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Effect of degradation on the chemical and biological properties of haptoglobin
42
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25545
E F F E C T OF D E G R A D A T I O N ON T H E CHEMICAL AND BIOLOGICAL P R O P E R T I E S OF H A P T O G L O B I N I. PRODUCTS OF T R Y P T I C D I G E S T I O N W'ANDA DOBRYSZYCKA AND ELW'IRA LISOWSI,2A
Department of Scientific Investigation of the 2VIedical Academy and Departmenl of Biochemistry, Institztte of Immunology and Experimental Therapy, Wroctaw (Poland) (Received September 22nd, 1965)
SUMMARY
Haptoglobin, an c%-sialomucoprotein of serum, was found to be an inhibitor of influenza virus haemagglutination. The inhibitory activities of the three main genetic types of haptoglobin were identical and found to be lO -I ~g per 4 haemagglutinating units. Haptoglobin was shown to be susceptible to trypsin (EC 3.4.4.4), as measured by the marked decrease in peroxidase activity of the complex of the trypsin-treated haptoglobin with haemoglobin, as well as by the corresponding rise in ninhydrin colour value. Hexose, hexosamine and sialic acid contents of trypsin-treated haptoglobin were determined, as well as biological activities, namely, the capacity to form a complex with haemoglobin having the properties of a "true" peroxidase and the ability to inhibit influenza virus haemagglutination. When trypsin-treated haptoglobin was fractionated on a column of Sephadex G-ioo, 3 glycopeptides were obtained; on a column of DEAE-Sephadex A-5o, 4 glycopeptides were obtained. It was shown that although tryptic digestion resulted in marked reduction in the activities examined, one glycopeptide was nevertheless found to retain the property of activating haemoglobin, and two glycopeptides inhibited haemagglutination to nearly the same degree as the native Hp. Analysis of the two glycopeptides indicated the absence of sialic acid.
INTRODUCTION
Hp is a well-defined acid c%-mucoprotein of serum, first identified by POLONOVSKI AND JAYLE1. Hp is characterized in part by its ability to bind Hb stoichiometrically and irreversibly. Hb is a pseudoperoxidase because its peroxidase activity is a logarithmic function of the concentration, whereas peroxidase activity of widely distributed plant and animal enzymes is linear within defined values. On the contrary the H p - H b complex exhibits catalytic activity of true peroxidase type and this has become the basis of the quantitative determination of Hp by JAYLE2. Abbreviations: Hp, haptoglobin; Hb, haemoglobin; HpT, trypsin-treated haptoglobin.
Biochim. Biophys. Acta, ~2t (I960) 42-5 °
PROPERTIES OF TRYPTIC GLYCOPEPTIDES OF HAPTOGLOBIN
43
As with many other mucoproteins, Hp was found to contain sialic acid susceptible to the action of neuraminidase. Removal of sialic acid from the Hp molecule neither affects formation of the H p - H b complex nor changes the peroxidase activity of the complex formed ~. Several mucoproteins with terminal sialic acid residues in their oligosaccharide chains are inhibitors of haemagglutination caused by influenza virus 4. It was therefore considered of interest to test Hp as a possible inhibitor of haemagglutination. The present report is concerned with the effect of degradation of the Hp molecule by trypsin (EC 3.4.4.4) on the phenomenon of "activation" of Hb as well as on the virus haemagglutination inhibitory properties of Hp. The main purpose of the study has been to define the chain portion indispensable to form with Hb a complex having the properties of a true peroxidase. At the same time virus haemagglutinin inhibition by the products of the tryptic digestion of Hp was examined.
MATERIAL AND METHODS Materials Hydrogen peroxide was obtained from AB Ferrosan, Malm6 (Sweden), Sephadex G-25, Sephadex G-ioo, Sephadex G-2oo and DEAE-Sephadex A-5o from Pharmacia, Uppsala (Sweden). DEAE-cellulose DE II "column chromedia" was a Whatman product. N-Acetylneuraminic acid was from L. Light & Co. The Lee strain (B) of the influenza virus and chicken erythrocytes were kindly supplied by Professor Z. SKURSKAfrom the Institute of Immunology and Experimental Therapy in Wroctaw. Trypsin, twice crystallized, was a Worthington product. Neuraminidase from Vibrio cholerae was supplied by Behringwerke. Ascitic fluids used for preparation of haptoglobin were obtained from patients of the First Clinic of Internal Diseases of the Medical Academy in Wroclaw. Hp was prepared from ascitic fluid by precipitation with ammonium sulfate. Material, precipitated by 40--55 % saturated ammonium sulfate (Hp2_ 1 and Hp2_2) or by 50-65 % saturated ammonium sulfate (HpI_I) according to LAURELL5, was collected, dialysed first against water, then against o.oi M acetate buffer (pH 4.7), and finally submitted to chromatography on DEAE-cellulose, as described by SMITH, EDMAN AND OWEN6. Further purification of Hp was effected by gel filtration on Sephadex G-2oo in o.I M Tris buffer (pH 8.0) containing o.I M NaC1 according to KILLANDER7. A column of 4.3 cm × 5o cm was used for a sample size of 4 ml, and a flow rate of 0.2 ml/min. After ultrafiltration, dialysis and lyophilization, only such fractions of haptoglobin were used for the present study that showed a purity of near IO o % as assayed by the peroxidase method of JAYLE2 (compared with the total protein estimation), and by starch-gel electrophoresis. Hb was prepared from horse erythrocytes by the method of McQUARRIE AND BENIAMSs. Analytical methods Hb was determined by JAYLE'S method 2 utilizing the specific peroxidase activity of the H p - H b complex formed. Protein was assayed by the tannin turbidimetric micromethod of MEJBAUMKATZENELLENBOGEN9, or in free Hp preparations by the absorbance at 278 m/~ using Biochim. Biophys. Acta, 121 (I966) 42-5 o
44
W. DOBRYSZYCKA, E. LISOWSKA
extinction coefficients for Hpl_ 1 of 1.15, for Hp2_ 1 of 1.16 and for Hp2 2 of 1.18, respectively 1°. The ninhydrin reaction was performed according to MOORE AND STEIN 11, using a final volume of 4 ml (i ml of sample, 0.6 ml of ninhydrin reagent and 2.4 ml of 60 7/'o propanol). L-Leucine was used as standard. Conditions for trypsin digestion were as follows. Samples contained o.5 % of trypsin, referred to Hp, dissolved in o.02 M NaHCO 3 (pH 7.5). Time of incubation 5-6 h at 37 °. After incubation the mixture was desalted by gel filtration on a Sephadex G-25 column (2.3 cm >l 30 cm) and lyophilized. Carbohydrates were assayed by the following methods: the indole method 12 employing galactose as standard for estimation of sugars, hexosamine excluded; hexosamine was determined by the method of RONDI~E AND MORGANla (after hydrolysis of the material with 0.5 N HC1 for 16 h at IOO~) using glucosamine hydrochloride as standard; sialic acid was estimated according to SVENYERI~OLM~ by the resorcinol method, N-acetylneuraminic acid being used as standard. For the determination of the inhibitory activity against influenza virus haemagglutinins, the Lee strain (B), inactivated by 3o-min incubation at 5 6 , was used. Virus haemagglutinin inhibitory assays were carried out according to TAMM A~D HORSFALL~5, using chicken erythrocytes. One haemagglutinating unit is that amount of virus which causes partial agglutination of o.o25 ml 5 % (v/v) chicken erythrocytes. Paper electrophoresis was carried out at pH 8.6 in v e r o n a l - a c e t a t e buffer. at pH 7.o in phosphate buffer and at p H 4-5 in acetate buffer.
