Differential effects of monovalent cations and alkanols on the thermal denaturation of sockeye and coho hemoglobins

Comp. Biochern. Physiol. Vol. 76B, No. 2, pp. 235-240, 1983

0305-0491/83 $03.00 + 0.00 © 1983 Pergamon Press Ltd

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DIFFERENTIAL EFFECTS OF MONOVALENT CATIONS AND ALKANOLS ON THE T H E R M A L D E N A T U R A T I O N OF SOCKEYE A N D COHO H E M O G L O B I N S JOHN P. HARRINGTON Department of Chemistry, University of Alaska, Anchorage, 3221 Providence Drive, Anchorage, AK 99508, USA

(Received 22 February 1983) Abstract--1. The thermostability of hemoglobins isolated from the coho, O. kisutch, and sockeye, O. nerka, salmon was investigated. Using stareh-gel electrophoresis and scanning densitometry, a quantitative determination of the effect of several monovalent cations and short-chain alkanols on the differential thermostabilities of the individual hemoglobin components of these fish was carried out. 2. Li ÷ was the most effective cation in destabilizing both the coho and sockeye hemoglobins. 3. Decreased resistance to thermal denaturation was directly related to the length of the alkyl chain of the alkanols. 4. Anodal hemoglobin components of both species studied are less resistant to thermal denaturation. 5. Urea denaturation of isolated anodal and cathodal hemoglobin components of the sockeye salmon indicate inherent differences in the stability of these components.

MATERIALS AND METHODS

INTRODUCTION

Recent comparative studies on the thermostability of hemoglobins isolated from a wide range o f h o m e o t h e r mic and non-homeothermic vertebrates clearly demonstrate that fish and amphibians exhibit lower thermostability when compared to birds, mammals, and reptiles (Tondo et al., 1980). In a more detailed study offish hemoglobins, Borgese et aL (1980, 1982) suggest that a m o n g different species of fish, both teleost and elasmobranchs, there are four categories of hemoglobin thermostability. Extensive variability was evident in these four categories as determined by the effect of pH, ionic strength, and temperature on these hemoglobins. The fact that most previous heat denaturation studies were carried out on total hemoglobin hemolysates made analysis o f the different factors contributing to increased or decreased thermostability difficult to delineate. Evidence that most species of fish contain multiple hemoglobin components makes it important to be able to determine quantitatively the differential thermostability of these individual hemoglobin components. A first attempt at this was carried out in a study which focused upon a related group of teleosts, the Pacific salmon, Oncorhynchus, wherein it was shown that several anodal components of the adult pink, O. gorbuscha and coho smolt, O. kisutch salmon are more easily heat denatured than the ~ t h o d a l components (Harrington, 1982). This initial study has now been extended to a determination of the differential effects of monovalent cations and shortchain alkanols on the thermal denaturation of two species of Pacific salmon, the sockeye, O. nerka and the coho, O. kisutch, each of which exhibits extensive hemoglobin multiplicity as determined by vertical starch gel electrophoresis.

Blood collection Blood samples were collected from the caudal vein of live fish and transferred to flasks containing cold 1~o NaC1. The suspension was centrifuged at 4°C and the supernatant was discarded. Packed red cells were washed at least three times with the above saline solution. The white cells were removed by suction. After the final wash the red cells were osmotically lysed by the addition of two volumes of cold deionized/distilled water. Cellular debris was removed by centrifugation at low speed for 10 min at 4°C and followed by centrifugation of the hemoglobin supernatant for 30 min at 10,000 rev/min. This latter centrifugation was repeated twice. Supematant was removed and an aliquot was taken for the determination of hemoglobin concentration by Drabkin's methods (1946).

