TIIE JOURKAL cm BIOLOGICAL Cm~rsmr Vol. Z-16, No. 15, Issue of August 10, pp. 4886-4893, 19X

Printed in U.S.A.

Optical Rotatory Dispersion of Hemoglobin and Polypeptides

EFFECT OF HALOTHANE*

(Received for publication, December 24, 1970)

L. H. LAASBERG AND J. HEDLEY-WHYTE

From the Department of Anaesthesia, Harvard Medical Xchool and Beth Israel Hospital, Boston, Massachusetts 02215

SUMMARY

Optical rotatory dispersion of ferri-, oxy-, and deoxyhemo- globin was significantly reduced by commonly used concen- trations of the anesthetic halothane, Z-bromo-Z-chloro-l , 1, l- trifiuoroethane, at physiological pH in the ZZO- to 300~nm region. There was no significant effect of halothane on the conformation of the CY chain whereas the helicity in the p chain of hemoglobin was significantly decreased in the pres- ence of halothane at 23’. These changes were reversed by re-equilibration of the hemoglobin solution with air at 37”. The effect of the d and 1 enantiomers of halothane on oxy- hemoglobin was the same as the racemic mixture. Poly-L- lysine in a helical configuration showed a small reduction in helicity on exposure to halothane, but poly-L-glutamic acid in both a helical and random coil configuration showed no signifi- cant change in configuration on exposure to halothane.

The interaction of anesthetics with proteins has been suggested as the molecular basis of general anesthesia (l), but at present the evidence that anesthetics reversibly affect protein structure is scanty. Gaseous hydrocarbons including cyclopropane have been shown to cause a change in optical rotation at 546 nm of bovine serum albumin and ,&lactoglobulin (2).

The Cotton effect shown by proteins and polypeptides in the far ultraviolet is very sensitive to conformational changes, and the amplitude at the trough of the Cotton effect is considered a measure of the a helix content in macromolecules (3, 4). We aimed to determine whether change in structure of hemoglobin occurred on exposure to halothane, 2.bromo-2-chloro-l , 1, l-tri- fluoroethane, the most commonly used anesthetic. In order to further elucidate the effect of protein chain configuration on optical rotation in the far ultraviolet, o( and /3 chains of hemoglo- bin and polypeptides were also studied in the presence and absence of clinically used concentrations of halothane. The polypeptides we chose, poly-r-lysine and poly-r-glutamic acid, are known to change from a! helical to random coil configuration pitch change in pH (5). The reason we investigated o( and /3

* This work was supported in part by Grant GM-15904 from the IUatiod Institute of General Medical Sciences. A preliminary account of this research was presented to the 3rd European Con- gress of Anesthesiology, Prague, on September 2, 1970.

chains of hemoglobin as well as ferrihemoglobin was to separate out the effects of heme rearrangement from change in helical configuration. The optical rotatory dispersion of oxyhemoglo- bin equilibrated with d and 1 enantiomers of halothane was also investigated to rule out preferential absorption of one or other enantiomer by part of the oxyhemoglobin.

