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Molecular weights of blood plasma fractions photooxidized in the presence of sensitizers
BIOCHIMICA ET BIOPHYSICA ACTA
495
MOLECULAR W E I G H T S OF BLOOD PLASMA FRACTIONS PHOTOOXIDIZED IN T H E PRESENCE OF SENSITIZERS ZDENI~K VODR.~KA AND OTAKAR MACH Institute o[ Hematology and Blood Trans]usion, Prague (Czechoslovakia)
(Received November 24th, 196o)
SUMMARY Sensitized photooxidation of human blood plasma fractions, in contrast to high frequency electromagnetic irradiation of them, did not cause molecular splitting. On the contrary, an increase of the molecular weight was observed. This increase was, however, due only to the aggregation of a part of the molecules and its degree depended markedly on certain reaction conditions, such as temperature and protein concentration. By conducting the reaction at low temperature and low protein concentration and by reducing the reaction time, it was possible, at any rate when human serum albumin, v-globulin and fibrinogen were used, to obtain photooxidized proteins of unchanged molecular weight. The fractions aggregated were separated b y precipitation with ammonium sulphate. In order tomaintain themolecular weight unchanged, sensitization by eosin seemed to be preferable to sensitization by methylene blue. The influence of photooxidation on sensitizer-protein interactions is discussed.
INTRODUCTION WEIL et al. 1-4 observed that irradiation of a series of enzyme solutions with visible light in the presence of suitable sensitizers caused oxidative destruction of aromatic and heterocyclic rings and oxidation of groups containing sulphur. In our previous work 5-9 we found that similar photooxidation reactions occurred also with blood proteins and we described the changes arising in the chemical composition of blood protein molecules. B y comparing the effects of sensitizers differing in their chemical composition and b y following the influence of certain factors on the course of the process, we arrived at, and proposed a reaction scheme s. The present work is a more detailed study of the influence of photooxidation upon the molecular size of the main fractions of human plasma-albumin, v-globulin and fibrinogen. Direct measurement of the molecular weights of photooxidized proteins was carried out in only a few instances. WEIL AND BUCI-IERT1, using the light scattering method, observed that the molecular weight of fl-lactoglobulin increased from 42 000 to 147 ooo, oxygen consumption amounting to 20 moles/mole of fl-lactoglobulin. The photooxidized ovalbumin and v-globulin showed a marked increase in the fractions Abbreviation: HSA, human serum albumin. Biochim. Biophys. Acta, 49 (1961) 495-5or
496
Z. VODRA~KA, O. MACH
with sedimentation coefficients higher than those of the native proteins 1°. On the other hand, the sedimentation analysis of photooxidized chymotrypsin did not reveal any increase in the molecular weight 11. On the contrary, the photooxidized molecules of chymotrypsin had entirely lost their ability to dimerize. In the remaining experiments only viscosity measurements were carried out to obtain information on the size and shape of photooxidized protein molecules. The viscosity usually increased 12-1~, but it remained, in m a n y instances, unchanged 2,11,12,14.
EXPERIMENTAL
The HSA and ~,-globulin employed were ethanol fractionated products (fraction V and II1,~). For further work preparations obtained by the ammonium sulphate salting out method were also used. The human fibrinogen was prepared b y the ammonium sulphate precipitation method of ASTRUP AND MOLLERTZ15. The photooxidation, which has already been described 5, was carried out by irradiation with a 25o-W projection bulb with tungstene wire situated at a Io-cm distance, the depth of the solution being 2-3 cm. The media used were a phosphate buffer (pH 7.1, /~ ~ o.I), and, in several instances, a borate buffer (pH 9.2, /~ ---- o.i). Sensitization was achieved with a 1. 3- lO -4 M solution of methylene blue corresponding to the m a x i m u m effectS, 8 or in some instances a 8. lO -4 M eosin solution. The degree of photooxidation is expressed in terms of the consumption of moles of oxygen/mole of the protein, determined by analysis which showed the decrease in the histidine content. It has already been explained Dthat the values were obtained from the dependence of the decrease in the photoreactive amino acids of individual proteins on the consumption of oxygen. Histidine was determined in acid hydrolysate after diazotation by the method oi MACPHERSON16 b y isolation as a mercury complex by the method of LANG~7. The values obtained by this method for the content of histidine in the individual proteins before photooxidation, namely, 3.48 for HSA, 2.75 for y-globulin and 2.63 for fibrinogen, are in good agreement with those published in the literature, namely, 3.50, 2.50, and 2.60 respectively. The measurements of the sedimentation coefficients were carried out with a Phywe ultracentrifuge at 48 ooo rev./min, at temperatures of 18-2o °, and with protein concentrations of o.6-1%. The values of the sedimentation coefficients were obtained by plotting logarithms of the boundary position against the time of