An analysis of the hemoglobins from some common turtles

An Analysis of the Hemoglobins from Some Common Turtles

BENNETT HORTON, RONALD FRASER, DANIEL DUPOURQUE, DAVID BAILEY AND AMOZ CHERNOFF? Uiziue~si ty of Tennessee Memoi in l Resetrich Ceiztei, nrrd thc. D q ~ n l t r n e n t of Zoology, Uniueisr ty of Tennessee, Kiioxuille, Tennessee 3791 6

ABSTRACT A chromatographic analysis of the blood from seven species of turtles, representing four families, has revealed that all animals contained muItiple hemoglobins. Members of the Emydidae family had two major components separated by a minor one. There was some variation to this pattern in the box turtles (Terrapenes). The musk, soft shelled, and the snapping turtles examined had three, five, and four hemoglobins respectively. In general, electrophoresis of these proteins in starch gels confirmed the results obtained from DEAE cellulose chromatography in minicolumns.

Hemoglobins from the painted and Cumberland turtles were found to be quite resistant to dimerization on aging and to alkali denaturation. These proteins from the box turtles were seen to be moderately resistant to both types of molecular modification, while those from the musk, the soft shelled, and the snapping turtles polymerized readily on aging and denatured readily in alkali.

We have known since the reports of Ramirez and Dessauer ('57) and Dessauer et al. ('57) that many adult turtles (perhaps all) possess more than one type of hemoglobin. These early workers utilized paper electrophoresis to separate these proteins. More recently, other techniques of higher resolution have been employed. Except in the work of Dozy et al. ('64), turtle hemoglobins were fractionated by starch and acrylamide gel electrophoresis (Manwell and Schlesinger, '66; Sullivan and Riggs, '67a). Dozy and co-workers, in Huisman's laboratory, separated the hemoglobins from adult turtles by DEAE cellulose chromatography, using a phosphate buffer gradient, as well as by starch gel electrophoresis.

Riggs and his group proved that the hemoglobins of some turtles can undergo either polymerization (Riggs et al., '64) or chain separation (Sullivan and Riggs, '67b) once the cells have been lysed. The extent of these modifications is dependent on such external factors as the pH, ionic strength of the buffer, temperature and time.

We hoped to minimize some of the sources of error in separating turtle hemoglobins through the use of a rapid chromatographic procedure capable of

high resolution, by holding denaturation to a minimum, by checking our results electrophoretically, and by examining the extent of polymerization through aging experiments using ultracentrifugation techniques and additional chromatographic methods. The stability of the hemoglobin to alkali denaturation was also determined. In this study we have evalu- ated the hemoglobin composition of turtles from four families, but have made no attempt to draw any conclusions regard- ing phylogeny.

MATERIAL AND METHODS

The turtles used in these experiments were collected locally and maintained in an animal facility during the course of the study. We had single specimens of the musk (Sternothernus carinatus, family Kinosternidae) and soft shelled (Amyda ferox spinzfera, family Trionychidae) turtles. We analyzed the hemoglobins of ten common snapping turtles (Chelydra ser-

1 This research has been supported in part by grant AM-13256 from the National Institutes of Health.

*The authors wish to express appreciation to Mr., David Packard of the Tennessee Fish and Game Com- mission from whom many of the turtles studied were obtained.

3 Recipient of Research Career Award 5K6-GM-3750 from The Institute of General Medical Sciences.

J. E X P . ZOOL., 180: 373-384 373

374 HORTON ET AL.

pentina, family Chelydridae) and of 24 members of the family Emydidae. Of these, five were box turtles (Terrapene carolina carolina), one was an elegant slider (Psuedemys scripta elegans), four were Cumberland sliders ( P . scripta troosti), and 14 were painted turtles (Chrysemys picta marginata). '

Hemoglobin preparation

Blood from each turtle was drawn di- rectly from the heart through a small hole drilled in the plastron. Drilling was unnecessary in the soft shelled and snapping turtles. After small samples were used in the preparation of smears and for the determination of blood indices, the remaining blood, ranging in volume from two tenths to 1 ml (depending on the size of the animal), was placed immediately into chilled vials containing a sterile isotonic solution with dextrose and citrate (ACD, solution A in Vacutainers, Becton, Dickinson and Co.). The blood samples and hemoglobin preparations were kept about 4" C during all phases of this study. The blood cells were washed three times in a 0.9% NaCl solution, then lysed with two volumes of deionized water. Lysing was facilitated by the addition of a n equal volume of CC14 and agitation of the sus- pension on a vortex mixer for a few minutes. The hemolysate from each animal was analyzed for hemoglobin composition both by chromatography on a DEAE cellulose column and starch gel electrophoresis. Sedimentation and alkali stability determinations were also made on the released hemoglobins.