MATERIALS AND METHODS

Human hemoglobin (lot No. V-1304, ?\/lann) was twice crystal- lized, dialyzed against 10 changes of glass-distilled water, and freeze-dried. Electrophoretically the hemoglobin was homo- geneous on cellulose polyacetate strips in a pH 9.1 Tris-EDTA- borate buffer system. Reduced hemoglobin was prepared by exposing the hemoglobin solution to vacuum for 15 min. The evacuated space above the hemoglobin solution was then filled with NB, and 15 mg of NazSz04 in 0.1 ml of II,0 was injected through a rubber cap into the hemoglobin solution. Oxyhemo- globin was obtained by saturating an aliquot of reduced hemo- globin solution with atmospheric air. The pH of oxy and reduced hemoglobin solutions was varied between 6.00 and 7.60. a! and /3 chains from human hemoglobin were prepared with p-hydroxymercuribenzoate for splitting the hemoglobiu molecule (6). The separation of ~11 and /3 chains was carried out in car- boxymethyl and diethylaminoethyl cellulose columus (7) and confirmed by electrophoresis. For solutions of met,hemoglobin the pH was varied between 2.1 and 11.5. The concentrations of protein were determined on a dry weight basis and by spectro- photometric techniques (8). For optical rotatory dispersion measurements, the various hemoglobins, and the o( and /3 chains were dissolved in either 0.02 M KH21’04 buffer or in 0.1 RI NaCl solution. Poly-n-lysinc (molecular weight 15,950, lot No. T5048, hfann) and poly-L-glutamic acid (molecular weight approxi- mately 15,000, lot No. 1455, Nutritional Hiochcmicals) were also used for ORDl measurements in concentrations of 0.02 to 0.04 g/100 ml. The concentrations of poly-L-lysine and poly- L-glutamic acid were determined solely from their dry weights. The pH of poly-L-glutamic acid was adjusted to 4.2 and 11.3, for poly-r&sine to 2.5 and 11.3. The $1 adjustments were done with 0.1 N NaOH and 0.1 N HCl solutions. All pH meas- urements were made with a Radiometer (Copenhagen) model pHY-27 pH meter. Methemoglobin and oxyhemoglobin, a! and 0 chains, buffer and polyamino acid solutions were divided into two portions. A lo-ml aliquot was equilibrated with a known

1 The abbreviation used is: ORD, optical rotatory dispcrsioa.

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Iasur~ of August 10, 1971 L. H. Laasberg ad J. Herlley-Whyte 4887

concentration of halothanc in air, the other with air alone. For deosyhemoglobin solutions Ss (washed with alkaline pyrogallol) was used instead of air. The halothane concentrations used for equilibrat~ion wci-e 8.8 mg/lOO ml (l.OyO v/r), 22 mg/lOO ml (2.5 y;), and 35.2 mg/lOO ml (4.0 yO). Equilibration was carried out at 23 and 37” for 4 hours in a trtnp”ature-controlled ( ~0.05”) wv:tt,tr bath.

To test the reversibility of the coiiformational change, the samples, which had been exposed to halothane, were re-equili- bratetl with room air at 23 and 37”. I\fter determination of halothane concentration, the gas phase was expelled and air was int,roduced into the syringe. The re-equilibration of hemoglobin solution with air was repeated eight times for 15 min and any residual halothane was determined at the end of each 15-min period (9). The ORD of the re-equilibrated hemoglobin solu- tions was then determined at 23”. Calibrated, leak-proof ground glass svringes served as equilibration containers. The syringes were equipped with three-way stopcocks for transferring the samples. The syringes were filled with halothane vapor and the halothane concentration determined prior to introduction of the liquid sample int,o the syringe. After 4 hours of equilibration the halot’hane concentration in the gas phase was determined again by gas chromatography. ,4 Hewlett-Packard Research gas rhromatograph model 5750 was used for the halothane determinations. The instrument was equipped with dual B-foot columns, which were packed lvith 3.8L:O (w/w) of silicone fluid XF-1125 on acid-washed and silanized Chromosorb W (80 to 100 mesh). A dual hydrogen flame ionization detector was used for quantitation of the halothane. The temperatures of injec- tion lrort, CO~UIIIJIS, and detector were 102, 50, and 150”, respec- tively, aid were kept constant. Reproducibility of the halo- thane determination was ~2% and the responses of the drtect’or t,o concentration of halothane were linear (9).

111 order to transfer the samples without either exposure to air or loss of halothane the stoppers of the ORD cells were equipped with controllable inlets and outlets. The gaseous phase was first introduced into the ORD cell to flush out air and then the liquid sample was introduced. For ORD determinations of reduced hemoglobin solutions the cell was flushed with Nz prior to filling the cell.