sedimentation. The diffusion measurements were carried out at IO ± 0.2 ° with an electrophoretic apparatus, model EMB-52. The Tiselius electrophoretic cell and the Philpot-Svensson optical system were used. Compensation was carried out at a rate of 2 ml/h. The evaluation was carried out is by the method of the m a x i m u m ordinate and area (D0,h) and the method of moments (D2,0). The intensity of the light scattered at a 9 °0 angle was measured with a simple photoelectric apparatus constructed by ~PONAR19 in a cylindric cell at a wave length of 436 m/~. The methylene blue was removed b y shaking the solution with a pulp made from Seitz pads. The samples were clarified by filtration through Seitz K pads under nitrogen pressure and then through sintered glass G 4. The concentrations of the solutions were determined b y weighing after evaporating and drying the samples for 24 Biochim. Biophys. Acta, 49 (1961) 495-5 ol
MOLECULAR WEIGHTS
OF PHOTOOXIDIZED
BLOOD PROTEINS
497
h at 105%The correction for light absorption b y the remaining methylene blue was calculated from the equation *° I = I~° To
where I is the corrected intensity and I,o the intensity measured at a 9 °0 angle. T o represents the ratio of the intensities of the light beams after passing through the given protein solutions with and without methylene blue, the readings being taken b y using the cell for light scattering at zero angle and a wave length of 436 m/~. The constants used in the calculations, taken from data in the literature on the refractive index increment 21, are as follows: 6.o8.1o -7 for HSA, 6,17.1o -7 for v-globulin and 6.12. lO -7 for human fibrinogen. Viscosity measurements were carried out at 25 ~ 0.05 ° in the Ostwald viscosimeter with a water flow time of 12o sec. The molecular weights were determined from the dependence of the reduced light intensity scattered at 9 °0 (Kc/R~o) on the concentration b y extrapolating to zero concentration (Mw) and were also calculated from diffusion and sedimentation coefficients by using the Svedberg formula (Ms,D), or, in some instances from diffusion coefficients and intrinsic viscosities (M~,D) by he method of POLSO~22 Mn,D = 6.16"Io
6
I
E~" D3
The influence of photooxidation upon the extent of binding of sensitizers to proteins was investigated b y the method of equilibrium dialysis using the cells b y KA6ENA (see ref. 23). The amount of the free dye was determined spectrophotometrically. RESULTS AND
DISCUSSION
The values of molecular weights of photooxidized proteins determined by the lightscattering method, the sedimentation-velocity method, and in the case of HSA also b y calculation from the viscosities by POLSON'S method are listed in Table I, where they are compared with those for the native proteins and proteins heated in the absence of sensitizers. The values of sedimentation and diffusion coefficients and of intrinsic viscosities agree with those taken from the literature, both for the ethanol fractionated albumin and the preparation obtained by the salting out method. Even the light-scattering method offers an acceptable molecular weight of 75 ooo to 81 ooo. TIMASHEFF AND GIBBS24 state that 80 ooo is an adequate average molecular weight for albumin which has neither been crystallized nor purified in any other way. The values for v-globulin are also in keeping with various data from the literature. In the case of fibrinogen the molecular weight of 240 ooo is low as compared with the value of 400 ooo generally presented. In a recent paper, however, SOWlNSKI et al. 25 also give low values for bovine fibrinogen, ranging from 222 ooo to 274 ooo, and for human fibrinogen ze, 246 ooo275 ooo. To obtain the molecular weights, the samples of HSA were photooxidized tilI the consumption of oxygen had reached the value of 3 ° moles/mole of protein, corresponding to t h e decrease of I I moles in the histidine content, and of 6 in that Biochim. Biophys. Acta, 4 9 (1961) 4 9 5 - 5 o 1 -
Z. V O D R ~ K A ,
498
O. MACH
TABLE
I
HUMANSERUM ALBUMIN V, e t h a n o l p r o d u c t . Albumin Sensitizer
V S S V S S V V*** S S
---MB MB E MB MB MB MB
Temperature
S, s u l p h a t e p r o d u c t . s°2o x Io 1~ Main%* comp.
---** 3° IO Io IO 3o 3° 30 42
4-43 4.48
95 98
4-57 4.35 4.8o
94 94 98
4.44 4.52
Sensitizers:MethyleneBlue(MB),Eosin(E). D,,o x Io 7 [*l] × ~o~"
90 80
5.68 5.74
4.1 4 .2
5.76 5.71 3.86 4.31 4.Ol
4.5
Mw
81000 75 o o o 75 o o o 900oo 82 o o o
6.5 5.05 6.0
Ms, D
MD,~I
75 5 0 0
81990 77560
73 I o o 813oo
190000 lZ 4 o o o 13o00o
lO 7 IOO
71 6 4 o 164000 1524oo 155 6 0 0
* H i g h e r c o m p o n e n t s w i t h s e d i m e n t a t i o n c o e f f i c i e n t s 6 . 2 - 8 . 8 S. ** H e a t e d f o r 9 h a t 3 ° 0 w i t h o u t s e n s i t i z e r . *** P h o t o o x i d i z e d a t p H 9 . 2 . HUMAN
Fraction y
Sens~izer
111, 2
--
S
---
111,2 111,2
-
111, 2 S S
~-GLOBULIN
II1,2, e t h a n o l p r o d u c t . S, s u l p h a t e p r o d u c t .
Temper~ure s°~o × ~o~3 Main%~ comp.
-
D2'° × z°7
7 .18
93
3.o6
227 ooo 312ooo
57 **~
-
Ms,D
175 ooo
-
-37**
MB MB E
Mw
IO IO IO
400 ooo 270 ooo 7.4 ° 7.32
74 74
*Higher components with the sedimentation r e v e a l s t r a c e s of t h o s e o f 19 S. * * H e a t e d f o r 8 h a t 37 °. * * * H e a t e d f o r I h a t 57 ° .