RESULTS
Inhibitory activity of Hp against i~fluenza virus haemagglutinatio~ Purified preparations of three main genetic types of Hp, namely Hpl-1, Hp~-I and Hp2- 2, were examined for inhibitory activity of haemagglutination caused bv the influenza virus. Complexes of the three types of Hp with Hb, prepared b y adding H b in an equimolar ratio to give fully saturated H p - H b complexes, were also examined for the virus haemagglutinin inhibition. All six preparations showed identical inhibitory activity, that is they were found to inhibit agglutination of chicken erythrocytes at a concentration of o.I/~g/4 haemagglutinating units of the virus. The enzymic release of the sialic acid residues from H p or from their complexes with Hb, b y means of neuraminidase from Vibrio cholerae, resulted in a reduction in the inhibitory activity. Haemagglutination was inhibited by the concentration of Hp of 25/~g/4 haemagglutinating units of the virus.
Tryptic digestion of Hp During a 6-h digestion of Hp by trypsin, quantitative ninhydrin determinations, as well as determinations of total protein and peroxidase activity of H p - H b complex, were carried out. The experimental results, given in Fig. I, show that after 6 t1 of tryptic digestion, peroxidase activity of H p - H b complex, as measured according to JAYLE~ and then calculated in milligrams of "active" Hp, was reduced to practically nil; total protein level, however, did not change during the experiment. In the same time there was a release of o.8 mequiv of leucine that accounted for about 13 o/~oof the peptide bonds split off. Biochim. Biophys. Acta, 12i (1966) 42-5 °
45
PROPERTIES OF TRYPTIC GLYCOPEPTIDES OF HAPTOGLOBIN
Chemical and biological changes following trypsin action on lip After incubation of Hp with trypsin, the mixture was desalted and separated from components of molecular weight less than 5000 by gel filtration on a Sephadex G-25 column. The main fraction was lyophilized and analysed for carbohydrate
o.6i-~ z. ._c ~ O.d
~6~.
>
0 •
o E D.3 .c
o o
~.~c~ -6._> i--- ~
I/ V
UO
J 1
I
2
I 3 "rime [h)
z ~
Fig. I. T r y p s i n action on H p as a function of time. Composition of the sample: IOO mg of H p dissolved in io ml o.o2 M NaHCOa containing 0. 5 mg of trypsin. W a t e r b a t h at 37 °. x - - x , total protein determined b y the t a n n i n micromethod°; O - - © , concentration of " a c t i v e " H p measured b y the peroxidase m e t h o d of JAYLE 2, 0 - - 0 , n i n h y d r i n colour value according to MOORE AND S T E I N 11. TABLE I CARBOHYDRATE CONTENT AND BIOLOGICAL ACTIVITIES OF NATIVE AND TRYPSINIZED HAPTOGLOBIN Q u a n t i t a t i v e d e t e r m i n a t i o n s of sialic acid NANA,hexose (as galactose) and h e x o s a m i n e as in METHODS. The inhibitory activity aganst influenza virus is given as the minimal a m o u n t (/zg) t h a t completely inhibits 4 h a e m a g g l u t i n a t i n g units of virus (see M E T H O D S ) . Active H p was measured b y the peroxidase m e t h o d of J A Y L E 2 and compared with protein content of the sample as determined spectrophotometrically.
Haptoglobin
Trypsinized haptoglobin
NANA, (%)
5.0
6.7
Hexose* (%)
lO.2
II.0
H e x o s a m i n e (%)
4.6
4.9
I n h i b i t o r y activity #/4 h a e m a g g l u t i n a t i n g units
o.12
4.o
Active Hp, per cent of the protein content of the sample
98.0
0.86
* As galactose.
content as well as for biological activities. The results, compared with these obtained for native Hp, are presented in Table I. As may be seen from Table I, tryptic digestion of Hp results in almost complete loss of peroxidase activity. Haemagglutinin inhibitory activity decreased 30 times. The contents of sialic acid (NANA), hexose and hexosamine were a little higher in HpT than in native Hp.