In addition to the racemic mixture of pure halothane, lot No. 04210.13 of :I 75:25 ratio d:l enaritiomrr [a]: + 0.7”, aild lot No. 04210.(’ of a 30:70 ratio d:l enantiomer [cz]E5 - 0.6”, was prepared by Dow, LIidland, Jlichigan (10). The halothane in these two samples was 97 and 96y0 pure by gasliquid chromatog- raphy: the chief impurit~ics were CHCK’F~ and CHBr2CF3. These einuitiomeric mixtures (concentration 35.2 mg in 100 ml of air) were equilibrated with oxyhemoglobin solutions, 0.02 M KIIJ’O,, and n-hesane as described for the racemic halothanr. The 0R.D measurements were made with a Cary 60 spectropola- rimeter. -1utomatic slit and sensitivity control were used to provide R spectral slit width of 10 A at 300 nm. The scan speed was kept at 1 x 12. Observed rotations (sobs) were measured at wave lengths from 220 to 300 nm. From these measurements specific rotation’s [&lx at the given wave lengths were calculated from the equation

(1)

where d = the length of the light pat’h through the sample (in decimeters) and c = concentration of protein or polypeptide in

grams per 100 ml of solution. The reduced mean residue ro- tation [vL’]~ was calculated with a I’DI’- computer from

Im’lx =

where JfAW is the mean residue weight of amino acids in the protein and polypeptide molecules and n is the refractive index of solvent. The refractive index values at the various wave lengths were extrapolated from tables (11). Pit 23” the largest difference in refractive index between solvents and water was 0.003 as measured at the sodium n-line with an Abbe’s refractometer. After squaring the n values for water in formula (a), the magni- tude of the difference between water and solvent has a negligible effect on Im’]X.

Optical rotatory dispersion measurements from 300 to 220 nm of the solvent solutions, (0.02 M KHJ’O., or 0.1 M NaCl) were always run on the solvent alone or with halothane before meas- urements on protein or polypeptide solutions. Throughout the study two halothane spectra of proteins or polypeptides always followed two control (unequilibrated with halothane) spectra of the same batch of protein or polypeptide. The optical rotatory dispersion from 300 to 220 nm of the solvent solution alone or

5000

0 tm’ 1~

- 5000

- 10000

.=2.1

*=11.5

0=7.47

200 240 280

X[mpl

FIG. 1. Optical rotatory dispersion for ferrihemoglobin in aquc- ons solution at different pH values. Mean values are shown of six measurements at pH 2.1, of 12 at 7.47, and of aeven at 11.5. Concentrations of ferrihemoglobin ranged from 0.016 to 0.140 g/100 ml; the length of the light path in the optical cell was either lcmorlmm. No significant difference in readings resulted from the use of different concentrations or different cells. Standard errors at [vL’]~~~ were ~t67 for pH 2.1, f136 for pH 7.47, and &15Y for pH 11.5. Changes in [m’]~ from 250 to 230 nm due to pH are highly significant (p < 0.001) between pH 7.47 and 2.1 and pH 11.5 and 7.47. (See the text, Footnote 3). The difference in [m’]h is significant (p < 0.001) between the pH 2.1 and 11.5 groups at 233 nm only.

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4888 ORD of Hemoglobin with Halothane Vol. 246, No. 15

Compound PH Control Halothane PC

Ferrihemoglobina 2.13 -5243 k 67 -4717 * 157 co.02

Ferrihemoglobina 7.45 -9662 + 93 -8462 t 74 <O.OOl

Ferrihemoglobina 11.50 -6132 + 159 -6104 t 155 >0.66

Oxyhemoglobina 7.45 -9028 iI 35 -8512 + 81 <O.OOl

Deoxyhemoglobina 7.45 -9662 + 50 -9009 t 63 <O.OOl

Alpha chain (H?YIB)~ 7.46 -7978 rt. 48 -7884 t 19 >0.07

Beta chain (HMB)a 7.44 -8443 k 138 -7555 rf: 150 <O.OOl

Poly-L-lysineb 11.30 -11648 rf: 129 -11032 ?Y 212 <O.OOl

Poly-L-glutamic acidb 4.20 -11871 I!I 270 -12166 ?I 399 >0.23

Oxyhemoglobina 7.40 -8910 k 51 -8240 AI 108 (d) co. 001

Oxyhemoglobina 7.40 -8910 t 51 -8307 +- 43 (e) co. 001

TABLE I

Mean [m']m f S.E. at 23"

a Hemoglobin and its subunits were dissolved in 0.02 M KH PO 2 4'

b Poly-L-lysine and poly-L-glutamic acid were dissolved in 0.1 M NaCl.

c P-values were derived from Student's paired variate analysis.