1.74
421 o o o
c o e f f i c i e n t of a b o u t lO, 5 S; t h e s u l p h a t e p r o d u c t
HUMAN FIBRINOGEN (SULPHATE PRODUCT) Sensitizer
Temperaure
-__ MB
-2 0 °* lO °
s°*° × z°*8
7.84 7.84
Main%*comp. D*'° × r°~
95
3-14 1.3o
Mw
240000 380000 500 ooo
Ms, D
242000 583 5 0 0
* A f t e r i i h a t 2 0 °.
of tyrosine 1°. With y-globulin and fibrinogen, samples photooxidized up to a consumption of about 7 ° moles of oxygen/mole of protein were employed. With y-globulin this consumption corresponds to a destruction of 15 moles of histidine, 16 of tyrosine, and 7 of tryptophan 1°. According to weight-average molecular weights obtained by using the lightscattering method, all the proteins under study, photooxidized at room temperature Biochim.
Biophys.
~4cta, 49 (1961) 4 9 5 - 5 o l
MOLECULAR WEIGHTS OF PHOTOOXIDIZED BLOOD PROTEINS
499
or at a slightly higher temperature, showed approximately twofold molecular weight increases (Table I). It was, however, evident from the molecular kinetic properties found by sedimentation analysis that with all the three proteins (HSA, y-globulin, and fibrinogen) the main fractions remained unchanged (Fig. I). Nevertheless there was a A 13
Fig. i. Sedimentation patterns. A, H S A photooxidized at 3 °o (5 ° moles of O~/mole) ; B, v-globulin photooxidized at IO ° (70 moles of O2/mole ).
marked increase in the amount of fractions with higher sedimentation coefficients, which were initially present in only negligible amounts. This was evidently due to secondary aggregation of protein molecules under the experimental conditions and it explained the increase in the weight-average molecular weights found by the lightscattering method. To verify the fact that photooxidation does not in itself cause any changes in molecular weights, an experiment with HSA was carried out to follow the influences of protein concentration, temperature and photooxidation time upon the amount of fractions with higher sedimentation coefficients. Whereas in photooxidation carried out at a temperature ranging from o to IO ° virtually no aggregated fractions are observed, at 4 °o 20 % of the HSA was already aggregated and the dependence-curve illustrating the increase with temperature in the aggregated fractions rose steeply (Fig. 2). Aggregation occurred at a temperature of 3 o°, the consumption of oxygen amounting to 25 moles/mole of protein (about 2.5 h is required), as can be seen from the time-dependence curve (Fig. 3). With further prolongation of the reaction time no more changes in weight-average molecular weights were observed. The extension of aggregation continued with rising protein concentration (Fig. 4) and was slightly lower when eosin was used as sensitizer instead of methylene blue. It was found that larger aggregation products could be separated from the solutions of photooxidized albumin by repeated precipitation with ammonium sulphate at 4 ° 9/0 saturation. The molecular weight of the remaining substance was normal. When the HSA-solution was heated at 3 °0 without sensitizer, no change in the molecular weight occurred, and it had not occurred even after IO h.,On the other hand, with y-globulin, a more labile protein, the molecular weight was nearly doubled after 8 h at 37°; and with fibrinogen the molecular weight kept on increasing even after standing for I I h at 20 °. After heating the y-globulin in solution for I h at 57 °, the Biochim. Biophys. Acta, 49 (1961) 495-5 ol
500
Z. V O D R ~ K A ,
O. MACH
molecular weight rose to 400 ooo. These observations show that photooxidation does not necessarily cause an increase of molecular weight, but that the aggregation observed with labile proteins is caused by the slight increase of the temperature during irradiation of their solutions while photooxidation proceeds. Although experiments M w. 10-3
20 m °/o
130
15 120
110
100
-I
10
I
I
.
20 30 40 T e m p e r a t u r e °C
Fig. ~.
90
80 i
Fig. 2. D e p e n d e n c e of t h e a m o u n t of t h e agg r e g a t e d p o r t i o n of H S A - - f o u n d b y s e d i m e n tation analysis---on the temperature during p h o t o o x i d a t i o n . H S A w a s p h o t o o x i d i z e d till 5 ° moles of O~/mole of p r o t e i n were c o n s u m e d .
~o
20
30
i
40 02/mole
50
Fig. 320 %
Fig. 3. D e p e n d e n c e of t h e w e i g h t - a v e r a g e of m o l e c u l a r w e i g h t of H S A on t h e degree of p h o t o o x i d a t i o n , e x p r e s s e d in t e r m s of o x y g e n consumption.
Fig. 4. D e p e n d e n c e of t h e a m o u n t of t h e aggreg a t e d p o r t i o n of H S A on t h e a l b u m i n concent r a t i o n in p h o t o o x i d a t i o n . H S A w a s p h o t o oxidized till 5o moles of O~/mole of t h e p r o t e i n were c o n s u m e d .