Paper electrophoresis of Hp T A scheme showing the results of electrophoresis of HpT in comparison with native Hp at different pHs is given in Fig. 2. Biochim. Biophys. $cta, 121 (1966) 42-5o
46
w. DOBRYSZYCKA, E. LISOWSKA
H p T was s e p a r a t e d at a l k a l i n e p H i n t o t w o f r a c t i o n s . T h e first e x h i b i t e d t h e e l e c t r o p h o r e t i c m o b i l i t y of t h e n a t i v e H p , t h e s e c o n d m i g r a t e d f a s t e r t o w a r d s t h e a n o d e . B o t h f r a c t i o n s c o u l d be d y e d in t h e s a m e w a y w i t h b r o m p h e n o l b l u e as w i t h n i n h y d r i n . T h e e l e c t r o p h o r e t i c p a t t e r n p e r f o r m e d at p H 7.o a f t e r H b h a d b e e n a d d e d to H p or to H p T , i l l u s t r a t e s t h e H b b i n d i n g c a p a c i t y of H p T . P a p e r strips were s t a i n e d w i t h b e n z i d i n e a n d h y d r o g e n p e r o x i d e in o r d e r to o b s e r v e H p - b o u n d H b (free H b r e m a i n e d on t h e line of a p p l i c a t i o n ) . A s m a l l a m o u n t of H b b o u n d w i t h H p T was f o u n d to h a v e t h e m o b i l i t y c h a r a c t e r i s t i c of t h e H p - H b c o m p l e x at this pH. I n t e r e s t i n g results w e r e o b t a i n e d on e l e c t r o p h o r e t i c s e p a r a t i o n of H p T at p H 4.5. O n p a p e r strips s t a i n e d w i t h b r o m p h e n o l b l u e o n l y 3 f r a c t i o n s w e r e p r e s e n t , b u t on the paper strip with the same sample applied and stained with ninhydrin, 9 fractions of d i f f e r e n t m o b i l i t i e s w e r e visible. The 9 fractions were cut out from the corresponding undyed electrophoretic p a p e r s t r i p o b t a i n e d at p H 4-5, t h e p r o t e i n was e l u t e d i n t o a few millilitres of dist i l l e d w a t e r a n d c o n c e n t r a t e d in a d e s i c c a t o r o v e r P 2 Q - A f t e r t h e a d d i t i o n of H b t h e c o n c e n t r a t e d e l u a t e s w e r e e x a m i n e d for p e r o x i d a s e a n d h a e m a g g l u t i n a t i o n inh i b i t o r y a c t i v i t y . F r a c t i o n s d - i (Fig. 2) w e r e f o u n d to be i n h i b i t o r s of h a e m a g g l u t i n a t i o n , c o m p l e x e s of t h e F r a c t i o n s e - g w i t h H b s h o w e d w e a k p e r o x i d a s e a c t i v i t y , a n d c a t h o d i c F r a c t i o n s a - e h a d no effect. ®
® I
1.2
2
1.0 4
:3~ OB 5
0
6
~ 0,6
8 m
go2 c
10 a
b
c
d
ef
g
h
[
20 Tube n u m b e r
30
Fig. 2. Paper electrophoretic patterns of native and of trypsinized Hp/HpT/ at different pH. i, 2, 3, 4, barbiturate buffer (pH 8.6) ; I, 2, Hp ; 3,4, HpT. I, 3, were stained with bromophenol blue and 2, 4, with o.2 % ninhydrin in acetone. 5, 6, Phosphate buffer (pH 7.o); 5, Hp with addition of an equimolar amount of Hb; 6, HpT with Hb. 5 and 6 stained with 2 °/o benzidine in 2o % acetic acid with hydrogen peroxide. 7,8,9, Io, Acetate buffer (pH 4.5); 7, 8, Hp; 9, io, HpT. 7,9 were stained with bromophenol blue and 8, IO, with ninhydrin. Electrophoresis was carried out for 6 h at 22o V. Location of the origin on all the strips is indicated by the arrow. The samples were applied 2 cm from the centre of strips toward the cathode. Details of trypsin digestion and preparation of samples as in NIETHODS. Fig. 3- Fractionation of trypsinized Hp on a Sephadex G-ioo colunln. 3° nlg of lyophilized preparation of Hp, after tryptie digestion and subsequent gel filtration on a Sephadex G-25 colunln, was dissolved in 2 nil of distilled water and applied to the column (2.3 × 30 cm). Effluent fractions of 4 nil were collected at 4 ° with a flow rate of io ml/h. Isolated fractions 7~9, lO-16 and 17-21 were called gtycopeptides l, II and I l l (Sephadex G-Ioo) respectively. Biochirn. Biophys. Acta, 12i (1966) 42-5 °
PROPERTIES OF TRYPTIC GLYCOPEPTIDES OF HAPTOGLOBIN
47
Fractionation of Hp T on Sephadex G-Ioo and DEAE-Sephadex A-5o columns H p T (30 mg in 2 ml of distilled water) was placed on a Sephadex G-Ioo column (2.3 cm x 30 cm) and submitted to gel filtration. A typical elution pattern is shown in Fig. 3. The separation was not satisfactory. Nevertheless the Fractions 7-9 (I), lO-16 (II) and 17-21 (nI) were lyophilized and stored for determination of carbohydrates and biological activities. The yield of the fractionation was as follows: I, I I rag; II, 7 rag; and III, 2 mg. H p T (90 mg in 2 ml of buffer) was placed on a DEAE-Sephadex column (0. 9 cm x 16 cm) buffered with 0.02 M phosphate buffer (pH 6.7) and eluted with increasing concentrations of NaC1 from o.I M to 0.5 M. A typical elution pattern is shown in Fig. 4.
IT
2-
m
1v
0.8 0.1M N(:]CI
0 0.6 m
~ a4 a2 I
2o
l-I
4b
O,2M NeCI
do
~to
Tube number
Fig. 4. F r a c t i o n a t i o n of H p T on a D E A E - S e p h a d e x A-5o column. 90 mg of lyophilized preparation of H p T was dissolved in 2 ml of o.2 1V[p h o s p h a t e buffer (pH 6.7) and applied to the column (0.9 cm × 16 cm) buffered with the same buffer and eluted with increased concentrations of NaC1, indicated b y arrows. Effluent fractions of 2 ml were collected at 4 ° w i t h a flow rate of IO ml/h. Isolated fractions: 3-20, 28-41, 52-62 and 70-80 were called glycopeptides I, II, I I I and I V (DEAE-Sephadex) respectively.