Chromatography

The miniature chromatography column for the separation of hemoglobins has been described previously (Horton and Chernoff, '70). In the present study we employed several modifications. Three columns, each 16 cm in length and with an inner diameter of 3.5 mm, were run simultaneously. These columns were jack- eted in series and cooled by a flow of water with a temperature of 4" C at the inlet and 9" C at the outlet.

DEAE cellulose (Schleicher and Schuell, no. 72, type 40, lot no. 2078) had been previously equilibrated against a Tris-HC1

buffer (0.05 M, pH 8.5). Coarse particles of this resin were added to each column to yield a settled mass measuring about 11 cm in length.

Three chambers of a Technicon Auto- grad were used to create a buffer gradient for the elution of the hemoglobins. Each chamber contained 225 ml of a Tris-HC1 buffer. The molarity and pH of the buffers in each chamber, respectively, beginning with the lead chamber, were 0.05 M, pH 8.5; 0.2 M, pH 6.5; and 0.8 M, pH 6.5. Shortly after leaving the lead chamber the buffer was split into three lines each of which led through one channel of a Buchler polystatic pump at a flow rate of 30 ml per hour per channel to the top of a column. From the bottom of each column the eluate flowed through a four channel (one used as a reference) Gilford recording spectrophotometer to a fraction collector capable of collecting four samples simultaneously. Absorbance was measured at 415 nm. The contents (1 ml of eluate) of every fifth tube were assayed for pH. We determined the relative hemoglobin content both by measurement of the areas under the absorbance curves traced on the recorder paper, and from pooled fractions of each hemoglobin by calculating the product of the volume and absorbance. Chromatographic separation of those hemoglobins designated "fresh was started about 30 to 45 minutes after the blood had been collected from the animals, and was completed within three hours.

Electrophoresis Hemoglobin samples were subjected to

vertical starch gel electrophoresis (Smith- ies, '59) at 4" C. The resolved bands were stained with 0-dianisidine and Amido Black 10B.

Aging studies

Samples of hemoglobins from represen- tative species of turtles were run on DEAE cellulose columns both at four hours and four or five days after obtaining the hemolysate. They were stored in a refrigerator at 4" C during the aging period.

Sedimentation coefficient determination Sedimentation coefficients of the hemo-

globins were estimated for both fresh sam-

TURTLE HEMOGLOBINS 375

ples and those aged for various intervals at either 4" C or 22" C. The hemoglobin concentration ranged from 5 to 10 mg per ml in each sample. Analyses were made in a Spinco Model E ultracentrifuge at 20" C at 60,000 rpm. The samples were suspended in phosphate buffer (0.1 M, pH 7.4). Photographs were taken through a red filter of hemoglobin boundaries in cells of 12 mm path length.

Alkali denaturation

The progress of denaturation of hemoglobins from the turtles in both 13.2 and 52.6 mM NaOH (25 and 100 p12 M NaOH in 3.8 ml) was followed with a Gilford recording spectrophotometer. The optical density was measured at 576 nm. The slopes of the lines or portions of lines were computer-derived using the formulae given by Crow, Davis and Mayfield ('60). Absorbance readings obtained at one min-

ute intervals were used to calculate the percentage of undenatured hemoglobin present, the log of which was used as the Y value in the regression formula, the time in minutes being the X value. Only consecutive points that had a correlation coefficient equal to or less than -0.995 were used.

RESULTS

The results of the chromatographic analyses are shown in figures 1 and 3 through 6. Quantitative results are sum- marized in table 1.