d 75/25 ratio d/l enantiomer. --

E 30/70 ratio d/l enantiomer. -’ -

0

Cm’lX

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-IOOOC

2

0 Control l Halothane

I I I I I

0 240 280

XCmpl

FIG. 2. Optical rotatory dispersion for ferrihemoglobin in aqueous solution at pH 7.45. Mean values are shown of 18 meas- urements and for another 18 measurements after equilibration with halothane. Three concentrations of halothane were used, 1, 2.5, and 4.0y0 v/v, but neither the concentration of halothane nor the concentration of ferrihemoglobin (range 0.016 to 0.14 g/100 ml) had any significant effect on [m’]h so each set of 18 measure- monts was meaned. The S.E. for [m’233] was f93 without halo- thane, and ~74 after equilibration with halothane. Changes due to halothane were significant (p < 0.001) from [rn’]2ao to [wL’]z~~.

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Issue of August 10, 1971 L. H. Laasberg and J. Hedley-Whyte 4889

-5000

- I 0000

2 .01

I

---

0 Control l Holothane

I I 0 240 280

X tmpl

Fro. 3. Optical rotatory dispersion for deoxyhemoglobin in aqueous solutions at pH 7.45. Mean values are shown of 12 meas- urements and for another 12 measurements made after equilibra- tion with halothane. Three concentrations of halothane were used, 1,2.5, and 4.0% v/v, but neither the concentration of halo- thane nor the concentration of deoxyhemoglobin (range 0.08 to 0.20 g/100 ml) had any significant effect on [m’]h so each set of 12 measurements was meaned. The S.E. for [wz’]z~~ was f50 with- out halothane and G3 after equilibration with halothane. Changes due to halothane were significant (p < 0.001) at 233 and 235 run.

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2

OControl l Holothane

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Xtmp I

FIG. 4. Optical rotatory dispersion for oxyhemoglobin in aque- ous solutions at pH 7.45. Mean values are shown of 12 measure- ments and for another 12 measurements made after equilibration with halothane. Three concentrations of halothane were used, 1,2.5, and 47, v/v, but neither the concentration of halothane nor the concentration of oxyhemoglobin (range 0.013 to 0.15 g/100 ml) had any significant effect on [m’]x so each set of 12 measurements was meaned. The S.E. for [wL’]x~ was f35 without halothane and ~81 with halothane. Changes due to halothane were significant (p < 0.001) at 233 nm and also at 235 nm (p < 0.005).

5000

FIG. 5. Optical rotatory dispersion for oxyhemoglobin in aque- ous solutions at pH 7.40; the effect, of d and 1 halothane. Mean values are shown of seven control measurements and for another two sets of seven measurements made after equilibration with 35.2 mg/lOO ml of air of either 75:25 or 30:70 ratio of d:l enan- tiomers of halothane (10). The concentration of oxyhemoglobin ranged from 0.061 to 0.064 g/100 ml of 0.02 M KHzP04. [WZ’]LW and [vL’]~,~ after equilibration with the d and I halothane were signifi- cantly different from control (p < 0.001, Table 1). There was no signifidant difference (p > 0.10) between the ORD of the oxyhemo- globin equilibrated with 30: 70 and 75: 25 d:Z mixtures of halothane enantiomers (35.2 mg/lOO ml of air). The ORD of oxyhemoglobin equilibrakd with racemic halothane (Fig. 4) was not significantly difIerent (p > 0.05) from the ORD after equilibration with either the 40: 70 or 75: 25 d:l mixtures of halothane enantiomers.

0

IrnllX

- 5000

-too00

2

,-

:ot

0 Control A 75/25 d/l Holothanc A 30/70 d/l Halothonc

I I 1 240 280

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4s90 ORD of Hemoglobin with Halothane

TABLE II Mean di$erence between [m’]z33 and [m’]luo + S.E. at S’S”

Vol. 246, Ko. 15

Compound

Ferrihemoglobina

Oxyhemoglobina

Deoxyhemoglobina

Alpha chain (HMB)a

Beta chain (HMB)a

Oxyhemoglobina

Oxyhemoglobina

Poly-L-lysineb

Poly-L-glutamic acidb

PH

7.45 -8952 !I 87

7.45 -8768 k 48

7.45 -9005 t 107

7.46 -7794 t 48

7.44 -7703 t 106

7.40 -8691 + 81

7.40 -8691 + 81

11.30 -10998 + 152

4.20 -11139 k 374

. ([m 1233-Im’1300)‘k

Control (C) Halothane (H)