Four peaks were obtained in this fractionation (I-IV). The fractions comprising individual peaks were lyophilized, desalted b y gel filtration on a Sephadex G-25 column and lyophilized once more. The yield of the fractionation was as follows: I, 3 ° mg; II, IO rag; I I I , 4 mg; and IV, 6 mg. Carbohydrate content, peroxidase activity and haemagglutination inhibitory activity were determined on the three fractions obtained b y means of fractionation from a Sephadex G-Ioo column as well as in the four fractions obtained from the DEAE-Sephadex column. Results of the determinations are assembled in Table II. Fraction I (Sephadex G-Ioo) and Fraction IV (DEAE-Sephadex) were found to have the ability to activate Hb, i.e. their complexes with Hb gave a characteristic increase in peroxidase activity, nearly the same for each fraction. The two fractions contained different amounts of sugars. Fraction IV (DEAE-Sephadex) contained only half the amount of hexose in Fraction I (Sephadex G-Ioo); moreover no NANA was found in the carbohydrate moiety of this glycopeptide. Peroxidase activities of Hb complexes with glycopeptide I(Sephadex G-Ioo) and with glyeopeptide IV (DEAESephadex) were much higher than the activity of H p T before fractionation, as calcuBiochim. Biophys. Acta, i 2 i (I966) 42-50
48
W. D O B R Y S Z Y C K A , E. L I S O W S K A
TABLE
II
CARBOHYDRATE OF
CONTENT
AND
BIOLOGICAL
ACTIVITIES
OF THE
PRODUCTS
OF
TRYPTIC
DIGESTION
HAPTOGLOBIN
C o n d i t i o n s of q u a n t i t a t i v e d e t e r m i n a t i o n s a s in T a b l e I. S e p a r a t i o n on Sephadex O-ioo and DEAE-Sephadex A - 5 o a s in t h e t e x t .
Expt. and number N A N A of fraction (°/o)
Sephadex
Hexose* (°//o)
Hexosamine (%)
of d i g e s t i o n p r o d u c t s
of H p
Inhibitory Active Hp, per cent activity of the protein (l*g/4 haernag- content of the sample glutinating units)
G-ioo
I I[ II~
5.1 5.1 o
lO.6 lo. 5 2.8
2 10 10
i8. 4 o o
25 5O-lOO o.8 0.8
o o i5.t
DEAE-Sephadex 1
ii.o
1[ l[I 1V
8. 5 o o
15.I 13. 5 6. 3 3.8
6,2 5,8 i.i 0.8
o
* As galactose.
lated per milligram of lyophilized preparation. Their peroxidase activities with Hb, however, were much smaller than that of native Hp. The strongest inhibitory haemagglutination activities were found in glycopeptide I (Sephadex G-Ioo) and in glycopeptides I I I and IV (DEAE-Sephadex). The activities were higher than that of H p T before fractionation but lower than that of native Hp. Glycopeptides I I I and IV (DEAE-Sephadex) contained no NANA and rather low amounts of hexose and hexosamine. On the other hand, glycopeptides I and I I (DEAE-Sephadex), which were rich in sugars, neither acted as Hb activators nor were haemagglutination inhibitors. DISCUSSION
It is now known that all inhibitors of influenza virus haemagglutination are glycoproteins, the carbohydrate groups of which are terminated by sialic acid residues. The enzymic release of sialic acid results in a reduction in the inhibitory titre 16. This opinion was confirmed in the experiment on the action of neuraminidase on Hp. However, in addition to the presence of sialic acid residues, mucoproteins must possess other structural features in order to qualify as efficient inhibitors. It has been suggested that such an essential factor might be the molecular size of sialomucoproteins. Strong inhibitors have molecular weights of the order of Io5-1o6. MORAWIECKI AND LISOWSKA17 stated that after polymerization of orosomucoid its virus inhibitory activity increased from 5" IO-I to 8. IO 4/*g/4 haemagglutinating units. Recently it has been firmly established that the phenotype HpI_ 1 produces a protein or proteins which appear homogeneous ; however both other types are believed to be serial polymers of basic units differing only slightly in amino acid composition 18. In the present work all the three main genetic types of H p exhibited indentical inhibitory influenza virus haemagglutination activity. Neither molecular Biochim. Biophys. Acta, 121 (1966) 4 2 - 5 °
PROPERTIES OF TRYPTIC GLYCOPEPTIDES OF HAPTOGLOBIN
49
size nor binding of Hb to Hp affected the inhibitory activity. Binding of Hb does not occur through the carbohydrate moiety of Hp, but it is the cause of the same inhibitory activities of Hps and of their complexes with Hb. In view of the opinion on the role of sialic acid in the mechanism of the inhibitory action of sialomucoproteins, it is of interest that some glycopeptides obtained by tryptic digestion of Hp that did not contain any NANA in their carbohydrate moiety (glycopeptides III and IV (DEAE-Sephadex) still exerted almost the same inhibition as native Hp containing 5 To of NANA and much higher inhibition than for example glycopeptide I (DEAE-Sephadex) which contained about IO % of NANA). It seems likely that other sugar replaces NANA in the combination of influenza virus with Hp and with the particulate cellular receptors at the surface of the red-blood cell. Hp resulted in a marked increase in the number of terminal NH2-groups (ninhydrin reaction) as well as in a corresponding loss of peroxidase and haemagglutination inhibitory activities. GOTTSCHALK AND FAZEKAS DE ST. GROTH 16 reported that sialoglycopeptides resulting from tryptic digestion of ovine submaxillary glycoprotein were devoid of measurable inhibitory activity. According to the results of the present paper, although the direct product of tryptic digestion (HpT) showed low inhibitory activity as well as peroxidase activity in the complex with Hb, however, some glycopeptides after separation were found to be almost as strong inhibitors as the native Hp and exhibited measurable peroxidase activity when complexed with Hb. During tryptic digestion, in spite of the fragmentation of the haptoglobin molecule, the level of protein as measured by the tannin micromethod 9 remained constant during the course of the experiment. Tannic acid precipitates polypeptides of molecular weight above 2000*. It may be concluded that the amount of peptides and glycopeptides of molecular weight below 2000 arising from Hp by tryptic action did not exceed i0 % of the total protein, this amount being less than the standard error of the method. CHEFTEL et al. TM isolated glycopeptides resulting from hydrolysis of Hp by proteolytic enzymes. Their results cannot be compared with those presented in this paper because conditions of hydrolysis and different methods of separation of glycopeptides were different. The mechanism of activation of Hb by the action of Hp has not been elucidated. Hp is believed to ensure the regularity of the kinetics of catalysis by the H p - H b complex--which may be described as an enzyme--especially proportionality between velocity and concentration of the peroxidase and applicability of the Michaelis equation to the enzyme. The physico-chemical basis of the phenomenon has been a subject of interest (see refs. 20, 21). In the present work only one glycopeptide, a product of the action of trypsin on Hp, was found to retain the ability to activate Hb (glycopeptide I (Sephadex G-IOO) corresponding to IV (DEAE-Sephadex)). It would be interesting to have information about the size and composition of the smallest fragment of Hp molecule able to change Hb into "true" peroxidase. In view of the experiments on the reductive cleavage of disulphide bonds of Hp 2~, as well as those of the present paper, it seems likely that in the formation of the H p - H b complex having the properties of true peroxidase, the main role is played not by the secondary structure but rather by the primary one, namely by some essential amino acid corn* P e r s o n a l c o m m u n i c a t i o n from W. MEJBAUM-I'{ATZENELLENBOGEN.