Elution patterns of the hemoglobins from the painted turtles and the closely related elegant and Cumberland slider turtles were identical. The major component, comprising 65 to 75% of the total hemoglobin, had an elution pH near 8.10. A minor component (about 7%) washed from the column near pH 7.64, and a

TABLE I

Per cent composition of hemoglobins in turtles

92 of hemoglobin (Mean values) Turtle type Four to five

Hb fraction Fresh Hb Four hours old days old

Box

Box

Box

A 59 (3) 1

B 11 C 30 A 62 (4) Bi 7.4 Bz 4.6 C 26 AI 19 (3)

26 7.2

Az 17 9.2 A3 26 42 B 12 21 C 26 21

Painted A 74 (19) 72 77 B 6.5 8 5 C 20 20 18

Cumberland A 61 (5) 64 66 B 8 7.6 1.4 C 31 28 33

Musk A 20 (2 ) 19 3.6 B 33 40 19 C 47 41 77

B 50 50 31 C 35 39 60 D 1-2 1-2 1-2 E 10 15 3.9

B 38 37 20 C 23 29 62 D 16 15 12

Soft shelled A 3.5 (2) 4.0 4.0

Snapping A 23 (9) 19 1.8 +3.7

1 Numhers in parenthesis in this column represent the number of chromatographic analyses made i n deriving the per cent composition of the hemoglobins in the "Fresh" column.

376 HORTON ET AL.

FRACTION NUMBER

Fig. 1 Hemoglobins of the painted turtle. DEAE-cellulose chromatograms of fresh hemoglobins (A) and of that aged four days (B). Ab- sorbance shown by solid line, pH shown by dotted line.

third hemoglobin, constituting between 20 and 30% of the total, was eluted near pH 7.45. Figure 2, which summarizes the results of electrophoretic fractionation on starch gel, confirmed the observation that there were three hemoglobins with the approximate concentrations and relative net charges indicated by chromatographic analysis. In addition, neither polymerization nor chain dissociation of the fresh hemoglobins of the painted turtle was demonstrable by centrifugation (fig. 7A). A single symmetrical peak indicated one component with a S2, , ,o of 4.4. After three days at room temperature, hemoglobin dimerization of about 25% of the molecules was found (fig. 7B) indicative of the relative resistance of this protein to polymerization. There were no

changes in the pattern of hemoglobins eluted from DEAE cellulose after the samples had been aged in the refrigerator for four days (see fig. 1). Figure 8 A and 8B show that the hemoglobins from the painted and Cumberland turtles are more resistant to alkali denaturation than are those from the snapping turtle and much more so than human Hb A. They are similar in this regard to human Hb F (fig. 9). In summary the three hemoglobins present in the painted, the elegant and the Cumberland slider turtles were found to be relatively resistant to molecular change. Hematological data for the three members of the Emydidae family are given in table 2. Cells from the painted and Cumberland turtles are shown in figure 10A and 10B. There appear to be areas in the erythrocytes of the Cumberland turtle with a low hemoglobin content.

Of the five box turtles tested only two had hemoglobins similar to those of the animals described above (fig. 3A). Quan- titation of the hemoglobins revealed that the first, or most basic, component was in slightly less concentration (60% of the total) while the middle, or minor, component had increased to about 1 2 % . The most acidic hemoglobin was in the same concentration, however, as the corre- sponding one in the painted, slider, and Cumberland turtles. The net charges of the three hemoglobins were essentially the same as those of other members of this family.

In contrast to the two box turtles mentioned above, two other box turtles showed two minor bands rather than one located between the major fractions (fig. 3B). The total amount of hemoglobin in the two peaks was about the same as that of the single minor fraction. The elution pattern of hemoglobins from these two turtles was otherwise the same in all respects to those

TABLE 2

Mean hematological data on turtle erythrocytes

Turtle type Hh Hct RBC/mm3 MCV MCHh MCHbC

gm % % x 103 P3 PPS %> Painted (13) 1 5.6 24 723 333 80 23 Cumberland (2) 5.4 19 500 3 75 104 27 Box (1 ) 4.8 29 570 508 84 17 Snapping (5) 5.5 21 396 539 135 25

I Numbers in parenthesis represent the numbers of turtles from which these data are derived.


Fig. 2 Starch gel electrophoretogram of turtle hemoglobins. All figures read from left to right. (A) painted, painted, box, box, human Hb A; (B) soft shelled, musk, snapping, box; (C) all are snapping turtles; (D) human Hb A, box, box, Cumberland. (A) stained with o-dianisidine, the others stained with Amido Black 10B. Note the intraspecies heterogeneity of the non-hemoglobin proteins located cathodically, particularly in (C).

mentioned previously. The remaining box turtle showed another variation in that the first, or major, fraction from the column was subdivided into three elution peaks (fig. 3C) . Both the minor and the most acidic hemoglobins from this animal were the same as those from the previously mentioned turtles with regard to both concentrations and the pH required for their elution. The amount of hemoglobin in the three subfractions combined equalled that of the single major component (about 62%).