Heat denaturation A determined amount of hemoglobin was volumetrically diluted to a final concentration of 0.2 g/dl with 0.05 M potassium phosphate buffer of appropriate pH. Equal volumes were placed in corex test tubes and except for the control (0 rain), the tubes were placed in a water bath thermostatically controlled at 50°C. At specific time intervals the tubes were removed from the water bath, placed in an ice bucket and centrifuged for 10 rain at 5000 rev/min. The hemoglobin concentration of the supernatant was determined by adding 0.5 ml of the supematant to 3.0 ml of Drabkin's reagent and measuring the absorbance at 540 nm. Thermostability was determined by the time required for 50~ precipitation of a 0.2 g/dl buffered hemoglobin solution at 50°C (tl/2). A measure of the kinetics of the process as well as extent of heat denaturation is evident from plots of the logarithm of the percentage hemoglobin remaining in the supernatant against time. Hemoglobin solutions containing various monovalent cations (chloride form) and short-chain alkanols were prepared by volumetric dilution using 1 and 0.5 M stock solutions, respectively. 235

236

J.P. HARRINGTON

Urea and LiC1 denaturation Absorbance measurements were made on a Cary 219 spectrophotometer. Hemoglobin concentrations were determined spectrophotometrically assuming a molar extinction coefficient per heme of 1.25 x 105 at 412 nm (Dilorio, 1981) for each of the salmon hemoglobins studied. Several cathodal fractions as well as the major anodal hemoglobin fractions were isolated by ion-exchange chromatography using DE-52 (diethyl aminoethyl cellulose) developed with a 0.05 M Tris-HC1 buffer, pH 8.4. Individual fractions were eluted from Kontes split columns with 0.5 M NaCI in the same buffer. Identification of each fraction was done by starch-gel electrophoresis as described below. For each series of measurements volumetric dilutions were made using common stock solutions of protein and concentrated denaturing agent in 5 ml stoppered volumetric flasks. Absorbance measurements were made after 2 hr equilibration for urea solutions and 10-15 min for LiC1 solutions at 25cC. Precipitation occurred at high concentrations of LiC1 ( > 6.0 M) if solutions were left standing for more than 30 min.

Electrophoresis and densitometry Vertical starch-gel electrophoresis was carried out on all hemoglobin samples, both controls and heat denatured, at pH 8.6 using a Tris-borate-EDTA buffer according to Smithies (1959). Gels were stained with o-toluidine. Quantitative analysis of heat denatured salmon hemoglobins were accomplished using a Quick Scan R & D Helena Scanning Densitometer in reflectance mode at 505 nm.

RESULTS

The extent and uniqueness of the hemoglobin multiplicity present in teleost fish is evident from the starch gel of four species of Pacific salmon and the Pacific cod (Fig. 1). All species of salmon exhibit several anodal and cathodal components. In an attempt to determine the effect o f specific monovatent cations and short-chain alkanols ( C r C 4 ) on the thermal denaturation of the sockeye and coho hemo-

globins, heat denaturations of these hemoglobins (total hemolysates) in the presence of these cations and alkanols were carried out as described above. The effect of the different monovalent cations and alkanols on the t~/2 evident from Fig. 2 are summarized in Table 1. Each monovalent cation used was effective in decreasing the stability of the sockeye and coho oxyhemoglobins. Li ÷ exhibited the largest destabilizing effect on these hemoglobins as evident in the tl/2 = 15 and 17 min compared to the heat denatured controls (absence of specific monovalent ion) of tt/2 = 37 and 33 min for the sockeye and coho hemoglobins respectively. In a similar manner, the effectiveness of increasing the length of the alkyl chain of the short-chain alkanols from a methyl group to a butyl group, leads to a corresponding destabilization at a much lower concentration (0.2 M) than any of the above monovalent cations. A t,,,2 = 9 min was determined in the presence of 0 . 2 M 1-butanol compared to fi/2 = 3 4 m i n for the control with the coho hemoglobins (Table 1). The specific destablizing effect of each monovalent cation is seen when a determination of the individual hemoglobin components remaining after heat denaturation is carried out by a combination of vertical starch gel electrophoresis and scanning densitometry. Figures 3 and 4 and Tables 2 and 3 clearly indicate that the anodal components of both the sockeye and coho are further destabilized specifically in the presence of Li+, N a ÷ , and Cs + ions. It is also evident that the most electronegative component of the coho and sockeye hemoglobins are also destabilized as the hydrophobicity of the denaturing agent is increased (Table 2). The Li ÷ ion has the greatest effect of the monovalent cations used in destabilizing the most anodal components of both the coho and sockeye hemoglobins. The increased destabilization of the above anodal components of the coho and sockeye suggested that

Fig. 1. Vertical starch-gel electrophoresis of the total hemolysates for several species of salmon and cod at pH 8.6, Tris-borate buffer.