Biochim. Biophys. Acta, 121 (1966) 4 2 - 5 °
5°
W. DOBRYSZYCKA, E. LISOWSKA
position and sequence in the peptide chain fragment of Hp necessary for binding and activating Hb. A further analytical approach to these interesting questions would appear worthwhile. REFERENCES I 2 3 4 5 6 7 8 9 lO It 12 13 14 15 16 17 18 19 20 21 22
M. POLONOVSKI AND M. F. JAYLE, Compt. Rend. Soc. Biol., 129 (1938) 457M. F. JAYLE, Bull. Soc. Chim. Biol., 33 (1951) 876]N/[.E. RAFELSON, Jr., L. CLOAREC, J. MORETTI AND M. F. JAYLE, Nature, 191 (1961) 279. S, FAZEKAS DE ST. GROTH AND A. GOTTSCHALK, Biochim. Biophys. Aeta, 78 (1963) 248. C. B. LAURELL, Clin. Chim. Acta, 4 (1959) 79. H. SMITH, P. EDMAN AND J. A. OWEN, Nature, 193 (I962) 286. J. KILLANDER, Biochim. Biophys. Acta, 93 (1964) I. E. B. MCQUARRIE AND H. N. BENIAMS, Proc. Soc. Exptl. Biol. Med., 86 (1954) 627. W. 1V~EJBAUM-KATZENELLENBOGEN,Acta Biochim. Polon., 2 (1955) 279. L. CLOAREC,Contribution ~ l'Etude Physico-Chimique des Haptoglobines Humaines, h/Iprinaeric R. F o u l o n et Cie., Paris, 1964, p. 6. S. MOORE AND W. H. STEIN, J. Biol. Chem., 211 (1954) 907. Z. DISCHE, in D. GLICK, Methods of Biochemical Analysis, Vol. 2, lnterscience, New York, 1955, p. 313 . C. J. M. RONDLE AND \V. T. J. MORGAN, Biochem. J., 61 (I955) 586. L. SVENNERHOLM, Biochim. Biophys. Acta, 24 (1957) 004i. TAMM AND F. L. HORSFALL, J. Expll. Med., 95 (1952) 71. A. GOTTSCHALK AND S. FAZEKAS DE ST. GROTH, Biochim. Biophys. Acta, 43 (196o) 513 • A. MORAWlECKI AND E. LISOWSKA, Biochem. Biophys. Res. Commun., 18 (1965) 606. L. CLOAREC, Contribution ~'~l'Etude Physico-Chimique des Haptoglobines Humaines, hnprinlerie R. F o u l o n et Cie, Paris, 1964, p. 39. R. I. CHEFTEL, L. CLOAREC, J. MORETTI AND M. F. JAYLE, Bull. Soc. Chim. Biol., 47 (1965) 385 . J. MORETTI AND J. YON, Biochim. Biophys. Acta, 46 (1961) 545. \¥. DOBRYSZYCKA, Arch. Immunol. Terapii Experimentalis, 14 (1966) 561. E. LISOXVSKAAND \¥. DOBRYSZYCKA, Abstracts Third Meeting .FEES, Meeting edition, P,,lish Scient. Public. \Varsaw, 1966, No. FI52, p. 256.
Biochim. Biophys. Acta, I2 t (1966) 42-5 o
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25545
E F F E C T OF D E G R A D A T I O N ON T H E CHEMICAL AND BIOLOGICAL P R O P E R T I E S OF H A P T O G L O B I N I. PRODUCTS OF T R Y P T I C D I G E S T I O N W'ANDA DOBRYSZYCKA AND ELW'IRA LISOWSI,2A
Department of Scientific Investigation of the 2VIedical Academy and Departmenl of Biochemistry, Institztte of Immunology and Experimental Therapy, Wroctaw (Poland) (Received September 22nd, 1965)
SUMMARY
Haptoglobin, an c%-sialomucoprotein of serum, was found to be an inhibitor of influenza virus haemagglutination. The inhibitory activities of the three main genetic types of haptoglobin were identical and found to be lO -I ~g per 4 haemagglutinating units. Haptoglobin was shown to be susceptible to trypsin (EC 3.4.4.4), as measured by the marked decrease in peroxidase activity of the complex of the trypsin-treated haptoglobin with haemoglobin, as well as by the corresponding rise in ninhydrin colour value. Hexose, hexosamine and sialic acid contents of trypsin-treated haptoglobin were determined, as well as biological activities, namely, the capacity to form a complex with haemoglobin having the properties of a "true" peroxidase and the ability to inhibit influenza virus haemagglutination. When trypsin-treated haptoglobin was fractionated on a column of Sephadex G-ioo, 3 glycopeptides were obtained; on a column of DEAE-Sephadex A-5o, 4 glycopeptides were obtained. It was shown that although tryptic digestion resulted in marked reduction in the activities examined, one glycopeptide was nevertheless found to retain the property of activating haemoglobin, and two glycopeptides inhibited haemagglutination to nearly the same degree as the native Hp. Analysis of the two glycopeptides indicated the absence of sialic acid.