Starch gel electrophoresis failed to resolve the subunits of either the major or the minor peaks (fig. 2). Otherwise the pattern of bands that developed was con- sistent with our results from chromatographic fractionation.

Figure 3D and 3E reveals that on aging for four days at 4" C the box turtle herno- globins yielded some differences in their chromatographic profile. There was a decline in the amount present in both the slow and fast major peaks with a corre- sponding increase in the hemoglobin in

378 HORTON

1000-

600

400

zoo

0

ET AL.

the minor peak. Coincident with this we found the peaks to be somewhat flattened with some confluence, suggesting an appreciable denaturation of these proteins. The double minor peak of the two turtles showing this feature was reduced to one peak when the hemoglobins were aged, while the three subunits of the major peak found in the animal persisted (figs. 3D, 3E).

There was no evidence of polymerized hemoglobin in fresh blood drawn from the box turtles. After 20 hours, however, at room temperature (22' C), about 60% of this protein was in the dimer form (fig. 7C). Thus polymerization occurs more readily in the box than in other turtles of the Emydidae family that we tested, Fig- ure 9 indicates that the hemoglobins of the box turtle are also somewhat more sensitive to alkaline denaturation than that of the Cumberland and elegant slider, or painted turtles.

Hematological information on the red blood cells from the box turtles is provided in table 2, while photographs of a few cells from two animals are present in figure 10C,D. Note that the chemical differences found in the hemoglobins from members of this species are matched by differences in gross morphology of the red blood cells from different animals. The turtle with the triple major component had red blood cells that are less elongated as shown in figure 10D. The blood indices of the box turtles are more difficult to obtain be- cause of the extreme resistance of the red blood cells of this species to hemolysis in determining the hemoglobin concentration. On occasion, blood diluted with van Kampen Zijlstra solution containing Sterox SE as a hemolytic agent plus the addition of a few drops of saponin followed by repeated freeze thawing still contained red sediment on centrifugation.

We found that the musk turtle had three chromatographically definable fractions with concentrations of 20, 33 and 4 7 % (fig. 4A). In this same order, the elution pHs of the hemoglobins were 8.04, 7.90 and 7.78. Thus, these three proteins are quite similar with regard to their net charges. Electrophoresis in starch gel failed to resolve the fractions adequately (fig. 2), again reflecting a similar net charge.

a 800z"'.... -

-

-

-

5 1000 P

a + > rn z !I 600-

400-

200- A

+ R 0

r 8 -

- 8 0

.......

0 '

4001 190 I -1 C I p . . ...q -1 8 oPH

FRACTION NUMBER

Fig. 3 Hemoglobins of the box turtles. DEAE- cellulose chromatograms of fresh hemoglobins from turtles with three, four and five components (A, B and C respectively), and of hemoglobins aged four days from turtles with four (D) and five (E) components. Absorbance shown by solid line, pH shown by dotted line.


a 0 + a 0

600

400-

200

0

....... I \ A ‘ 000

B - .. - 8 0

PH

803r”’.

-

-

I

.... ..... ... ...... PH 600

5 $ 400 7 0

4 200 +

L W 0

_I 10001 ,90

FRACTION NUMBER

Fig. 4 Hemoglobins of the musk turtle. DEAE- cellulose chromatography of fresh hemoglobin ( A ) and of that aged four days (B). Absorbance shown by solid line, pH shown by dotted line.

When the hemoglobin sample from the musk turtle had been aged for four days in a cold room, the chromatographic elution profile changed quite markedly. The most basic component was reduced in concentration from 20 to 3.6%. The middle fraction similarly decreased from 33 to 19% of the total, while the concentration of the most acidic hemoglobin rose to 77%. This gross shift in molecular composition suggests to us that the third (most acidic) fraction may be an artifact caused by aging. More will be said about this point later.