Stability of salmon Hb

237

(b)

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1.7-

E= .c:

"\

1.6

_.1 1.5 -

~

=

,

1,4 1.3-

I 5

I 10

r 15

I 20

I 25

I I I 30 5 Time (rain)

I 10

I 15

I 20

I 25

I 30

I

Fig. 2. Effect of alkanols and monovalent cations on the thermostability of coho, O. kisutch, salmon hemoglobin at 50°C. (a) O, control; O, 0.2 M methanol; I-q, 0.2 M ethanol; V, 0.2 M l-propanol; A, 0.2 M 1-butanol. (b) O, control; 0 , 0.5 M CsCI; V, 0.5 M NaCI; and 0 , 0.5 M LiC1. Hemoglobin concentration was 0.2 g/dl.

further study regarding the unfolding of these proteins was warranted on these components. Hence, the individual hemoglobin components of the sockeye salmon were isolated. Isothermal denaturation of two cathodal (components 1 and 2) and the major anodal (component 4) components of the sockeye were carried out using urea and LiC1 as the denaturants. The effects of these denaturants on the absorbance spectra in the Soret region of the oxyhemoglobins from each of these components is shown in Fig. 5. A lower denaturation midpoint, DU2 3.6 M, was determined for the anodal component as compared to 6.0 M for both cathodal components studied. Similar results were obtained with LiC1. Likewise, it is evident that LiC1 is more effective than urea in unfolding both the anodal and cathodal components. Interestingly, the rate of autoxidation of the cathodal component 1 was more r a p i d than anodal component 4 at pH 7.3 as evident from Fig. 6. =

Table 1. Effect of several monovalent cations and alkanols on the rate of heat denaturation (tj/2, min.) for the coho and sockeye salmon (t = 50°C) Coho Hb Sockeye Hb Control CsCI(0.5 M) LiCI(0.5 M) NaCI(0.5 M) MeOH(0.2 M) EtOH(0.2 M) PrOH(0.2 M) BuOH(0.2 M)

33 21 15 18 36 32 19 9

39 25 15 23 29 27 17 8

DISCUSSION Since many species of fish contain multiple hemoglobin components as all the species of salmon, Oncorhynchus, do, a quantitative determination of individual hemoglobin components is essential in delineating the differential destabilizing effects. An initial study (Harrington, 1982), using a combination of electrophoresis and scanning densitometry, permitted a determination of the individual hemoglobin components from one species of fish that are more susceptible to denaturation under different conditions. Heat denaturation of the total hemolysates of the sockeye and coho salmon in the presence of several monovalent cations (Li+, Na ÷ and Cs ÷) indicates that Li ÷ is very effective in destabilizing the more electronegative (anodal) components of both the sockeye (component 4) and coho (components 7 and 8) hemoglobin systems. The Na ÷ and Cs ÷ ions appear to be effective in destabilizing these components of the coho to a greater extent than for the sockeye hemoglobin system (Table 2). These results point to a substantial difference in the thermal stability between these anodal components and the other components of the sockeye and coho hemoglobin systems. The enhanced effect of the Li ÷ is in agreement with previous studies wherein this cation has been shown to dehydrate proteins (Bull and Breese, 1970) as well as being the most effective to salt-in the peptide groups and showing the lowest tendency to salt-out hydrophobic groups (Nandi and Robinson, 1972a,b). Increasing the length of the alkyl chain of the short chain alkanols from a methyl to a butyl group leads