INTRODUCTION
Hp is a well-defined acid c%-mucoprotein of serum, first identified by POLONOVSKI AND JAYLE1. Hp is characterized in part by its ability to bind Hb stoichiometrically and irreversibly. Hb is a pseudoperoxidase because its peroxidase activity is a logarithmic function of the concentration, whereas peroxidase activity of widely distributed plant and animal enzymes is linear within defined values. On the contrary the H p - H b complex exhibits catalytic activity of true peroxidase type and this has become the basis of the quantitative determination of Hp by JAYLE2. Abbreviations: Hp, haptoglobin; Hb, haemoglobin; HpT, trypsin-treated haptoglobin.
Biochim. Biophys. Acta, ~2t (I960) 42-5 °
PROPERTIES OF TRYPTIC GLYCOPEPTIDES OF HAPTOGLOBIN
43
As with many other mucoproteins, Hp was found to contain sialic acid susceptible to the action of neuraminidase. Removal of sialic acid from the Hp molecule neither affects formation of the H p - H b complex nor changes the peroxidase activity of the complex formed ~. Several mucoproteins with terminal sialic acid residues in their oligosaccharide chains are inhibitors of haemagglutination caused by influenza virus 4. It was therefore considered of interest to test Hp as a possible inhibitor of haemagglutination. The present report is concerned with the effect of degradation of the Hp molecule by trypsin (EC 3.4.4.4) on the phenomenon of "activation" of Hb as well as on the virus haemagglutination inhibitory properties of Hp. The main purpose of the study has been to define the chain portion indispensable to form with Hb a complex having the properties of a true peroxidase. At the same time virus haemagglutinin inhibition by the products of the tryptic digestion of Hp was examined.
MATERIAL AND METHODS Materials Hydrogen peroxide was obtained from AB Ferrosan, Malm6 (Sweden), Sephadex G-25, Sephadex G-ioo, Sephadex G-2oo and DEAE-Sephadex A-5o from Pharmacia, Uppsala (Sweden). DEAE-cellulose DE II "column chromedia" was a Whatman product. N-Acetylneuraminic acid was from L. Light & Co. The Lee strain (B) of the influenza virus and chicken erythrocytes were kindly supplied by Professor Z. SKURSKAfrom the Institute of Immunology and Experimental Therapy in Wroctaw. Trypsin, twice crystallized, was a Worthington product. Neuraminidase from Vibrio cholerae was supplied by Behringwerke. Ascitic fluids used for preparation of haptoglobin were obtained from patients of the First Clinic of Internal Diseases of the Medical Academy in Wroclaw. Hp was prepared from ascitic fluid by precipitation with ammonium sulfate. Material, precipitated by 40--55 % saturated ammonium sulfate (Hp2_ 1 and Hp2_2) or by 50-65 % saturated ammonium sulfate (HpI_I) according to LAURELL5, was collected, dialysed first against water, then against o.oi M acetate buffer (pH 4.7), and finally submitted to chromatography on DEAE-cellulose, as described by SMITH, EDMAN AND OWEN6. Further purification of Hp was effected by gel filtration on Sephadex G-2oo in o.I M Tris buffer (pH 8.0) containing o.I M NaC1 according to KILLANDER7. A column of 4.3 cm × 5o cm was used for a sample size of 4 ml, and a flow rate of 0.2 ml/min. After ultrafiltration, dialysis and lyophilization, only such fractions of haptoglobin were used for the present study that showed a purity of near IO o % as assayed by the peroxidase method of JAYLE2 (compared with the total protein estimation), and by starch-gel electrophoresis. Hb was prepared from horse erythrocytes by the method of McQUARRIE AND BENIAMSs. Analytical methods Hb was determined by JAYLE'S method 2 utilizing the specific peroxidase activity of the H p - H b complex formed. Protein was assayed by the tannin turbidimetric micromethod of MEJBAUMKATZENELLENBOGEN9, or in free Hp preparations by the absorbance at 278 m/~ using Biochim. Biophys. Acta, 121 (I966) 42-5 o
44
W. DOBRYSZYCKA, E. LISOWSKA
extinction coefficients for Hpl_ 1 of 1.15, for Hp2_ 1 of 1.16 and for Hp2 2 of 1.18, respectively 1°. The ninhydrin reaction was performed according to MOORE AND STEIN 11, using a final volume of 4 ml (i ml of sample, 0.6 ml of ninhydrin reagent and 2.4 ml of 60 7/'o propanol). L-Leucine was used as standard. Conditions for trypsin digestion were as follows. Samples contained o.5 % of trypsin, referred to Hp, dissolved in o.02 M NaHCO 3 (pH 7.5). Time of incubation 5-6 h at 37 °. After incubation the mixture was desalted by gel filtration on a Sephadex G-25 column (2.3 cm >l 30 cm) and lyophilized. Carbohydrates were assayed by the following methods: the indole method 12 employing galactose as standard for estimation of sugars, hexosamine excluded; hexosamine was determined by the method of RONDI~E AND MORGANla (after hydrolysis of the material with 0.5 N HC1 for 16 h at IOO~) using glucosamine hydrochloride as standard; sialic acid was estimated according to SVENYERI~OLM~ by the resorcinol method, N-acetylneuraminic acid being used as standard. For the determination of the inhibitory activity against influenza virus haemagglutinins, the Lee strain (B), inactivated by 3o-min incubation at 5 6 , was used. Virus haemagglutinin inhibitory assays were carried out according to TAMM A~D HORSFALL~5, using chicken erythrocytes. One haemagglutinating unit is that amount of virus which causes partial agglutination of o.o25 ml 5 % (v/v) chicken erythrocytes. Paper electrophoresis was carried out at pH 8.6 in v e r o n a l - a c e t a t e buffer. at pH 7.o in phosphate buffer and at p H 4-5 in acetate buffer.