Figure 7D shows that dimerization of hemoglobin was evident in fresh blood of the musk turtle. It must be emphasized that by the time the blood cells, freshly drawn from the turtle, had been washed three times, lysed, the hemoglobin sample brought up to speed in the centrifuge, and spun for 94 minutes, about three hours had elapsed. When analyzed at five and one-half hours, about 70% of the total hemoglobin appeared to be in the dimer form (fig. 7E). We infer from these obser- vations that more than one of the hemoglobins of the musk turtle polymerizes readily. These proteins are also rather easily denatured in a basic medium as

+ a 600p ..... I

200u70 0 0 20 40 60 80

h 0

FRACTION NUMBER

Fig. 5 Hemoglobins of the soft shelled turtle. DEAE-cellulose chromatogram of the fresh hemoglobin (A) and of that aged five days (B). Absorb- ance shown by solid line, pH shown by dotted line.

seen in figure 9. Of the hemoglobins analyzed, only human Hb A apparently was more sensitive to hydroxide treatment,

We eluted five hemoglobin fractions from the DEAE cellulose columns charged with fresh samples from the blood of the soft shelled turtle, Amyda ferox spinifera (fig. 5). From most basic (first off the column) to the most acidic, the relative concentrations of these hemoglobins were 3.5, 50, 35, about 1-2, and 10%. Respec- tively, the elution points were 8.15, 8.04, 7.75, 7.72, and 7.51. Figure 5 also reveals that the hemoglobins from this turtle, when aged for four days in the cold, changed in relative concentrations. The effects of aging were particularly notice- able in the second or major molecular species which was reduced to about 30% of the total, and in the third fraction, which rose in amount to become the major component constituting 60% of the total hemoglobin. The pH values at which the aged hemoglobins were eluted from the DEAE cellulose columns were the same as those required to free the fresh ones. The electrophoretic behavior of the hemoglobins from the soft shelled turtle is shown in figure 2. A discrepancy in the

380 HORTON

f a -

- ....

I 000 -... 800 ~

600 -

-

-

ET AL.

80

PH

70

m z ; I 000,

4001 , 4 ..... ... . . .,.

ZOO 70

0 0 20 40 60 80

FRACTION NUMBER

Fig. 6 Hemoglobins of the snapping turtle. DEAE-cellulose chromatogram of fresh hemoglobin (A) and of that aged for four days (B). Ab- sorbance shown by solid line, pH shown by dotted line.

profile of the more acidic fractions, com- pared with that derived from chromatography, is evident.

Regions in the red blood cells from the soft shelled turtle in which there appears to be relatively little hemoglobin are seen in figure 10F.

There were four chromatographically distinct hemoglobins found in the snapping turtle, C k l y d r a serpentina (fig. 6).

The third component was seen as a shoul- der on the trailing edge of the peak of the second, or major, fraction. Respectively, the fractions were eluted at pH 8.19, 7.79, 7.68, and 7.50, and quantitatively repre- sented 23, 38, 23, and 16% of the total hemoglobin present. Figure 2C shows that the electrophoretic profile of the hemoglobins of these snapping turtles is com- parable to that obtained from our chromatographic data. When the hemoglobins had been aged for four days at 4" C, differences in the chromatograms were noted. There was a decrease in the relative concentration of all components except the third (or minor) one. This fraction rose to 62% of the hemoglobin in the sample, thus becoming the major type. At the same time a new fraction in very low concentration and with much the same elution point as that of the most basic one found in fresh blood, became evident. The significance of this fraction, comprising about 2 or 3% of the total hemoglobin, is not apparent. We suspect, however, that the third fraction found in the fresh blood of this species represents a modified hemoglobin.

We found the sedimentation coefficient for fresh snapping turtle hemoglobin at 20" C to be 4.4, indicative of the presence of the molecule only in the monomeric form. After two hours at this same temperature a second peak appeared with a coefficient of 6.5. After 24 hours all of the hemoglobin appears to be in this

Fig. 7 Schlieren photographs of hemoglobin boundaries during centrifugation at 20°C and at 60,000 rpm. Phosphate buffer, 0.1 M, pH 7.4 was the suspending medium. Each photograph was taken at 94 minutes into a run. Hemoglobin: (A) fresh from painted turtle; (B) after 72 hours at 25' C from painted turtle; (C) after 20 hours at 22' C from box turtle; (D) fresh from musk turtle; (E) after four hours at 22" C from musk turtle.

