracy and precision must be made from the theoretical basis of the method and from indirect evidence.

Meaningful application of the F rle method requires the absence of ex- traneous isoleucine, leucine, and phenylalanine. It is especially vital that nonhemoglobin components which are known to be present in a hemol- yzate and to contain proportionally high amounts of isoleucine (13) be absent from the pooled fractions of F. + A,. When these nonhemoglobin components were isolated by CM-Sephadex chromatography (14) and then rechromatographed on DEAE-Sephadex under normal conditions, they were in part eluted in the first 50 ml of effluent and in part retained

DETERMINATION OF PETAL HEMOGLOBIN 239

at the top of the column; none was observed in the position of F0 + A,. Further evidence for the absence of extraneous amino acids comes in- directly from the F,, + A, of samples that contain no A0 and hence no A,; the results cluster closely around 100% and do not go above as they would if extraneous isoleucine were present.

The accuracy of an amino acid analysis by automatic methods is gen- erally considered to be t-3% (15, 16). In the range of 10 to 100% Hb-F, in the F. + A, zone, the ratio of isoleucine to leucine will lie between 1: 90 and 1: 9 and that of isoleucine to phenylalanine between 1: 37.5 and 1:3.75. When control mixtures within this range were analyzed, the ratios were within &3% of theory. Duplicate analyses of individual hydrol- yzates also agreed within these licits (for example, see H.W. and J.A. of Table 1). The choice of leucine and phenylalanine as the bases of cal- culation is dictated by their identical or nearly identical numbers of residues in the a@ and ay subunits. Although the content of leucine is slightly different (35 and 36 residues), the use of the average in the cal- culation introduces no appreciable error into the result. The calculation of results uses the average of isoleucine-leucine and isoleucine-phenyl-

TABLE 1 Results of Determinations on the Same ‘&mple in Both Laboratories by the FI le Method

‘; f,, in FO f A, “; F in hemolyzate

Subject o/o Fo + Al Aug. Pas. Aug. Pas.

E.G. 28.9 69 72 19.9 20.8 R.G. 20.7 88 90 18.2 18.6

E.W. 7.8 31 34 2.4 2.6

C.W. 8.6 60 75 5.2 6.5 8.H. 15.5 91 95 14.1 14.i

R.L.R. 42.8 68 i2 29.1 30.8

*M.P. 9.7 41 49 4.0 4.8 C.We. 21.6 86 92 18.6 19.9

B.H. 35.5 74 78 26.3 2r mc .I H.L. 11.2 56 56 6.3 6 .::

H.W.* 1 6.7 92 6.2 98 6.6

J.A.” 1 19.x 57 11.3 15.1 74 11.2

B.L.6 j 14.0 14 3 0

15.7 14 2.2 T.H.b 1 6.0 24 1.4

15.1 10.5 1 .(i

0 Duplicate analyses of hydrolyeate of a single sample.

b Individual analyses from duplicate chromatograms or samples of blood.

240 SCHROEDER ET AL.

alanine ratios although calculation on the basis of either ratio usually agrees within 3%.

In order to test precision between laboratories, the final concentrated and dialyzed sample of F, + A, was divided, hydrolyzed in individual laboratories, and analyzed. Table 1 records the results. Although the num- ber of significant figures in Table 1 is inappropriate to the probable ac- curacy, they have been given to permit better comparison. The samples encompass a wide range of % F, + A, as well as ,% F in F. + A,. The agreement is generally within 5% although it should be noted that results from Pasadena usually are higher than those from Augusta. Data from J.A. and T.H. serve as examples of the difference in the percentage of F, + A, as a result of aging of the sample. Despite the difference, du- plicates by the FILe method are in excellent agreement.

The accuracy of the F rle method is also dependent upon the accuracy of the determination of F. + A, in the hemolyzate by chromatography. Recovery of hemoglobin from DEAE-cellulose chromatography averages 98% (17) and comparable results are obtained with DEAE-Sephadex chromatography. Quantitation involves integration under the chroma- tographic peaks and should not be subject to appreciable error.

In Figure 2, data are compared for 59 samples that have been examined by the three methods. Values from the chromatographic analysis (FChrom.) and from the alkali denaturation method (FA.=.) are plotted-against those from the File procedure. Determination by the File method generally re- sults in values that are lower than those by the Fchrom. method and higher than those by the FA.=. method. The FChrom. data are higher than the File data by a roughly constant additive amount whereas the FA.,,. data are a roughly constant percentage of the File data. The lines have been drawn on the basis of a least-squares treatment of the data. The intercept of the Fchrom. vs. Fn, data is 6% and the slope is almost 1 (0.97). The quantity of F, + A, in the normal adult in whom F,, is 1% or less is 5 to 8% (18). The intercept at 6% is, therefore, a realistic estimate of the average amount of A, in F, + A, by chromatography. On the other hand, the FA.=. results are very nearly directly proportional to the F,,, values; the intercept is 0.26% and the slope of the line and therefore the proportion- ality factor is about 0.8 (0.79). These relationships-the constant additive factor between the Fn, and FChrom. data in contrast to the proportionality between File and FA.D. data-are indicative of the greater accuracy of the File method.