RESULTS
Inhibitory activity of Hp against i~fluenza virus haemagglutinatio~ Purified preparations of three main genetic types of Hp, namely Hpl-1, Hp~-I and Hp2- 2, were examined for inhibitory activity of haemagglutination caused bv the influenza virus. Complexes of the three types of Hp with Hb, prepared b y adding H b in an equimolar ratio to give fully saturated H p - H b complexes, were also examined for the virus haemagglutinin inhibition. All six preparations showed identical inhibitory activity, that is they were found to inhibit agglutination of chicken erythrocytes at a concentration of o.I/~g/4 haemagglutinating units of the virus. The enzymic release of the sialic acid residues from H p or from their complexes with Hb, b y means of neuraminidase from Vibrio cholerae, resulted in a reduction in the inhibitory activity. Haemagglutination was inhibited by the concentration of Hp of 25/~g/4 haemagglutinating units of the virus.
Tryptic digestion of Hp During a 6-h digestion of Hp by trypsin, quantitative ninhydrin determinations, as well as determinations of total protein and peroxidase activity of H p - H b complex, were carried out. The experimental results, given in Fig. I, show that after 6 t1 of tryptic digestion, peroxidase activity of H p - H b complex, as measured according to JAYLE~ and then calculated in milligrams of "active" Hp, was reduced to practically nil; total protein level, however, did not change during the experiment. In the same time there was a release of o.8 mequiv of leucine that accounted for about 13 o/~oof the peptide bonds split off. Biochim. Biophys. Acta, 12i (1966) 42-5 °
45
PROPERTIES OF TRYPTIC GLYCOPEPTIDES OF HAPTOGLOBIN
Chemical and biological changes following trypsin action on lip After incubation of Hp with trypsin, the mixture was desalted and separated from components of molecular weight less than 5000 by gel filtration on a Sephadex G-25 column. The main fraction was lyophilized and analysed for carbohydrate
o.6i-~ z. ._c ~ O.d
~6~.
>
0 •
o E D.3 .c
o o
~.~c~ -6._> i--- ~
I/ V
UO
J 1
I
2
I 3 "rime [h)
z ~
Fig. I. T r y p s i n action on H p as a function of time. Composition of the sample: IOO mg of H p dissolved in io ml o.o2 M NaHCOa containing 0. 5 mg of trypsin. W a t e r b a t h at 37 °. x - - x , total protein determined b y the t a n n i n micromethod°; O - - © , concentration of " a c t i v e " H p measured b y the peroxidase m e t h o d of JAYLE 2, 0 - - 0 , n i n h y d r i n colour value according to MOORE AND S T E I N 11. TABLE I CARBOHYDRATE CONTENT AND BIOLOGICAL ACTIVITIES OF NATIVE AND TRYPSINIZED HAPTOGLOBIN Q u a n t i t a t i v e d e t e r m i n a t i o n s of sialic acid NANA,hexose (as galactose) and h e x o s a m i n e as in METHODS. The inhibitory activity aganst influenza virus is given as the minimal a m o u n t (/zg) t h a t completely inhibits 4 h a e m a g g l u t i n a t i n g units of virus (see M E T H O D S ) . Active H p was measured b y the peroxidase m e t h o d of J A Y L E 2 and compared with protein content of the sample as determined spectrophotometrically.
Haptoglobin
Trypsinized haptoglobin
NANA, (%)
5.0
6.7
Hexose* (%)
lO.2
II.0
H e x o s a m i n e (%)
4.6
4.9
I n h i b i t o r y activity #/4 h a e m a g g l u t i n a t i n g units
o.12
4.o
Active Hp, per cent of the protein content of the sample
98.0
0.86
* As galactose.
content as well as for biological activities. The results, compared with these obtained for native Hp, are presented in Table I. As may be seen from Table I, tryptic digestion of Hp results in almost complete loss of peroxidase activity. Haemagglutinin inhibitory activity decreased 30 times. The contents of sialic acid (NANA), hexose and hexosamine were a little higher in HpT than in native Hp.
Paper electrophoresis of Hp T A scheme showing the results of electrophoresis of HpT in comparison with native Hp at different pHs is given in Fig. 2. Biochim. Biophys. $cta, 121 (1966) 42-5o
46
w. DOBRYSZYCKA, E. LISOWSKA
H p T was s e p a r a t e d at a l k a l i n e p H i n t o t w o f r a c t i o n s . T h e first e x h i b i t e d t h e e l e c t r o p h o r e t i c m o b i l i t y of t h e n a t i v e H p , t h e s e c o n d m i g r a t e d f a s t e r t o w a r d s t h e a n o d e . B o t h f r a c t i o n s c o u l d be d y e d in t h e s a m e w a y w i t h b r o m p h e n o l b l u e as w i t h n i n h y d r i n . T h e e l e c t r o p h o r e t i c p a t t e r n p e r f o r m e d at p H 7.o a f t e r H b h a d b e e n a d d e d to H p or to H p T , i l l u s t r a t e s t h e H b b i n d i n g c a p a c i t y of H p T . P a p e r strips were s t a i n e d w i t h b e n z i d i n e a n d h y d r o g e n p e r o x i d e in o r d e r to o b s e r v e H p - b o u n d H b (free H b r e m a i n e d on t h e line of a p p l i c a t i o n ) . A s m a l l a m o u n t of H b b o u n d w i t h H p T was f o u n d to h a v e t h e m o b i l i t y c h a r a c t e r i s t i c of t h e H p - H b c o m p l e x at this pH. I n t e r e s t i n g results w e r e o b t a i n e d on e l e c t r o p h o r e t i c s e p a r a t i o n of H p T at p H 4.5. O n p a p e r strips s t a i n e d w i t h b r o m p h e n o l b l u e o n l y 3 f r a c t i o n s w e r e p r e s e n t , b u t on the paper strip with the same sample applied and stained with ninhydrin, 9 fractions of d i f f e r e n t m o b i l i t i e s w e r e visible. The 9 fractions were cut out from the corresponding undyed electrophoretic p a p e r s t r i p o b t a i n e d at p H 4-5, t h e p r o t e i n was e l u t e d i n t o a few millilitres of dist i l l e d w a t e r a n d c o n c e n t r a t e d in a d e s i c c a t o r o v e r P 2 Q - A f t e r t h e a d d i t i o n of H b t h e c o n c e n t r a t e d e l u a t e s w e r e e x a m i n e d for p e r o x i d a s e a n d h a e m a g g l u t i n a t i o n inh i b i t o r y a c t i v i t y . F r a c t i o n s d - i (Fig. 2) w e r e f o u n d to be i n h i b i t o r s of h a e m a g g l u t i n a t i o n , c o m p l e x e s of t h e F r a c t i o n s e - g w i t h H b s h o w e d w e a k p e r o x i d a s e a c t i v i t y , a n d c a t h o d i c F r a c t i o n s a - e h a d no effect. ®
