Membrane biochemical indicator of Nox exposure

Journal of Membrane Science, 5 (1979) 265-273 o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

MEMBRANE

BIOCHEMICAL

INDICATOR

265

OF NO, EXPOSURE

HIDEAKI MATSUOKA, MASUO AIZAWA and SHUICHI SUZUKI Research Laboratory of Resources Utilization, Tokyo Institute of Technology, Midori-ku, Yokohama 227 (Japan)

Nagatsuta,

(Received August 30, 1978; accepted in revised form March 5, 1979)

Summary For the evaluation of NO, exposure by a biochemical indicator, .a hemoglobin membrane was prepared by immobilizing hemoglobin into agarose gel attached to the surface of a cellulose acetate membrane. The membrane was fixed in a spectrophotometric cell which was designed to enable the membrane-bound hemoglobin to maintain a constant water content and to react with gaseous NO,. A drastic change in the absorption spectrum of the hemoglobin membrane resulted from NO, exposure, which was closely related to the interconversion of oxyhemoglobin to methemoglobin. NO, exposure caused the mole-fraction of methemoglobin to increase. The hemoglobin membrane appears to be a useful new biochemical indicator of NO, exposure.

Introduction Increasing attention is being paid, in environmental evaluation, to the use of biochemical indicators, including cultured tissues, cells, organelles, enzymes and other biologically active substances [ 11. These indicators often provide more specific and detailed information than biological indicators, which involve the use of living animals and plants [2]. In this investigation we took hemoglobin as an example of a biochemical indicator for the evaluation of air pollutants, because it is very reactive with gaseous molecules such as OZ, CO, and NO,. Although considerable work has been done with blood, little has been done to assess the value of hemoglobin alone as a potential biochemical indicator. Specific physiological damage has been attributed to the oxidants present in the atmosphere, especially nitrogen oxides and ozone. Of the nitrogen oxides, NO2 is a free-radical molecule and one of the most hazardous species [3]. Both in viuo and in vitro studies aimed at elucidating the effects of NO, have shown that NO, may interact with hemoglobin derivatives, which include oxyhemoglobin (HbOz), deoxyhemoglobin (Hb), and methemoglobin (MetHb) [ 3-71. The physiological ratio of each derivative is found to change with exposure to NO,, the ratio of MetHb to HbO? content increasing in the process [3,5]. These observations suggest that the effects of NOz could be

266

evaluated according

by monitoring the MetHb level. MetHb is produced to the following reaction:

from HbOl

HbOz NO?_ MetHb Due to its availability and simplicity, we employed a spectrophotometric method for the determination of the MetHb ratio. In order to develop a simple biochemical indicator, hemoglobin was immobilized in a membrane form, The hemoglobin membrane was incorporated into a spectrophotometric cell which enabled the hemoglobin to react directly with gaseous NO2 at constant water content, similar to the state of mucous biomembranes. Although a great number of immobilization techniques have been developed recently, few investigations have been made in which immobilized bioactive substances react with a gas. The newly developed device should be practical for direct exposure to gas. The performance of the hemoglobin membrane and its feasibility as a biochemical indicator of NO1 is described in this paper. Methods

and Materials

1. Preparation of a hemoglobin membrane Hemoglobin was prepared from freshly drawn bovine blood supplied by a slaughterhouse. The plasma was removed by washing with physiological saline solution and the erythrocytes were hemolyzed to yield hemoglobin according to Drabkin’s method [ 81. Fifteen milligrams of agarose (for electrophoresis, Nakarai Co., Ltd., Kyoto) were dissolved in 1.0 ml of phosphate buffer at pH = 7.0, in the temperature range of 80-9O”C. After cooling the solution to 45-5O”C, an appropriate concentration of hemoglobin solution was homogeneously mixed with the agarose solution in a volume ratio of 1:2. One hundred ~1 of the resultant solution were immediately cast on a cellulose acetate membrane (1 cm2 and 50-pm thick). Gelation took place on this membrane at about 25°C to yield a l.O-mm hemoglobin-agarose layer, or “hemoglobin membrane”. 2. Construction of a photometric cell coupled with the hemoglobin membrane A photometric cell consisting of the hemoglobin membrane and a water compartment was assembled as shown in Fig.1. One interface of the hemoglobin membrane was exposed to the atmosphere while the opposite interface was in contact with water through the cellulose acetate membrane. The water content of the hemoglobin membrane was constant since evaporation from the membrane surface was balanced by diffusion from the water compartment through the cellulose acetate membrane. The thickness of the hemoglobin membrane ranged from 200 to 250 pm at steady state. The hemoglobin content of the membrane was expressed in nanomoles/cm’ membrane.