The accuracy and precision of the method may also be assessed by analyzing the F, + A, of samples in which Hb-A is absent and hence Hb-A,, too. Cases in point are sickle-cell anemia patients and double heterozygotes for Hb-S and a variety of other inherited hematological

DETERMINATION OF FETAL HEMOGLOBIN 241

FIG. 2. Values for determination of Hb-F by the F.o. and Fchrom. values by the F1l. method.

36-

32-

26-

; 16-

z ii

a l2-o

o FChrom.

*FAD.

0 4 8 12 16 20

Percent FII,

methods vs.

conditions. The minor component Hb-S1 falls in the position of Ao and does not contaminate Fo. In “F, + A,” analyses of 24 such samples, % File in F, + A1 ranged from 81 to 105 and only two samples were below 87. The average was 92.5%. It must be realized that the average result is equivalent to 3.7 of 4.0 residues of isoleucine per my subunit. This would be an acceptable analytical value in the analysis of protein espe- cially when, as in the case of isoleucine, the particular amino acid is in low amount.

Note Added in Proof: Recent experiments show that 72-hr hydrolyzates are to be preferred over 24-hr hydrolyzates. If longer hydrolysis had been used in the above determinations, the average probably would have been 96-97s.

How reliable is the method when the amount of Hb-F is low as in normal adults? Results of the application to 12 presumably normal adults are listed in Table 2. The answers are approximately those that are usually quoted for adults. In the absence of any absolute standards by which to test the method, an estimate of the accuracy and precision of these data cannot be made.

242 SCHROEDER ET AL.

TABLE 2 Percentage of Hb-F in Normal Individuals by the FII, Method

Subject Sex % Fo + AI To F in Fo + A,

% FO in hemolyrate % FAD.

E.G. R.H. M.S. F.S. J.P. J.S. M.P. H.C. Je.P. Ju.P. F.M. M.M.

M 3.4 45 1.5 0.8 F 3.8 28 1.1 0.7 F 4.3 14 0.6 0.9 M 6.8 9 0.6 0.8 F 12.0 7.5 0.9 0.6

F 6.1 5 0.3 1.1 F 8.5 3.5 0.3 0.6 F 9.7 2 0.2 0.6 M 7.0 3 0.2 0.6 F 11.4 4 0.45 0.7

M 6.9 13 0.9 0.6

F 6.1 23 1.4 0.5

Chromatographically isolated zones of Hb-Ao, Hb-So, and Hb-S, should be devoid of isoleucine. When such samples have been analyzed, traces of isoleucine have always been apparent. When calculation is made the results would suggest the presence of significant and unreasonable amounts of Hb-F. Because Hb-Ao, Hb-So, and Hb-S, precede Hb-F, on the column, the traces of isoleucine cannot be caused by “tailing” of the Hb-F. zone into succeeding zones. The traces of isoleucine in these components may be due to small amounts of nonhemoglobin proteins.

CONCLUSIONS

More than 200 analyses for Hb-F have now been made by the File method. It is a time-consuming procedure that was designed for accuracy rather than for routine use and screening. Although an exact assessment of accuracy is not possible, consideration of the theoretical bases of the method and of the possible sources of error suggests that results by the File method may be accurate to ~10% when Hb-F accounts for a rea- sonable proportion of the total hemoglobin. Whether the accuracy is this high when the Hb-F is very low in the hemolyzate cannot be stated with much assurance.

SUMMARY

The described analytical method for human fetal hemoglobin is espe- cially applicable when the amount is intermediate between the very low percentage in the adult and the very high percentage in the newborn infant. The procedure requires the chromatographic isolation of a zone which contains a mixture of hemoglobin F and a minor component of

Dli?r@RMINATiOd OF S’tiTAL HEMOGLOBIN 243

hemoglobin A: the proportion of F in the zone is determined by amino a&d analysis for isoleucine. Consideration of sources of error and the help of model experiments suggest that the method may be accurate to +-lo% when hemoglobin F accounts for a reasonable proportion of the total hemoglobin.

ACKNOWLEDGMENTS

We thank Mrs. N. Bouver and Mr. P. Lerch for making the column chroma- tographic analysis.

This investigation was supported in part by grants (HE-O2.%8 and HE-05168) from the National Institutes of Health, U. S. Public Health Service.

This is @ntribution No. 3947 from the Division of Chemistry and Chemical Engineering, California Institute of Technology.

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