15
10
./ t 0.5
f
I 10
1 1.5 g / m l • 102
I 20
Fig. 4.
with HSA indicate that photooxidized proteins aggregate more readily, it is possible (by carrying out the reaction at a sufficiently low temperature or by removing the aggregated product) to prepare photooxidized albumin with its molecular weight unchanged. Methylene blue seemed to promote the aggregation of photooxidized proteins, although with HSA no difference between the sensitization by eosin and that by methylene blue was found in the course of the reactions 8, In this connection it is worth-while to mention the influence of photooxidation upon the extension of interaction between methylene blue and HSA. Whereas the native protein does not bind the methylene blue virtually at all 27, the photooxidized proteins form stable complexes with this dye, which cannot be completely removed, even b y long term dialysis. Biochim. Biophys. Acta, 49 (1961) 495-5Ol
MOLECULAR WEIGHTS OF PHOTOOXIDIZED BLOOD PROTEINS
501
It is of interest that the relation between the amount of methylene blue bound to a weight unit of HSA and the degree of photooxidation follows a dependence similar to that of weight-average molecular weight. The opinion that methylene blue is bound to the aggregates being formed is also confirmed by precipitation with ammonium sulphate. Most of the methylene blue was then contained in precipitates, whereas the supernatant, containing the molecules of photooxidized HSA with normal molecular weight, was nearly colourless. In addition, the possibility cannot be excluded that the methylene blue is responsible for the albumin aggregation, v-globulin and fibrinogen, however, do not bind methylene blue either in the native or photooxidized state and eosin is bound to the albumin ~3, the influence of photooxidation showing no marked effect. All the photooxidation experiments were carried out at pH 7.I and with HSA, for comparison, also at pH 9.2. No changes in the amino acid composition of HSA were observed during photooxidation at these two pH's. The change in pH influenced only the reaction rate. The weight-average molecular weight found for HSA photooxidized at pH 9.2 and at 3 °0 amounts to 15o ooo-19o ooo, which represents an increase similar to that observed after photooxidation at pH 7.1. ~ P O N A R A N D LOgTICK~ ~s, however, reported that in borate buffer (such as that used in this work) aggregation of native albumin occurs, probably because complexes with the borate are formed. They recorded, by the light-scattering method, a molecular weight of 19o ooo. REFERENCES 1 L. WEIL AND A. R. BUCHERT, Arch. Biochem. Biophys., 34 (1941) I ; Federation Proc., z I (1952) 307. 2 L. WEIL, S. JAMES AND A. R. BUCHERT, Arch. Biochem. Biophys., 46 (1953) 266. a L. WEIL AND T. S. SEIBLES, Arch. Biochem. Biophys., 54 (1955) 368. 4 L. WEIL, A. R. BUCHERT AND J. MAHER, Arch. Biochem. Biophys., 4o (1952) 245. 5 Z. VODR~.~KA AND R. PRAUS, Chem. listy, 49 (1955) 127; Collection Czechoslov. Chem. Communs., 20 (1955) 598 . 6 R. PRAUS, Chem. listy, 49 (1955) 921; Collection Czechoslov. Chem. Communs., 2o (1955) 1269. 7 Z. VODR~KA AND J. ~PONAR, Chem. listy, 49 (1955) lO69; Collection Czechoslov. Chem. Communs., 20 (1955) 1281. 8 Z. VODR/~KA AND J. ~PONAR, Chem. listy, 51 (1957) 1649; Collection Czechoslov. Chem. Communs., 23 (1957) 229. 9 Z. VODR~KA AND K. PI~ISTOUI'ILOV,t,,Chem. listy, 51 (1957) 1657; Collection Czechoslov. Chem. Communs., 23 (1958) 752. 10 H. SMETANA AND n . SHEMIN, J. Exptl. Med., 73 (194 I) 223. 11 R. EGAN, H, O. MICHEL, R. SCHLUETER AND B. J. JANDORF, Arch. Biochem. Biophys., 66 (1957) 366. 12 T. V. VENKgTERN, Biokhimiya, 18 (1953) 96. 13 Z.'VODR/~KA, Chem. listy, 53 (1959) 829. 14 Z. VODR/~KA, Fotodynamickf~ ]ev., St. Zdrav. Nakl., Praha, 1957, p. 42. 15 I. ASTRUP AND S. MOLLERTZ, Arch. Bioehem. Biophys., 4 ° (1952) 346. le H. T. MACPHERSON, Biochem. J., 36 (1942) 59. 17 K. LANG, z . physiol. Chem., Hoppe Seyler's, 222 (1933) 3. 18 H. P. LUNDGREEN AND W. H. WARD, in D. M. GREENBERG, A m i n o Acids and Proteins, T h o m a s , S p r i n g f i e l d , 1951, p. 331. 19 j . ~PONAR, Chem. listy, 53 (1959) 429 2o N. BENHAMOU AND Cx. WEIL, Biochim. Biophys. Acta, 24 (1957) 548. 21 G. E. PERLMANN AND L. G. LONGSWORTH, J. Am. Chem. Soc., 7° (1948) 2719. ~2 A. POLSON, Biochim. Biophys. Acta, 21 (1956) 185. 23 Z. VODR/~KA, Collection Czechoslov. Chem Communs., 25 (196o) 41o. 24 S. N. TIMASHEFF AND R. G. GIBBS, Arch. Biochem. Biophys., 7° (1957) 547. 25 R. SOWlNSEI, L. OHARENKO AND V. L. KOENIG, J. Am. Chem. Soc., 81 (1959) 6193. ~s R. SOWlNSKI, V. FREILING AND V. L. KOENIG, Makromol. Chem., 31 (196o) 152. 27 Z. VODR~KA AND J. ~PONAR, Chem. listy, 49 (1955) lO64; Chem. listy, 50 (1956) 860; Collection Czeehoslov. Chem. Communs., 20 (1955) 1275 ; CollectionCzechoslov. Chem. Communs., 22 (1957) 1241. ~8 j . ~PONAR AND C. LOgTICK¢/, Collection Czechoslov. Chem. Communs., 25 (196o) 159.