Difference C-H

-8080 + 159 -872

-7928 L 198 (c) -840

-8516 k 91 -489

-7637 k 25 -157

-6773 + 137 -930

-7990 + 139 (d) -701

-8097 _+ 64 (e) -594

-10356 t 219 -642

-10976 ?- 415 -163

P

<O.OOl

<0.002

<O.OOl

0.014

<O.OOl

<O.OOl

<O.OOl

<O.OOl

0.68

;kThe difference between control and halothane groups is significant (P < 0.002), except for a-chain and poly-L-glutamic acid.

a Hemoglobin and its subunits were dissolved in 0.02 M KH2P04.

b Poly-L-lysine and poly-L-glutamic acid were dissolved in 0.1 M NaCl.

c Racemic halo thane .

d 75125 ratio d/l halothane. --

e 30/70 ratio d/l halothane. --

with halothane was then repeated. A comparison of the solvent spectra was used to determine base-line drift. We also deter- mined the ORD of racemic halothane at concentrations of 1900, 95, and 47 mg/lOO ml of n-hexane and the 75:25 d:l and 30:70 d :I enantiomeric mixtures at concentrations of 1840, 184, and 36.8 mg/lOO ml of n-hexane. For these measurements with n-hexane we used an 0.2-cm ORD cell.

RESULTS

In Fig. 1 ORD spectra for ferrihemoglobin at pH 2.1, 7.47, and 11.5 are shown; the conformation of hemoglobin is $1 sensitive (Table I). The reduced mean residue rotation for ferri-, oxy-, deosghemoglobin, and /3 chains of hemoglobin were significantly reduced at 233 nm, when these protein solutions were equilibra- ted with either 8.8, 22, or 35.2 mg of halothane per 100 ml, over- all mean 3.2% v/v (27.9 mg in 100 ml of air or nitrogen).* The

2 We used these three concentrations of halothane in the gaseous phase during equilibration of the protein solutions but there were ILL) significant differences between ORL> changes caused by the thrc,t, halothane concentrations. Therefore t,he OID values ob- taincd after equilibration with halothanc have been meaned in Figs. 2 to 4 and 6 to 8. The predominantly cl or I enantiomers were only used at a concentration of 35.2 mg/lOO ml air. All references to halothane are to racemic halothane unless otherwise specified.

mean residue rotation [m’],,, of ferrihemoglobin in the presence of halothane at pH 7.45 is 11% different from that of control ferri- hemoglobin solution (Fig. 2). Halothane added to NaCl and KH2P04 solutions did not cause any alteration in ORD spectra. Halothane raised the mean [~~‘I233 value of reduced hemoglobin 7.2% from -9662 to -9009 (Fig. 3). The [w& of oxyhemo- globin was raised 6.1% on exposure to halothane (Fig. 4). The 75:25 ratio d:l enantiomers of halothane raised [V&S of oxy- hemoglobin 8.1%; the corresponding rise with the 30:70 d:Z

ratio was 7.2% (Fig. 5). There is no statistical difference be- tween the effects of the two isomeric mixtures on the ORD of oxyhemoglobin, nor do these effects caused by the two enantio- mers differ significantly from the 6.1% change found with the racemic mixture. The changes of [m’]233 caused by halothane in ferri-, deoxy-, and oxyhemoglobin solutions are significant at physiological pH (p < O.OOl), by paired variate analysis (Table I). These changes are also seen when the optical rotatory dis- persion change is expressed as [wL’]~~~ - [m’]300 (Table II).

To evaluate whether the changes occurred only in the intact hemoglobin molecule the effect of halothane upon cx and fl chains was determined. Halothane raised the [m’1233 value of fl chains by 10.5y0 (Fig. 6), whereas the effect of halothane upon the a! chain was insignificant (p < 0.05) (Fig. 7). Halothane did not change the pH of t,he hemoglobin solutions.