238

J.P. HARRINGTON

Fig. 3. Vertical starch-gel electrophoresis of the effect of several monovalent cations (0.5 M) on the thermostability of coho, O. kisutch, hemoglobin components.

to a similar destabilization of the total hemolysates of these hemoglobin systems. This effect is achieved using a lower concentration of these alkanols (0.2 M). The action of these alkanols is also most effective in denaturing the more electronegative hemoglobin components suggesting that the number and/or presence of specifically located hydrophobic contacts within these components are more important in main-

taining the native conformation of these hemoglobins (Fig. 2). The fact that both electrostatic and hydrophobic agents destabilize the same anodal components indicates that certain inherent differences exist between these components and the more electrooositive ones. These differences may involve varying amino acid composition of the globin chains constituting these components as well as alterations in the

Fig. 4. Vertical starch-gel electrophoresis of the effect of several monovalent cations (0.5 M) on the thermostability of sockeye, O. nerka, hemoglobin components.

Stability of salmon Hb

239

Table 2. Determination of hemoglobin components after heat denaturation for the coho salmon by scanning densitometry Hb Components

e

Control Control (heat denatured) CsCI(0.5 M) NaCI(0.5 M) LiCI(0.5 M) Methanol(0.2 M) Ethanol(0.2 M) 1-Propanol(0.2 M) 1-Butanol(0.2 M)

I

2

3

4

5*

6

7

8# •

1 I 24 27 34 3 1 7 12

2 3 22 16 20 5 2 10 13

10 17 23 26 21 14 16 18 21

16 23 22 26 19 24 23 23 21

19 21 2 2 3 18 25 17 19

18 14 5 3 3 16 16 15 12

9 4 1 0 0 4 4 6 2

25 17 1 0 0 17 14 4 0

*Origin for samples. Component 8 contains more than one Hb component as indicated by a shoulder in the scan profile.

Table 3. Determination of hemoglobin components after heat denaturation for the sockeye salmon by scanning densitometry Hb Components

•

Control Control (heat denatured) CsCI(0.5 M) NaCI(0.5 M) LiCI(0.5 M) Methanol(0.2 M) Ethanol(0.2 M) 1-Propanol(0.2 M) l-Butanol(0.2 M)

1

2

3*

4

5

34 33 24 29 32 40 39 48 66

25 32 23 37 39 38 41 46 34

7 9 37 22 29 8 7 6 0

27 18 16 12 0 13 13 0 0

7 8 0 0 0 1 0 0 0

*Origin for samples.

(a)

0

-

(b)

z~

-_ 0

1.2

zx

1.1 1.0 x

0.9

'~

0.8 o

0.7

A

0.6 0.5 I

I

J

1 2 3

I 4

I 5

I 6

Urea (m/I)

I 7

I 8

1 9

I

I

I

1 2 3

I

I

I

I

I

I

4

5

6

7

8

9

LiCI (m/I)

Fig. 5. The denaturation and of individual anodal (component 4) and cathoda] (components 1 and 2) hemoglobins of the sockeye, O. nerka by urea (a) and LiC1 (b) followed by changes in absorbance at 412 nm Soret band. O, Cathodal, component 1; • cathodal, component 2; and ~., anodal, component s,. All solutions were buffered by 0.05 M Tris-HCl, pH 7.3. Hemoglobin concentrations used were 5.9-7.6 tt M.

240

J, P. HARRINGTON

2.0 kcal/mol have been determined for the cathodal 2.0

1.9 -r" >. x o

1.8

._1 1.7

--

1.6-45

90

135

180

225

270

315

and anodal components of the sockeye salmon respectively. The value for the cathodal components is similar to that obtained with urea for human hemoglobin (Elbaum et al., 1974) as well as the multisubunit hemoglobin of the earthworm, L. terrestris, (Herskovits and Harrington, 1975). Finally, the large difference in the kinetics of autoxidation between the cathodal (component 1) and the anodal component wherein the fomer component is more readily oxidized by exposure to air, but is more stable to thermal denaturation, requires additional investigation. Denaturation and precipitation of human hemoglobins are usually initiated by some form of oxidation. These studies are presently underway.