267

Fig.1. Spectrophotometric

cell for a hemoglobin membrane.

3. Preparation and determination of NO, Nitrogen oxides (NO, ) were prepared by the thermal lead nitrate near 180°C: Pb(N03)2

decomposition

of

--f PbO + 2N02 + l/2 O2

It was shown by IR spectroscopy that the predominant species in NO, was NOz. Thus, the concentration of NO, was approximated by that of dissolved NOz, which was determined by Saltzman’s method [9]. Saltzman’s method is based on the calorimetric determination of a diazo dye produced by the reaction of NOz with N-( 1-naphthyl)-ethylenediamine and sulpnanilic acid. Saltzman’s coefficient, defined as the NO-JN02 ratio, in a solution, was estimated to be 0.72. NO, gas to be assayed was introduced from an NO, reservoir, through a glass capillary, into a sample of Saltzman’s solution containing 0.2 mJ4 N-(1-naphthyl)-ethylenediamine, 30 m.M sulphanilic acid, and 0.88 M acetic acid. After incubation at 30°C for 30 min, an aliquot of the resulting solution was analyzed spectrophotometrically at 545 nm. 4. Exposure of the hemoglobin membrane to NO, The hemoglobin membrane was exposed to NO, in the continuous NO, flow system depicted in Fig.2. NO, test gas was extracted from the reservoir with the adjusting and sampling syringes, and was then diluted with dried air in the diluting vessel. A peristaltic pump fed the sample gas into the

268 Adjusting

syrjnge

Saltzman’s

Fig.2. Schematic diagram of the continuous flow system for NO, exposure: For the exposure of a hemoglobin membrane to NO,, the glass capillary was replaced by a small glass tube (6-mm diameter). The gas flow rate was controlled at 10 ml/min with a peristaltic pump.

reaction vessel. After 30 minutes exposere to NO,, the hemoglobin was taken out for spectrophotometric determination.

membrane

Results

1. Spectrophotometric

characteristics of a hemoglobin solution

Oxyhemoglobin (HbO*) has absorbance peaks at 542 and 577 nm, while methemoglobin (MetHb) absorbs at 500 and 630 nm [4]. Although a hemoglobin solution contains various hemoglobin derivatives, its major components in the present preparations may be considered to be HbOz and MetHb. Hence, the characteristic state of the hemoglobin solution used is given by the mole fractions of HbOz and MetHb. In this investigation, the MetHb mole fraction, x, was approximated by [ MetHb]

jij= [ HbOJ

+ [ MetHb]

(1)

The concentrations of HbOz and MetHb, [ HbOJ and [ MetHb] , were calculated from the absorbances at 542 and 500 nm according to eqn. (2): f gf4t02

MetHb ES42

HbO2 es00

MetHb Go0

(2)

269

where 0Dsd2 and ODsO,, are the optical ively, and e is the millimolar extinction mJo2

14.4;

EF;Oz

E542

=

eg;m

= 6.60; ey;;m

[HbO,] = 0.0936

densities at 542 and 500 nm, respectcoefficient (m.W’ cm-‘) [4].

= 5.05; = 9.04.

0DsG2 - 0.0683

ODsoo (3)

[MetHb]

= -0.0523

OD542 + 0.1488

OD500

Gaseous NO, was introduced through a capillary into a 50 pM hemoglobin solution at 30°C for a period of 30 min. Figure 3 shows the absorption spectrum of the hemoglobin solution before and after exposure to NO,. The concentration of NO, was found to be 14.4 pM by Saltzman’s method. The absorbances at 542 and 577 nm decreased whereas those at 500 and 630 nm increased. Isosbestic points appeared at 523 and 590 nm. The spectroscopic analysis suggests that the MetHh mole fraction of the solution increased from 0.54 to 0.71 due to exposure to NO,.