Biochim. Biophys. Acta, 49 (1961) 495-5 ol
495
MOLECULAR W E I G H T S OF BLOOD PLASMA FRACTIONS PHOTOOXIDIZED IN T H E PRESENCE OF SENSITIZERS ZDENI~K VODR.~KA AND OTAKAR MACH Institute o[ Hematology and Blood Trans]usion, Prague (Czechoslovakia)
(Received November 24th, 196o)
SUMMARY Sensitized photooxidation of human blood plasma fractions, in contrast to high frequency electromagnetic irradiation of them, did not cause molecular splitting. On the contrary, an increase of the molecular weight was observed. This increase was, however, due only to the aggregation of a part of the molecules and its degree depended markedly on certain reaction conditions, such as temperature and protein concentration. By conducting the reaction at low temperature and low protein concentration and by reducing the reaction time, it was possible, at any rate when human serum albumin, v-globulin and fibrinogen were used, to obtain photooxidized proteins of unchanged molecular weight. The fractions aggregated were separated b y precipitation with ammonium sulphate. In order tomaintain themolecular weight unchanged, sensitization by eosin seemed to be preferable to sensitization by methylene blue. The influence of photooxidation on sensitizer-protein interactions is discussed.
INTRODUCTION WEIL et al. 1-4 observed that irradiation of a series of enzyme solutions with visible light in the presence of suitable sensitizers caused oxidative destruction of aromatic and heterocyclic rings and oxidation of groups containing sulphur. In our previous work 5-9 we found that similar photooxidation reactions occurred also with blood proteins and we described the changes arising in the chemical composition of blood protein molecules. B y comparing the effects of sensitizers differing in their chemical composition and b y following the influence of certain factors on the course of the process, we arrived at, and proposed a reaction scheme s. The present work is a more detailed study of the influence of photooxidation upon the molecular size of the main fractions of human plasma-albumin, v-globulin and fibrinogen. Direct measurement of the molecular weights of photooxidized proteins was carried out in only a few instances. WEIL AND BUCI-IERT1, using the light scattering method, observed that the molecular weight of fl-lactoglobulin increased from 42 000 to 147 ooo, oxygen consumption amounting to 20 moles/mole of fl-lactoglobulin. The photooxidized ovalbumin and v-globulin showed a marked increase in the fractions Abbreviation: HSA, human serum albumin. Biochim. Biophys. Acta, 49 (1961) 495-5or
496
Z. VODRA~KA, O. MACH
with sedimentation coefficients higher than those of the native proteins 1°. On the other hand, the sedimentation analysis of photooxidized chymotrypsin did not reveal any increase in the molecular weight 11. On the contrary, the photooxidized molecules of chymotrypsin had entirely lost their ability to dimerize. In the remaining experiments only viscosity measurements were carried out to obtain information on the size and shape of photooxidized protein molecules. The viscosity usually increased 12-1~, but it remained, in m a n y instances, unchanged 2,11,12,14.
EXPERIMENTAL
The HSA and ~,-globulin employed were ethanol fractionated products (fraction V and II1,~). For further work preparations obtained by the ammonium sulphate salting out method were also used. The human fibrinogen was prepared b y the ammonium sulphate precipitation method of ASTRUP AND MOLLERTZ15. The photooxidation, which has already been described 5, was carried out by irradiation with a 25o-W projection bulb with tungstene wire situated at a Io-cm distance, the depth of the solution being 2-3 cm. The media used were a phosphate buffer (pH 7.1, /~ ~ o.I), and, in several instances, a borate buffer (pH 9.2, /~ ---- o.i). Sensitization was achieved with a 1. 3- lO -4 M solution of methylene blue corresponding to the m a x i m u m effectS, 8 or in some instances a 8. lO -4 M eosin solution. The degree of photooxidation is expressed in terms of the consumption of moles of oxygen/mole of the protein, determined by analysis which showed the decrease in the histidine content. It has already been explained Dthat the values were obtained from the dependence of the decrease in the photoreactive amino acids of individual proteins on the consumption of oxygen. Histidine was determined in acid hydrolysate after diazotation by the method oi MACPHERSON16 b y isolation as a mercury complex by the method of LANG~7. The values obtained by this method for the content of histidine in the individual proteins before photooxidation, namely, 3.48 for HSA, 2.75 for y-globulin and 2.63 for fibrinogen, are in good agreement with those published in the literature, namely, 3.50, 2.50, and 2.60 respectively. The measurements of the sedimentation coefficients were carried out with a Phywe ultracentrifuge at 48 ooo rev./min, at temperatures of 18-2o °, and with protein concentrations of o.6-1%. The values of the sedimentation coefficients were obtained by plotting logarithms of the boundary position against the time of sedimentation. The diffusion measurements were carried out at IO ± 0.2 ° with an electrophoretic apparatus, model EMB-52. The Tiselius electrophoretic cell and the Philpot-Svensson optical system were used. Compensation was carried out at a rate of 2 ml/h. The evaluation was carried out is by the method of the m a x i m u m ordinate and area (D0,h) and the method of moments (D2,0). The intensity of the light scattered at a 9 °0 angle was measured with a simple photoelectric apparatus constructed by ~PONAR19 in a cylindric cell at a wave length of 436 m/~. The methylene blue was removed b y shaking the solution with a pulp made from Seitz pads. The samples were clarified by filtration through Seitz K pads under nitrogen pressure and then through sintered glass G 4. The concentrations of the solutions were determined b y weighing after evaporating and drying the samples for 24 Biochim. Biophys. Acta, 49 (1961) 495-5 ol
MOLECULAR WEIGHTS
OF PHOTOOXIDIZED
BLOOD PROTEINS
497
h at 105%The correction for light absorption b y the remaining methylene blue was calculated from the equation *° I = I~° To
where I is the corrected intensity and I,o the intensity measured at a 9 °0 angle. T o represents the ratio of the intensities of the light beams after passing through the given protein solutions with and without methylene blue, the readings being taken b y using the cell for light scattering at zero angle and a wave length of 436 m/~. The constants used in the calculations, taken from data in the literature on the refractive index increment 21, are as follows: 6.o8.1o -7 for HSA, 6,17.1o -7 for v-globulin and 6.12. lO -7 for human fibrinogen. Viscosity measurements were carried out at 25 ~ 0.05 ° in the Ostwald viscosimeter with a water flow time of 12o sec. The molecular weights were determined from the dependence of the reduced light intensity scattered at 9 °0 (Kc/R~o) on the concentration b y extrapolating to zero concentration (Mw) and were also calculated from diffusion and sedimentation coefficients by using the Svedberg formula (Ms,D), or, in some instances from diffusion coefficients and intrinsic viscosities (M~,D) by he method of POLSO~22 Mn,D = 6.16"Io
6
I
E~" D3
The influence of photooxidation upon the extent of binding of sensitizers to proteins was investigated b y the method of equilibrium dialysis using the cells b y KA6ENA (see ref. 23). The amount of the free dye was determined spectrophotometrically. RESULTS AND
DISCUSSION
The values of molecular weights of photooxidized proteins determined by the lightscattering method, the sedimentation-velocity method, and in the case of HSA also b y calculation from the viscosities by POLSON'S method are listed in Table I, where they are compared with those for the native proteins and proteins heated in the absence of sensitizers. The values of sedimentation and diffusion coefficients and of intrinsic viscosities agree with those taken from the literature, both for the ethanol fractionated albumin and the preparation obtained by the salting out method. Even the light-scattering method offers an acceptable molecular weight of 75 ooo to 81 ooo. TIMASHEFF AND GIBBS24 state that 80 ooo is an adequate average molecular weight for albumin which has neither been crystallized nor purified in any other way. The values for v-globulin are also in keeping with various data from the literature. In the case of fibrinogen the molecular weight of 240 ooo is low as compared with the value of 400 ooo generally presented. In a recent paper, however, SOWlNSKI et al. 25 also give low values for bovine fibrinogen, ranging from 222 ooo to 274 ooo, and for human fibrinogen ze, 246 ooo275 ooo. To obtain the molecular weights, the samples of HSA were photooxidized tilI the consumption of oxygen had reached the value of 3 ° moles/mole of protein, corresponding to t h e decrease of I I moles in the histidine content, and of 6 in that Biochim. Biophys. Acta, 4 9 (1961) 4 9 5 - 5 o 1 -
Z. V O D R ~ K A ,
498
O. MACH
TABLE
I
HUMANSERUM ALBUMIN V, e t h a n o l p r o d u c t . Albumin Sensitizer
V S S V S S V V*** S S
---MB MB E MB MB MB MB
Temperature
S, s u l p h a t e p r o d u c t . s°2o x Io 1~ Main%* comp.
---** 3° IO Io IO 3o 3° 30 42
4-43 4.48
95 98
4-57 4.35 4.8o
94 94 98
4.44 4.52
Sensitizers:MethyleneBlue(MB),Eosin(E). D,,o x Io 7 [*l] × ~o~"
90 80
5.68 5.74
4.1 4 .2
5.76 5.71 3.86 4.31 4.Ol
4.5
Mw
81000 75 o o o 75 o o o 900oo 82 o o o
6.5 5.05 6.0
Ms, D
MD,~I
75 5 0 0
81990 77560
73 I o o 813oo
190000 lZ 4 o o o 13o00o
lO 7 IOO
71 6 4 o 164000 1524oo 155 6 0 0
* H i g h e r c o m p o n e n t s w i t h s e d i m e n t a t i o n c o e f f i c i e n t s 6 . 2 - 8 . 8 S. ** H e a t e d f o r 9 h a t 3 ° 0 w i t h o u t s e n s i t i z e r . *** P h o t o o x i d i z e d a t p H 9 . 2 . HUMAN
Fraction y
Sens~izer
111, 2
--
S
---
111,2 111,2
-
111, 2 S S
~-GLOBULIN
II1,2, e t h a n o l p r o d u c t . S, s u l p h a t e p r o d u c t .
Temper~ure s°~o × ~o~3 Main%~ comp.
-
D2'° × z°7
7 .18
93
3.o6
227 ooo 312ooo
57 **~
-
Ms,D
175 ooo
-
-37**
MB MB E
Mw
IO IO IO
400 ooo 270 ooo 7.4 ° 7.32
74 74
*Higher components with the sedimentation r e v e a l s t r a c e s of t h o s e o f 19 S. * * H e a t e d f o r 8 h a t 37 °. * * * H e a t e d f o r I h a t 57 ° .