Time (min)

REFERENCES

Fig. 6. Kinetics of autoxidation of the anodal (O) and cathodal component 1 (&) hemoglobins of the sockeye, O. nerka for room air at 25°C in 0.05 M Tris-HCl, pH 7.3 buffer.

amino acid residues in contact with the heme moiety from one hemoglobin component to another. Additional studies of the isolated individual globin chains, leading to their amino acid composition and the determination of sequence data, will be necessary to clarify this situation. The extent of these differences is further evident when individual hemoglobin components of the sockeye salmon, two cathodal fractions (components 1 and 2) and the major anodal fraction (component 4), were exposed to increasing concentrations of two

denaturing agents, urea and LiCI. Isothermal denaturation at 25°C was followed by alterations in the Soret region (390--450rim) which is sensitive to the heme environment (Fig. 5). Large differences are clearly indicated in protein stability between these components as evident from the lowering of the denaturation midpoint from 6.0 M for the two cathodal components to 3.6 M for the anodal component in the presence of urea. Likewise, in the presence of LiC1 the denaturation midpoint is lowered from 4.6 to 3.9 M for the cathodal (component 1) and the anodal components 4, respectively. A recent study by Martin et al. (1979) showing the effects of urea on the Soret spectra of several fish hemoglobins indicates that considerable differences exist between species. Denaturation midpoints ranged from about 4.0 to 9.0 M urea. Estimations of the free energy of unfolding in the absence of denaturant (urea), -AC,82° ---# , may be deter-

mined using a linear relationship between AG, and denaturant concentration. Free energies of 3.7 and

Borges¢ T. A., Borges¢ J. M., Harrington J. P. and Nagel R. L. (1980) Thermostability of fish hemoglobins. Biol. Bull. 159, 448. Borgese T. A., Harrington J. P., Borgese J. M. and Nagel R. U (1982) Thermostability of fish hemoglobins. Comp. Biochem. Physiol. 72B, 7-11. Bull H. R. and Breese K. (1970) Water and solute binding by proteins. Archs Biochem. Biophys. 137, 299-305. Dilorio E. (1981) Preparation of derivatives of ferrous and ferric hemoglobin. In Methods in Enzymology 76 (Edited by Antonini E., Rossi-Bernardi L., and Chiancone E.), pp. 47-72, Academic Press, New York. Drabkin D. L. (1946) The crystallographic and optical properties of the hemoglobin of man in comparison with that of other species. J. biol. Chem. 164, 703-723. Ellbaum D., Pandofelli E., and Herskovits T. T. (1974) Denaturation of human and Glycera dibranchiata hemoglobins by the urea and amide classes of denaturants. Biochemistry 13, 1278-1284. Harrington J. P. (1982) Thermostability of salmon hemoglobins. Comp. Biochem. Physiol. 73B, 919-922. Herskovits T. T. and Harrington J. P. (1975) Solution studies on heine proteins: subunit structure, dissociation and unfolding of Lumbricus terrestris hemoglobin by the ureas. Biochemistry 14, 4964-4971. Martin J. P., Bonaventura J., Fyhn H. J., Garlick R. L., and Powers D. A. (1979) Structural and functional studies of hemoglobins isolated from amazon stingrays of the genus Potamotrygon. Comp. Biochem. Physiol. 62A, 131-138. Nandi P. K. and Robinson D. R. (1972a) The effects of salt on the free energy of the peptide group. J. Am. chem. Soc. 94, 1299-1308. Nandi P. K. and Robinson D. R. (1972b)The effects of salt on the free energies of non-polar groups in model peptides. J. Am. chem. Soc. 94, 1308-1315. Smithies O. (1959) An improved procedure for starch-gel electrophoresis: further variations in the serum protein of normal individuals. Biochem. J. 71, 585-587. Tondo C. U., Mendez H. M. and Reischl E. (1980) Thermostability of hemoglobins in homeothermic and nonhomeothermic vertebrates. Comp. Biochem. Physiol. 6611, 151-154.