0.

O.D.

0.

0.

460

500

540

Wavelength

580

620

(nm)

Fig.3. Spectral change of a hemoglobin solution due to NO, exposure: [Hbt] = 50 &M, phosphate buffer (pH 7.0, I = O.l), [NO; ] = 14.4 PM; - - - -before NO, exposure (Xi = 0.54)7 after NO, exposure (H,, = 0.71).

270

To regenerate HbOz, 10 ~1 of 5% Na,Sz04 were added to 10 ml of the hemoglobin solution exposed to NO,. After incubation at 30°C for 30 min, the solution was oxygenated by aeration for 30 min. The absorption spectrum indicated that HbOz was regenerated by the dithionite. When the solution was again exposed to NO,, the absorbances at 500 and 630 nm increased in a manner similar to that described above. These results suggest that the hemoglobin--NO, reaction is primarily an oxidation of HbOz to MetHb. 2. Response of the hemoglobin membrane to NO, A hemoglobin membrane, containing 80-140 nanomoles of hemoglobin per square centimeter of membrane, was fixed to the spectrophotometric cell. The inner compartment was filled with water in order to keep the water content of the hemoglobin membrane constant. After equilibration, the absorption spectrum of the hemoglobin membrane was measured. The peaks attributed to HbOz and MetHb appeared in the same regions as those of a hemoglobin solution. It is noted that no appreciable change resulted from the immobilization of hemoglobin. The spectrum indicated that the hemoglobin membrane contained 0.6-0.7 mole fraction of MetHb. The hemoglobin membrane was exposed to gas containing 2.9 ppm NO,, 2.0

1.6

1.2 O.D.

460

500

540

Wavelength

580

620

660

(nm)

Fig.4. Spectral change of a hemoglobin membrane due to NO, exposure: [Hb,] = 140 (nanomoles/cm2 membrane), [NO,] = 2.9 ppm, LO ml/min, 30 min; - - - before NO, exposure (Xi = 0.666) after NO, exposure (X,, = 0.760).

271

which had a flow rate of 10 ml/min for 30 min. The absorption spectrum before and after exposure to NO, is shown in Fig.4. Exposure to NO, caused the spectrum of the hemoglobin membrane to change in a manner similar to that of a hemoglobin solution. The absorbances at 540-542 and 577 nm decreased whereas those at 500 and 630 nm increased. The MetHb mole fraction increased from 0.686 to 0.760. 3. NO, monitoring by means of the hemoglobin membrane The autoxidation of the immobilized hemoglobin due to exposure to air was examined. The hemoglobin membrane was exposed to air for 80 min, to NO, gas for 30 min, and then to air at a flow rate of 10 ml/min. The mole fraction of MetHb changed as is shown in Fig.5. A slight increase in MetHb resulted from air exposure. However, the autoxidation may have been negligible as compared to the oxidation by NO,. To monitor gaseous NO,, the hemoglobin membrane was exposed to NO, at a constant flow rate of 10 ml/min for a period of 30 min. The concentration of NO, gas was varied. Each measurement was performed with a different membrane. The MetHb mole fraction was determined before and after exposure to NO,. Fig.6 shows the relationship between - -.the ratio {Ax/(l-xi)} /[Hb,] and the NO, concentration, where AX, Xi, and [Hbt] are the increase in MetHb mole fraction, the initial MetHb mole fraction, and the total hemoglobin concentration, respectively. The concentration of NO, was determined by Saltzman’s method, as described above. The ratio {AX/(l-xi) } /[Hbt] increased linearly with the concentration of NO, in the range of O-10 ppm.

0.6

0.51

-

I -60

I

I

I

1

-30

0

30

60

Time

(min)

Fig.6. Exposure of a hemoglobin membrane to air and NO,: [Hb,] = 140 (nanomoles/ air = 10 ml/min. NO, = 2.9 ppm, 10 ml/min, 30 min; cm* membrane) B

272 l-i Lii (x 1o-3) o 10 2 8 E 2 q Y

8

-

6

-

0

h

0

2

4 [NO21

6

8

10

(ppm)

Fig.6. Relationship between the increase of MetHb mole fraction and NO, concentration: [Hb,] = 80-140 (nanomoles/cm’ membrane), [NO,] = 10 ml/min, 30 min.