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Issue of August 10, 1971 L. II. Laasberg and J. Hetdley-Whyte 4891

0

[rn’lA

r

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l Haiothone

J I I --- 200 240 280

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FIG. (i. Optical rotatory dispersion for /3 chain (p-hydroxy- merc~~ribellzoate) in aqueolls solutions at pH 7.44. Mean values are shown of 10 measurements and for another 10 measurements made after equilibration with halothane. The concentrations of halothalle used were 2.5 and 4yL v/v. The concentration of p chain in the aaueous solution ranged from O.OB to 0.08 g/100 ml. The solut,iorls of p chain were equilibrated with air andair-halo- thane mixtures at, 23”. The S.E. for [m’]pa~ was ~138 without halothane and &I50 with halothane. Changes due to halothane were significant (p < 0.001) at 235 and 233 nm. The p value for differences of [n~‘]h between 260 nm and 235 nm ranged from 0.04 to 0.008.

Poly-L-glutamic acid and poly-L-lysine in disordered conforma- tion at pH 11.3 and 2.5, respectively, did not exhibit any signifi- cant change of their ORL) pattern when exposed to halothane. When these polypeptides were in LY helical configuration there was

a decrease in the negativit’y of [rn’lzSS of poly-L-lysine from - 11648 to -11032 at pII 11.3 (p < O.OOl), but the [m’JzS3 of poly-L-glu- tamic acid changed only from -11871 to -12166 at pH 4.2 on equilibration with halothane (p > 0.23, Table I).

The effect of halot,hane on the ORD spectra was not reversed in hemoglobin solutions which were repeatedly re-equilibrated with room air at 23”, regardless of the low (3.2 mg/lOO ml) re- sidual concentration of halothane in t,he sample. By contrast after the re-equilibration procedure had been carried out at 37”, the [m’],,, was insignificantly different from the preanesthetic

[TYL’]~~~ (Fig. 8).3 The halothane concentrations in the gas phase and liquid phase’ after re-equilibration at 37” were 0.08 and 0.07 mg per 100 ml, respectively, and thus approximately one-fortieth

3 For detailed t,ables of all ORD measurements and levels of significance order NAPS Document 01538 from ASIS National Auxiliary Publications Service, y0 CCM Information Corporation, 909 Third Avenue, New York, New York 10022, remitting $2.00 for microfilm or $5.00 for photocopies.

Im’l x

0 Control

l Halothane

-1

I I 200 240 280

ArmpI

FIG. 7. The mean values of optical rotatory dispersion for 01 chains (p-hydroxymercuribenzoate) in aqueous solutions at pH 7.4G are shown for 11 measurements and for another 11 measure- ments made after equilibration with halothane. The concentra- t,ions of halothane used were 2.5 and4.0% v/v. The concentrat,ion of 01 chain in t,he solution ranged from 0.105 to 0.300 g/100 ml. The solutions of a chain were equilibrated with air and air-halo- thane mixtures at 23”. The [m’] value was not significantly affected either by the halothane concentration or by the concen- tration of protein. The S.E. for [m’]233 was 148 without halo- thane and &19 with halothane. Changes due to the presence of halothane were not statistically significant between 300 and 220 nm.

of the residual concentration after re-equilibration with room air at 23”.

The nonreversal of the [m’lza3 value after re-equilibration at 23” is probably caused by the high boiling point (+50.2”) of halo- thanc and the slow release of the compound from the hemoglobin solution. Fig. 9 shows typical spectra of ferrihemoglobin at pH 7.40: halothane can be seen to raise [m’]233 13.5%, an 11% change was observed at pH 7.45 (Fig. 2, Table I). When the optical ro- tatory dispersion of the solvent systems was compared before and after each set of four spectra the mean change in base-lines at 233 nm was 1.1 millidegrees, less than 1% of the observed rotation values. The complete details of variation in base-line are avail- able on request.3 The largest change in base-line was 3 millide- grees.