TURTLE HEMOGLOBINS 38 1

heavy form. At 4" C a second peak (SzO,w =6.4) appeared after 24 hours of aging, while after 72 hours about 60% was di- merized. Fresh hemoglobin (in phosphate buffer, pH 7.4) to which iodoacetamide was added (1.4 mg/100 ml final concentration) remained in the monomeric form after one week of aging in the cold. If, however, iodoacetamide was added to the hemoglobin that had been aged for one week at a similar temperature (4" C), the hemoglobin had a single sedimentation coefficient of 7.0.

Figure 8 indicates that some of the hemoglobin from snapping turtles is alkali labile, relative to those of the painted and Cumberland species, although more stable than human Hb A. At least one of the hemoglobins seems to be resistant to alkali denaturation.

Some data on the red blood cells of snapping turtles are given in table 2. Figures 10G and 10H reveal large inclu- sions (parasites?) in some of the erythrocytes of an albino snapping turtle. This albino turtle appeared in other respects like the other snapping turtles. There is also evidence of vacuolization in these cells.

In the snapping turtles, and to some extent in the box turtles, there was a heterogeneity of nonhemoglobin proteins seen in the starch gel electrophoretogram stained with Amido Black (fig. 2). These are most prominent cathodic to the origin.

DISCUSSION

We found that all seven species of turtles analyzed in this study contained more than one type of hemoglobin. Indeed, the work of others, particularly Riggs and his group (Sullivan and Riggs, '67a) has

Fig. 8 Alkali denaturation of hemoglobins. Denaturation in NaOH 13.2 m molar (A), and 52.6 m molar (B). Solid lines are drawn from linear regression data. Broken lines are drawn from the origin to the one minute value. Data on (1) the slope of the line, (2) the standard error, (3) the graph (whether A or B), and (4) the pertinent time interval follows (respectively and i n parenthesis) each hemoglobin source. Values are derived from regression equations. m , painted turtle ( - 0.0072, 0.0020, A 6 1 4 ; -0.0413, 0.0097, B 1-14); A, Cumberland turtle ( - 0.0326, 0.0053, B 1-14); 0 , snapping turtle (-0,0124, 0.0029, A 7-14; -0.2074, 0.0172 B 1-6); A , human Hb A (-0,1394, 0.0158, A 14).

L

L

c

382 HORTON ET AL.

L .

M I N U T E S

given substance to the generalization that turtles possess multiple hemoglobins. The number that we found varied from three in certain Emydidae and the musk turtle to five in both the soft shelled and one of the box turtles examined. In general we have been able to identify in each of these animals at least one fraction more than has been previously reported. These are minor components, frequently overlooked or not resolved for one reason or another in electrophoretic fractionations.

At the same time we should realize that what is seen may well represent a minimum number of hemoglobins, The elution profile is dependent on several conditions, such as ion content and pH of the buffers used, nature of the supporting medium, flow rate, etc. It is conceivable that several molecular species may have net charges so similar as to be unresolved in both electrophoresis and chromatography. This would be particularly likely when the two fractions differ widely in concentration with the minor component being covered by the major fraction. Dimeriza- tion of hemoglobins may lead to chromatographically or electrophoretically distinct fractions which represent no genetic differences. We have presented some evidence that a few of the minor components found in fresh blood may have arisen by this procedure, and thus are artifacts. After aging, hemoglobins found to have quantitative changes chromatographically also contain dimers.

There was no evidence of dissociation of hemoglobin molecules in blood taken from the turtles we examined. We did not, however, subject the hemoglobins to fluids of particularly Low pH. Nor have we found any polymerization beyond the dimer con- dition (2 entire hemoglobin molecules),

Fig. 9 Alkali denaturation of hemoglobins. Denaturation in 13.2 m molar (A) and in 52.6 m molar (B) NaOH. Solid lines are drawn from linear regression data. Broken lines are drawn from origin to the one minute value. Data on (1) the slope of the line, (2) the standard error, (3) the graph (whether A or B), and (4) the pertinent time interval follows (respectively and in parenthesis) each hemoglobin source. Values are derived from regression equations. A, box turtles.

14); 0 , musk turtles ( - 0.0258, 0,001 7, A 2-7; - 0.0156, 0.0036, A 8-14); H, human cord blood (-0.0109, 0.0013 A 7-14; -0.0356, 0.0053 B 1-12).

(-0.0103, 0.0034 A 1-14; -0.0600, 0.0090 B 1-


Fig. 10 Photographs of turtle erythrocytes. Wright’s stain. All photographs are at X 1000. A. painted, B. Cumberland, C and D. box, E. musk, F. soft shelled, G., H. and I. snapping turtles.