Discussion Hemoglobin was found to be very sensitive to NO, exposure either in solution or in membrane form. Since NOz is the major component of NO,, the interaction of NOz with hemoglobin is primarily discussed here. It has been indicated that NO, reacts directly with both the heme and the globin moieties of the hemoglobin molecule, and also binds to the iron atoms. ESR studies by several investigators have shown that NO1 reacts with hemoglobin solutions to produce a broad resonance at ca. g = 2, with a nitrogen hyperfine coupling of 16 Gauss plainly observable on the high-field side of the signal. This evidence suggests the possibility that NOz was reduced, with the formation of a NO-hemoglobin complex subsequent to the NOz-hemoglobin reaction. In the present investigation, NO, seemed to react reversibly with hemoglobin in the lower NO, concentration range. Oxyhemoglobin was regenerated from the hemoglobin exposed to NO, by dithionite. However, an irreversible reaction might take place in the higher NO, concentration range. The regeneration of HbOz from the hemoglobin exposed to NO% was partly possible. Spectroscopic analysis indicated that the oxidation of HbOz to MetHb was the predominant process in the reaction of NOz with hemoglobin. Furthermore, ferrous ions are capable of reducing nitrite to NO. Nitrogen dioxide

273

also reacts with water to generate 2N02 + Hz0 + HNOz + H’ + NO,

nitric acid and NO, as follows: (4)

3HN02 + H+ + NO; + 2N0 + HZ0 These facts suggest that NO2 oxidizes HbOz to MetHb at lower NO, concentrations, and that a NO-hemoglobin complex may form with an increase in NO, concentration. The generation of MetHb may result from the reduction of NO* by the heme iron. The heme iron, Fe*+, may be oxidized to Fe3+, which causes the hemoglobin to lose its oxygen-binding ability. In concentrated NO,,not only NO2 but also NO may attach to the porphyrin ring and the globin moieties of hemoglobin. The hemoglobin membrane was found to be a useful biochemical indicator of NO, exposure. This membrane was specially designed on the model of a mucous biomembrane, which is capable of maintaining a constant moisture. Gaseous NO, can react directly with hemoglobin on the membrane surface. The diffusion of water through the membrane and the evaporation of water from the membrane surface are balanced at steady state. The immobilized hemoglobin can react with NO, while retaining its conformation and function. The hemoglobin membrane offers another advantage in that gaseous NO, may be accumulated in the membrane phase. Gaseous NO, was monitored in the concentration range of O-10 ppm by this method. Therefore, the hemoglobin membrane can be used as a biochemical indicator for environmental evaluation. References 1 W.A. Thomas, Indicators of Environmental Quality, Plenum Press, New York, 1972. 2 S. Matsunaka, Shihyo Seibutsu (Biological Indicators), Kodansha, Tokyo, 1975. 3 Industrial Pollution Control Association of Japan, Comprehensive Report - 1976 International Meeting on Environmental Quality Standards for Nitrogen Oxides; Address: 18 Shibanishikubo, Sakuragawa-cho, Minato-ku, Tokyo, 105, Japan. 4 O.W. van Aasendelft, The Spectrophotometry of Hemoglobin Derivatives, Royal Van Gorcum, Ltd., Assen, The Netherlands, 1970. 5 J.R. Rowlands, E.M. Gause and S. Antonio, Arch. Inter. Med. Symp., 9 (1971) 234. 6 H. Kon, J. Biol. Chem., 243 (1968) 4350. 7 E.G. Moore and Q.H. Gibson, J. Biol. Chem., 251 (1976) 2788. 8 D.L. Drabkin, J. Biol. Chem., 164 (1964) 703. 9 Japanese Standards Association, J.I.S. K 0516-(1976), NO and NO, Standard Samples; Address: 4-l-24 Akasaka, Minato-ku, Tokyo 107, Japan.