Neither 1900 mg of racemic halothane nor the 75:25 nor the 30: 70 d:l mixture of its enantiomers dissolved in 100 ml of n-hex- ane caused any discernible ORD from 600 to 250 nm; the corre- sponding [rn’]~~ values were +5, +12, and -9 millidegrees; for [VZ’]SZ~ +21, S.6, and -6 millidegrees: below 220 nm the masi- mum shifts from base-line were -35, +160, and -108 millide- grees, respectively. Of these three halotha.ne solutions, 184 mg

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4892 ORD of Hemoglobin with Halothane Vol. 246, No. 15

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0 -- i- [ml 1 x

/

OControl n Recovery

Army1

FIG. 8. Optical rotatory dispersion curves are shown for ferri- hemoglobin at pH 7.4; 0 control; W, exposed to halothane 22 mg/lOO ml at 37” for 4 hours and re-equilibrated eight times with room air each time for 15 min at the same temperature. The hemoglobin concentration was 0.016 e/100 ml and the final residual halothane concentration in the-sample was 0.07 mg/lOO ml. There is no significant difference between the [m’]~ of control samples and those after re-equilibration. This indicates that changes in hemoglobin conformation due to halothane are re- versible.

FIG. 9. Two typical optical rotatory dispersion spectra of ferri- hemoglobin (0.082 g/100 ml) are shown after eauilibration with 35.2 mg of halotha~e/lOO ml of air at pH 7.4 (&rves 3 and 4). Two spectra of the same batch of ferrihemoglobin solution that were not equilibrated with halothane are also shown (Cures 1 and 2). Throughout the study, two halothane spectra always followed two control (unequilibrated with halothane) spectra. The horizontal tracing (approximately over 0.5) represents four patterns obtained with buffer solutions. Panel 1 shows KHzPO, (0.02 M) without halothane, Panels 2 and S, show KHgPOd after rquilibration with 35.2 mg of halothane/lOO ml of air, and Panel 4, like 1, shows KH,PO* without halothane. All eight recordings were made from 300 t’o 200 nm and were run consecutively. Full scale (shown as 0.0 to 1.0) represents 0.4”. An0.2-cm cell was used. The horizontal line at 0.057 has been emphasized to demonstrate the difference between cont.rol curves and halothane curves.

-I

in 100 ml of n-hexane did not cause more than f2 millidegrees rotation for the d isomer and -2 millidegrees for the I isomer from 600 to 220 nm; below 220 nm the maximum shifts were +30 and -9 millidegrees. Of the halothane mixtures, 47 mg/ 100 ml of n-hexane caused no observable optical rotation at wave lengths from 400 to 215 nm. The maximum concentration of halothane in all our protein, polypeptide or buffer samples was approximately 19.5 mg/lOO ml. We thus conclude the changes in rotation observed with hemoglobin solutions were not caused by possible interference of the halothane per se.

DISCUSSION

When ferri-, oxy-, and deoxyhemoglobin solutions are exposed to the most commonly used concentrations of the anesthetic halo- thane their mean residue rotations are significantly raised. The mean residue rotation of the a! chain of hemoglobin is almost unaffected by halothane; the change occurs in the fl chain. Does halothane have any effect on hemoglobin structure similar to li- gands such as CO or 02? The effect of ligands upon the con- formation of myoglobin as determined by ORD is insignificant (12-14). Carbon monoxide has a negligible effect on the con- formation of cy and /3 chains of hemoglobin (15) ; these studies suggest that such ligand effect, is not primarily responsible for the changes we observed when ferri-, deoxy-, and oxyhemoglobin solutions were equilibrated with halothane. Rearrangements of the heme groups are most unlikely as the cause of the ORD changes we observed, since the changes in [&I233 of @ chains and intact hemoglobin are comparable. This hypothesis is supported by reports that halothane does not change the oxy- hemoglobin dissociation curve (16).

cr and fi chains of hemoglobin can have different reactivities with azides and thiocyanates (17), which suggests that interac- tions of the a: andp chainswith other compounds may also proceed independently. It is unlikely that the changes observed in the ORD characteristics of oxy-, deoxy-, and methemoglobin on exposure to halothane were influenced by the spectral shifts of hemoglobin which occur in the 400 to 430-nm range with differ- ent ligands (17), because oxy-, deoxy-, and metmyoglobin have the same [m’]233 value regardless of the large differences in the Soret region spectra (12-14). Moreover, the [m’],,, values for oxy- and carboxyhemoglobin are not significantly different de- spite large differences in Soret region spectra (15).