1.74
421 o o o
c o e f f i c i e n t of a b o u t lO, 5 S; t h e s u l p h a t e p r o d u c t
HUMAN FIBRINOGEN (SULPHATE PRODUCT) Sensitizer
Temperaure
-__ MB
-2 0 °* lO °
s°*° × z°*8
7.84 7.84
Main%*comp. D*'° × r°~
95
3-14 1.3o
Mw
240000 380000 500 ooo
Ms, D
242000 583 5 0 0
* A f t e r i i h a t 2 0 °.
of tyrosine 1°. With y-globulin and fibrinogen, samples photooxidized up to a consumption of about 7 ° moles of oxygen/mole of protein were employed. With y-globulin this consumption corresponds to a destruction of 15 moles of histidine, 16 of tyrosine, and 7 of tryptophan 1°. According to weight-average molecular weights obtained by using the lightscattering method, all the proteins under study, photooxidized at room temperature Biochim.
Biophys.
~4cta, 49 (1961) 4 9 5 - 5 o l
MOLECULAR WEIGHTS OF PHOTOOXIDIZED BLOOD PROTEINS
499
or at a slightly higher temperature, showed approximately twofold molecular weight increases (Table I). It was, however, evident from the molecular kinetic properties found by sedimentation analysis that with all the three proteins (HSA, y-globulin, and fibrinogen) the main fractions remained unchanged (Fig. I). Nevertheless there was a A 13
Fig. i. Sedimentation patterns. A, H S A photooxidized at 3 °o (5 ° moles of O~/mole) ; B, v-globulin photooxidized at IO ° (70 moles of O2/mole ).
marked increase in the amount of fractions with higher sedimentation coefficients, which were initially present in only negligible amounts. This was evidently due to secondary aggregation of protein molecules under the experimental conditions and it explained the increase in the weight-average molecular weights found by the lightscattering method. To verify the fact that photooxidation does not in itself cause any changes in molecular weights, an experiment with HSA was carried out to follow the influences of protein concentration, temperature and photooxidation time upon the amount of fractions with higher sedimentation coefficients. Whereas in photooxidation carried out at a temperature ranging from o to IO ° virtually no aggregated fractions are observed, at 4 °o 20 % of the HSA was already aggregated and the dependence-curve illustrating the increase with temperature in the aggregated fractions rose steeply (Fig. 2). Aggregation occurred at a temperature of 3 o°, the consumption of oxygen amounting to 25 moles/mole of protein (about 2.5 h is required), as can be seen from the time-dependence curve (Fig. 3). With further prolongation of the reaction time no more changes in weight-average molecular weights were observed. The extension of aggregation continued with rising protein concentration (Fig. 4) and was slightly lower when eosin was used as sensitizer instead of methylene blue. It was found that larger aggregation products could be separated from the solutions of photooxidized albumin by repeated precipitation with ammonium sulphate at 4 ° 9/0 saturation. The molecular weight of the remaining substance was normal. When the HSA-solution was heated at 3 °0 without sensitizer, no change in the molecular weight occurred, and it had not occurred even after IO h.,On the other hand, with y-globulin, a more labile protein, the molecular weight was nearly doubled after 8 h at 37°; and with fibrinogen the molecular weight kept on increasing even after standing for I I h at 20 °. After heating the y-globulin in solution for I h at 57 °, the Biochim. Biophys. Acta, 49 (1961) 495-5 ol
500
Z. V O D R ~ K A ,
O. MACH
molecular weight rose to 400 ooo. These observations show that photooxidation does not necessarily cause an increase of molecular weight, but that the aggregation observed with labile proteins is caused by the slight increase of the temperature during irradiation of their solutions while photooxidation proceeds. Although experiments M w. 10-3
20 m °/o
130
15 120
110
100
-I
10
I
I
.
20 30 40 T e m p e r a t u r e °C
Fig. ~.
90
80 i
Fig. 2. D e p e n d e n c e of t h e a m o u n t of t h e agg r e g a t e d p o r t i o n of H S A - - f o u n d b y s e d i m e n tation analysis---on the temperature during p h o t o o x i d a t i o n . H S A w a s p h o t o o x i d i z e d till 5 ° moles of O~/mole of p r o t e i n were c o n s u m e d .
~o
20
30
i
40 02/mole
50
Fig. 320 %
Fig. 3. D e p e n d e n c e of t h e w e i g h t - a v e r a g e of m o l e c u l a r w e i g h t of H S A on t h e degree of p h o t o o x i d a t i o n , e x p r e s s e d in t e r m s of o x y g e n consumption.
Fig. 4. D e p e n d e n c e of t h e a m o u n t of t h e aggreg a t e d p o r t i o n of H S A on t h e a l b u m i n concent r a t i o n in p h o t o o x i d a t i o n . H S A w a s p h o t o oxidized till 5o moles of O~/mole of t h e p r o t e i n were c o n s u m e d .