Again, the pH of our solutions was not sufficiently high to permit further polymerization. We agree with Riggs et al. (‘64) that polymerization begins at the time of cell lysis; there is no evidence that this phenomenon occurs naturally within the erythrocytes.

In addition to appreciable changes in hemoglobins with aging detected chro-

matographically and by ultracentrifugation, figure 9 indicates that one or more hemoglobins from musk, soft shelled and snapping turtles are particularly sensitive to alkaline denaturation. At the same time, hemoglobins from members of one family (painted, elegant slider, Cumber- land slider, and box turtles) are relatively resistent to both dimerization and to de-

384 HORTON ET AL.

naturation. It seems tempting to suggest a causal relationship between the two phenomena, but there is no direct evidence that would warrant such a generalization.

Our results of aging experiments have shown a generalized flattening with some confluence of the components in the chromatograms of hemoglobins from some turtles. These same hemoglobins were found to be readily denatured in alkali suggesting that denaturation may occur readily in the hemoglobins from these animals.

We can say that the difficulty encoun- tered in classifying the Terrapenes (Carr, ’52) is matched by a similar diversity in red blood cell morphology, in the number of hemoglobins and in the types of red cell nonhemoglobin proteins which these turtles possess. Any generalizing on this observation, however, would require much more information than we have presented here.

We are left in a quandary concerning the effect that diet may have on the hemoglobin composition of these animals. This must surely be a problem to anyone who approaches the maintenance of animals with considerable ignorance. The effects of a laboratory environment on turtles is also difficult to assess. These turtles were housed in a windowless room. Perhaps these animals, which normally hibernate, are profoundly affected by such conditions. Certainly the appearance of the erythrocytes of some of the turtles suggests that the animals may be in an iron deficient state. It seems reasonable that anemia may develop in the turtles eating a diet of questionable merit. We have no knowledge whether a reduction in hemoglobin content in the cells is

shared uniformly by all fractions, or whether certain ones are affected more than others. We suspect that the diet of turtles may determine to some extent the percentage composition but not the diversity of hemoglobins.

LITERATURE CITED

Carr, A. 1952 Handbook of Turtles. Cornell U. Press, Ithaca, N. Y.

Crow, E. L., F. A. Davis and M. W. Maxfield 1960 Statistics Manual. Dover Publication, Inc., New York, New York, pp. 164-165.

Desauer, H. C., W. Fox and J. R. Ramirez 1957 Preliminary attempt to correlate paper-electro- Dhoretic minration of hemoglobin with Dhvloe- eny in Amphilia and Reptiria. Arch. Bibchem. Biophys., 71: 11-16.

Dozy, A. M., C. A. Reynolds, J. M. Still and T. H. J. Huisman 1964 Studies on animal hemoglobin. I . Hemoglobins in turtles. J. Exp. Zool., 1 5 5 : 343-348.

Horton, B., and A. Chernoff 1970 Miniature column chromatography of hemoglobins. J. Chromatog., 47: 493498.

Manwell, C., and C. V. Schlesinger 1966 Poly- morphism of turtle hemoglobin and geograph- ical differences in the frequency of variants of Chrysemys picta “slow” hemoglobin - An ex- ample of “temperature anti-adaptation?” Comp. Biochem. Physiol., 18: 627-637.

Ramirez, J. P., and H. C. Dessauer 1957 Isola- tion and characterization of two hemoglobins found in the turtle, Pseudemys scripta elegans. Proc. SOC. Exp. Biol. Med., 96: 690-684.

Riggs, A,, B. Sullivan and J. R. Agee 1964 Poly- merization of frog and turtle hemoglobins. Proc. Nat. Acad. Sci., 51: 1127-1133.

Smithies, 0. 1959 An improved procedure for starch gel electrophoresis: further variations in the serum proteins of normal individuals. Bio- chem. J., 71 : 585-595.

Sullivan, B., and A. Riggs 1967a Structure, function and evolution of turtle hemoglobins. 11. Electrophoretic studies. Comp. Biochem. Physiol., 23. 499458.

1967b The subunit dissociation prop- erties of turtle hemoglobins. Biochim. Biophys. Acta, 140: 274-283.

Documents

An analysis of the hemoglobins from some common turtles