The finding that there was no significant change in the ORD spectra of cx chains of hemoglobin on exposure to halothane sug- gests that changes occur in the secondary structure of the p chain of hemoglobin. In the complete hemoglobin molecule halothane may also affect inter- and intrachain interactions, but changes in the ORD patterns of globular proteins cannot yet be fully corre- lated with specific changes in their secondary, tertiary, and qua- ternary structure.

We determined the optical rotatory dispersion of halothane from 300 to 220 nm but the insignificant changes obtained do not account for the rotational changes of hemoglobin and poly-L-ly- sine on exposure to halothane. In the highest concentrations that we used for equilibration with proteins, 35.2 mg/lOO ml of gas (4.0%), neither racemic halothane nor the d nor 1 isomers caused change in the ORD pattern of NaCl, KH~POI, hexane, or poly-L-glutamic acid solutions. Of course it is just possible that London interactions between halothane and the fl chain of hemo- globin or the Q: helix of poly-I&sine, cause the halothane-protein

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Issue of August 10, 1971 L. H. Laasberg and J. Hedley-Whyte 4893

(or polypeptide) complex to have a slightly different ORD spec- trum from the protein or polypeptide without halothane. The ORD findings with 2-chloroethanol, an optically inactive com- pound do not support this poasibilit,y (18). Our results with 75 :25 d: 1 and 30 : 70 d: 1 halothane when equilibrated with oxyhe- moglobin do not suggest that d and 1 enantiomers interact differ- ently with hemoglobin (p > 0.1 at 235 to 233 nm, Fig. 5). How- ever, when pure d- and I-halothane becomes available the differ- ences between their interactions with hemoglobin and its subunits should be clearly identified.

The changes in [m’],,, that we observed at 23” probably apply at temperatures up to approximately 50” since variation of tem- perature between 0 and 50” has been fomld to have no effect on [v&~ of hemoglobin solutions (18). It has been postulated (18) that interaction of hemoglobin with 2-chloroethanol at low con- centrations (up to 20% v/v) in aqueous solutions is followed by marked conformational changes primarily related to a decrease in a! helical content. The helical content remained low with increasing 2-chloroethanol concentration except when 2-chloro- ethanol was increased to 90% v/v; then the helical content of hemoglobin, always estimated from changes in [v&~ and by the Moffitt-Yang equation, returned to its original value. These findings as well as ours show that small halogenated organic molecules when present in low concentrations in aqueous hemo- globin solutions at physiological pH may cause conformational changes in the secondary structure of hemoglobin and its sub- uuits. These changes are ahnost certainly not attributable to changes in the ORD of either 2-chloroethanol or halothane per

se. Our finding that a predominantly dextrorotatory mixture had the same effect on the ORD of oxyhemoglobin as a pre- dominantly levorotatory mixture supports this conclusion. The small changes we found in the [m’]?33 values of poly-L-lysine solutions suggest that the amino acid composition of the poly- peptide chain plays a role in the interaction of halothane with macromolecules.

The significant reduction in [~&a suggests that the 01 helical content of the 0 chains of hemoglobin, and poly-L-lysine are de- creased by concentrations of halothane that are often used for general anesthesia. This finding is of interest in view of the fact that microtubules which are thought to have an Q! helical configu- ration are known to reversibly disarray on exposure to general anesthetics (19). It will be interesting t,o see if these observa-

tions lead to an increased understanding of the mechanisms whereby general anesthetics exert their actions.

Aclcnowledgments-We thank Dr. Elizabeth Simons for intro- ducing us to the techniques of ORD and for fruitful discussions. We wish to thank Professor Ellis Cohen of Stanford University for the 75 : 25 d: 1 and 30: 70 d: 1 enantiomer mixtures of halothane. These mixtures were donated to Professor Cohen by Dr. F. Y. Edamura (10). Pure halothane was donated by Ayerst Inc., New York, New York. We also wish to thank Mrs. C. Becker and Mrs. S. Molea for help in the preparation of manuscript and Mr. C. Becker for help with the computer analysis.

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L. H. Laasberg and J. Hedley-WhyteHALOTHANE

Optical Rotatory Dispersion of Hemoglobin and Polypeptides: EFFECT OF

1971, 246:4886-4893.J. Biol. Chem. 

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