The chemiluminescence from the reaction of hemoproteins with inorganic peroxides

840

Journal of Luminescence 4O&4l (1988) 840—841 North-Holland, Amsterdam

THE CHEMILUMINESCENCE FROM THE REACTION OF HEMOPROTEINS WITH INORGANIC PEROXIDES

Yaning LIU, member of the Chinese Society of Biophysics; Basic Lab, The Xidiaoyutai Hospital,

No.30, West Diaoyutai,

Xinhua Zl-IAO. Beijing, Chits.

Chemiluminescence from hemoproteins oxidation are composed of two components: the dimol emission of the singlet oxygen and the excited carbonyls, accompanied by the destruction of por~yrin rings.

The biological importance of hemoproteins is NV

cytochrome C

not inferior to DNA. The development of rapid and simple measurement for hemoproteins is certainly of great clinical and biological value.

0.2 0. ~

talase

erritin

It has been reported that in the chemilumi— 0.1 nescent reactions of luminol and lipid peroxides, 1’2. Rut the hemoprotelns act as catalysts instability of luminol has also been noticed3.

50

100

150 t(sec.)

200

250

Furthermore, the pure organic peroxides are hard to come by.

FIGURE 2

We found that when hemin and hemorpoteins

Chemiluminescent emission dynamic Curves cC cy— tochrome C, catalase, ferritin and water blank.

react with inorganic peroxides (sodium perborate or H 202)

in alkaline solution, low—level chemi—

luminescences could be detected )Fig.1,2).

Measured with our home—made luminometer, the luminosity sequence of hemoproteins and control

MV

materials was as follows (the integral time was 6 myoglobin

set to 100 seconds):

hemoglobin

myoglobin > hemoglobin > horseradish per— oxidase > cytochrome C > hemln > catalase > ferrltin > water blank 3 FeSO4 > FeCl3 >

24

hematoporphyrin 2

~

+

iron ion

by OMA—2two multichannel spectroscopy and Determined cutting filters, bands appa~red in the emission spectra: a red band with a peak around 630 nm and a blue band with a peak around 4240 cm

hemin

)Fig.3). 50

100 t(sec.)

150

The red band must be the dm01

emission of

singlet oxygen, as these chemiluminescences could be strongly quenched by a_-carotene. The FIGURE 1 Chemiluminescent emission dynamic curves of myo— glcbin, hemoglobin, horseradish peroxidase and hemin. 0022—23l3/88/$03.50 © Elsevier Science Publishers By. (North-Holland Physics Publishing Division)

blue band is supposed to be the luminescence from the excited carbonyls,

which might be .1 produced through the Dioxetane mechanism

Y. Liu, X. Zhao

/

841

Chemiluminescence from the reaction of hemoproteins

The ground state On the 3d orbits

of iron

ion is paramagnetic.

there are spin—unpaired

elec-

trons, their spin—spin coupling is very weak. After the iron ions being complexed into por—

mt.

both phyrmns, iron their and porphyrin unpaired are electrons markedly are enhanced. involved in the bigger rings conjugations and become highly delocalized, thus the reactivities of

400

500

600

700

Therefore, hems and hematoporphyrin behaved quite differently during oxidation.

stave length (nm)

Good linear relationships between concentration and luminosity were obtained, both for

FIGURE 3 OMA—2 emission spectrum of myoglobin oxidation.

myoglobin at ng level and for horseradish per—

The scavengers of hydroxyl radicals could

oxidase at pg level (the correlation coeffient was 0.9926 and 0.9975, respectively(. These

partially inhibit these chemiluminescences,

chemiluminescences may be helpful to the search

indicating the participation of active oxygen

for new luminescent assays. For example, serum

other than the singlet oxygen.

myoglobmn determination is valuable to the rapid 5 and, diagnosis of acute myocardial infarction if the luminometer is sensitive enough, the

The blank luminescence of sodium perborate in alkaline solution could also be quenched by fl—carotene to great extent and inhibited by

luminescent assay for horseradish peroxidase

hydroxyl radical scavengers to some extent, so

may be a safe substitute for the traditional

the singlet oxygen might partially come from

ELISA, protecting the operator from contacting

the Arneson mechanism

4

the highly carcinogenic substrate, i.e., the

During oxidation the Soret bands of hemin

3,3’—diamincbenzidine(DAR).

and hemoproteins decreased gradually, representing the destruction of porphyrin rings.

ACKNOWLEDGEMENT

The excited carbonyls might come from porphy—

We would like to thank Prof. Lin Me Chun and

rins and their adjacent groups. The luminosity

Cheng Bo Ji for their comments on ferriporphy—

differences among various hemoproteins might

rin and excited carbonyls, as well as Dr. Liu

account for the different amounts of excited

Jing Qing for her help in OMA—2 measurement.

carbonyls produced during oxidation. The fact that different hemoproteins emitted light at different rates when they were cxi—

REFERENCES !.

dized, might be caused by the different expo—

E. Cadenas et ml., Low level chemilumines— cence of biological systems, in: Free Radicals in Riology, ed. W.A. Fryer (Academic Press, Vol.VI, 1984) pp.231—242.

sure states of heme groups to solvents. The chemiluminescence induced by the mixture of hematoporphyrin and iron ion (ferrous or ferric( was much weaker than those of hemin

2. T. Olsson et al., Clin. Chim. Acta, pp.31—40.

138(1984)

3. W.R. Seitz et al., Anal. Chem. 48(1976(1003. and hemoproteins, and there was no apparent porphyrin destruction during oxidation. The complex compound of ferriporphyrin

i.

R.M. Arneson, Arch. (1976(352.

Biochem. Biophys. 136

must play

a unique role in these chemiluminescences.

5. J.P. Chapelle, Eur. Heart J. 3(1982)122.