15
10
./ t 0.5
f
I 10
1 1.5 g / m l • 102
I 20
Fig. 4.
with HSA indicate that photooxidized proteins aggregate more readily, it is possible (by carrying out the reaction at a sufficiently low temperature or by removing the aggregated product) to prepare photooxidized albumin with its molecular weight unchanged. Methylene blue seemed to promote the aggregation of photooxidized proteins, although with HSA no difference between the sensitization by eosin and that by methylene blue was found in the course of the reactions 8, In this connection it is worth-while to mention the influence of photooxidation upon the extension of interaction between methylene blue and HSA. Whereas the native protein does not bind the methylene blue virtually at all 27, the photooxidized proteins form stable complexes with this dye, which cannot be completely removed, even b y long term dialysis. Biochim. Biophys. Acta, 49 (1961) 495-5Ol
MOLECULAR WEIGHTS OF PHOTOOXIDIZED BLOOD PROTEINS
501
It is of interest that the relation between the amount of methylene blue bound to a weight unit of HSA and the degree of photooxidation follows a dependence similar to that of weight-average molecular weight. The opinion that methylene blue is bound to the aggregates being formed is also confirmed by precipitation with ammonium sulphate. Most of the methylene blue was then contained in precipitates, whereas the supernatant, containing the molecules of photooxidized HSA with normal molecular weight, was nearly colourless. In addition, the possibility cannot be excluded that the methylene blue is responsible for the albumin aggregation, v-globulin and fibrinogen, however, do not bind methylene blue either in the native or photooxidized state and eosin is bound to the albumin ~3, the influence of photooxidation showing no marked effect. All the photooxidation experiments were carried out at pH 7.I and with HSA, for comparison, also at pH 9.2. No changes in the amino acid composition of HSA were observed during photooxidation at these two pH's. The change in pH influenced only the reaction rate. The weight-average molecular weight found for HSA photooxidized at pH 9.2 and at 3 °0 amounts to 15o ooo-19o ooo, which represents an increase similar to that observed after photooxidation at pH 7.1. ~ P O N A R A N D LOgTICK~ ~s, however, reported that in borate buffer (such as that used in this work) aggregation of native albumin occurs, probably because complexes with the borate are formed. They recorded, by the light-scattering method, a molecular weight of 19o ooo. REFERENCES 1 L. WEIL AND A. R. BUCHERT, Arch. Biochem. Biophys., 34 (1941) I ; Federation Proc., z I (1952) 307. 2 L. WEIL, S. JAMES AND A. R. BUCHERT, Arch. Biochem. Biophys., 46 (1953) 266. a L. WEIL AND T. S. SEIBLES, Arch. Biochem. Biophys., 54 (1955) 368. 4 L. WEIL, A. R. BUCHERT AND J. MAHER, Arch. Biochem. Biophys., 4o (1952) 245. 5 Z. VODR~.~KA AND R. PRAUS, Chem. listy, 49 (1955) 127; Collection Czechoslov. Chem. Communs., 20 (1955) 598 . 6 R. PRAUS, Chem. listy, 49 (1955) 921; Collection Czechoslov. Chem. Communs., 2o (1955) 1269. 7 Z. VODR~KA AND J. ~PONAR, Chem. listy, 49 (1955) lO69; Collection Czechoslov. Chem. Communs., 20 (1955) 1281. 8 Z. VODR/~KA AND J. ~PONAR, Chem. listy, 51 (1957) 1649; Collection Czechoslov. Chem. Communs., 23 (1957) 229. 9 Z. VODR~KA AND K. PI~ISTOUI'ILOV,t,,Chem. listy, 51 (1957) 1657; Collection Czechoslov. Chem. Communs., 23 (1958) 752. 10 H. SMETANA AND n . SHEMIN, J. Exptl. Med., 73 (194 I) 223. 11 R. EGAN, H, O. MICHEL, R. SCHLUETER AND B. J. JANDORF, Arch. Biochem. Biophys., 66 (1957) 366. 12 T. V. VENKgTERN, Biokhimiya, 18 (1953) 96. 13 Z.'VODR/~KA, Chem. listy, 53 (1959) 829. 14 Z. VODR/~KA, Fotodynamickf~ ]ev., St. Zdrav. Nakl., Praha, 1957, p. 42. 15 I. ASTRUP AND S. MOLLERTZ, Arch. Bioehem. Biophys., 4 ° (1952) 346. le H. T. MACPHERSON, Biochem. J., 36 (1942) 59. 17 K. LANG, z . physiol. Chem., Hoppe Seyler's, 222 (1933) 3. 18 H. P. LUNDGREEN AND W. H. WARD, in D. M. GREENBERG, A m i n o Acids and Proteins, T h o m a s , S p r i n g f i e l d , 1951, p. 331. 19 j . ~PONAR, Chem. listy, 53 (1959) 429 2o N. BENHAMOU AND Cx. WEIL, Biochim. Biophys. Acta, 24 (1957) 548. 21 G. E. PERLMANN AND L. G. LONGSWORTH, J. Am. Chem. Soc., 7° (1948) 2719. ~2 A. POLSON, Biochim. Biophys. Acta, 21 (1956) 185. 23 Z. VODR/~KA, Collection Czechoslov. Chem Communs., 25 (196o) 41o. 24 S. N. TIMASHEFF AND R. G. GIBBS, Arch. Biochem. Biophys., 7° (1957) 547. 25 R. SOWlNSEI, L. OHARENKO AND V. L. KOENIG, J. Am. Chem. Soc., 81 (1959) 6193. ~s R. SOWlNSKI, V. FREILING AND V. L. KOENIG, Makromol. Chem., 31 (196o) 152. 27 Z. VODR~KA AND J. ~PONAR, Chem. listy, 49 (1955) lO64; Chem. listy, 50 (1956) 860; Collection Czeehoslov. Chem. Communs., 20 (1955) 1275 ; CollectionCzechoslov. Chem. Communs., 22 (1957) 1241. ~8 j . ~PONAR AND C. LOgTICK¢/, Collection Czechoslov. Chem. Communs., 25 (196o) 159.
Biochim. Biophys. Acta, 49 (1961) 495-5 ol