® I
1.2
2
1.0 4
:3~ OB 5
0
6
~ 0,6
8 m
go2 c
10 a
b
c
d
ef
g
h
[
20 Tube n u m b e r
30
Fig. 2. Paper electrophoretic patterns of native and of trypsinized Hp/HpT/ at different pH. i, 2, 3, 4, barbiturate buffer (pH 8.6) ; I, 2, Hp ; 3,4, HpT. I, 3, were stained with bromophenol blue and 2, 4, with o.2 % ninhydrin in acetone. 5, 6, Phosphate buffer (pH 7.o); 5, Hp with addition of an equimolar amount of Hb; 6, HpT with Hb. 5 and 6 stained with 2 °/o benzidine in 2o % acetic acid with hydrogen peroxide. 7,8,9, Io, Acetate buffer (pH 4.5); 7, 8, Hp; 9, io, HpT. 7,9 were stained with bromophenol blue and 8, IO, with ninhydrin. Electrophoresis was carried out for 6 h at 22o V. Location of the origin on all the strips is indicated by the arrow. The samples were applied 2 cm from the centre of strips toward the cathode. Details of trypsin digestion and preparation of samples as in NIETHODS. Fig. 3- Fractionation of trypsinized Hp on a Sephadex G-ioo colunln. 3° nlg of lyophilized preparation of Hp, after tryptie digestion and subsequent gel filtration on a Sephadex G-25 colunln, was dissolved in 2 nil of distilled water and applied to the column (2.3 × 30 cm). Effluent fractions of 4 nil were collected at 4 ° with a flow rate of io ml/h. Isolated fractions 7~9, lO-16 and 17-21 were called gtycopeptides l, II and I l l (Sephadex G-Ioo) respectively. Biochirn. Biophys. Acta, 12i (1966) 42-5 °
PROPERTIES OF TRYPTIC GLYCOPEPTIDES OF HAPTOGLOBIN
47
Fractionation of Hp T on Sephadex G-Ioo and DEAE-Sephadex A-5o columns H p T (30 mg in 2 ml of distilled water) was placed on a Sephadex G-Ioo column (2.3 cm x 30 cm) and submitted to gel filtration. A typical elution pattern is shown in Fig. 3. The separation was not satisfactory. Nevertheless the Fractions 7-9 (I), lO-16 (II) and 17-21 (nI) were lyophilized and stored for determination of carbohydrates and biological activities. The yield of the fractionation was as follows: I, I I rag; II, 7 rag; and III, 2 mg. H p T (90 mg in 2 ml of buffer) was placed on a DEAE-Sephadex column (0. 9 cm x 16 cm) buffered with 0.02 M phosphate buffer (pH 6.7) and eluted with increasing concentrations of NaC1 from o.I M to 0.5 M. A typical elution pattern is shown in Fig. 4.
IT
2-
m
1v
0.8 0.1M N(:]CI
0 0.6 m
~ a4 a2 I
2o
l-I
4b
O,2M NeCI
do
~to
Tube number
Fig. 4. F r a c t i o n a t i o n of H p T on a D E A E - S e p h a d e x A-5o column. 90 mg of lyophilized preparation of H p T was dissolved in 2 ml of o.2 1V[p h o s p h a t e buffer (pH 6.7) and applied to the column (0.9 cm × 16 cm) buffered with the same buffer and eluted with increased concentrations of NaC1, indicated b y arrows. Effluent fractions of 2 ml were collected at 4 ° w i t h a flow rate of IO ml/h. Isolated fractions: 3-20, 28-41, 52-62 and 70-80 were called glycopeptides I, II, I I I and I V (DEAE-Sephadex) respectively.
Four peaks were obtained in this fractionation (I-IV). The fractions comprising individual peaks were lyophilized, desalted b y gel filtration on a Sephadex G-25 column and lyophilized once more. The yield of the fractionation was as follows: I, 3 ° mg; II, IO rag; I I I , 4 mg; and IV, 6 mg. Carbohydrate content, peroxidase activity and haemagglutination inhibitory activity were determined on the three fractions obtained b y means of fractionation from a Sephadex G-Ioo column as well as in the four fractions obtained from the DEAE-Sephadex column. Results of the determinations are assembled in Table II. Fraction I (Sephadex G-Ioo) and Fraction IV (DEAE-Sephadex) were found to have the ability to activate Hb, i.e. their complexes with Hb gave a characteristic increase in peroxidase activity, nearly the same for each fraction. The two fractions contained different amounts of sugars. Fraction IV (DEAE-Sephadex) contained only half the amount of hexose in Fraction I (Sephadex G-Ioo); moreover no NANA was found in the carbohydrate moiety of this glycopeptide. Peroxidase activities of Hb complexes with glycopeptide I(Sephadex G-Ioo) and with glyeopeptide IV (DEAESephadex) were much higher than the activity of H p T before fractionation, as calcuBiochim. Biophys. Acta, i 2 i (I966) 42-50
48
W. D O B R Y S Z Y C K A , E. L I S O W S K A
TABLE
II
CARBOHYDRATE OF
CONTENT
AND
BIOLOGICAL
ACTIVITIES
OF THE
PRODUCTS
OF
TRYPTIC
DIGESTION
HAPTOGLOBIN
C o n d i t i o n s of q u a n t i t a t i v e d e t e r m i n a t i o n s a s in T a b l e I. S e p a r a t i o n on Sephadex O-ioo and DEAE-Sephadex A - 5 o a s in t h e t e x t .
Expt. and number N A N A of fraction (°/o)
Sephadex
Hexose* (°//o)
Hexosamine (%)
of d i g e s t i o n p r o d u c t s
of H p
Inhibitory Active Hp, per cent activity of the protein (l*g/4 haernag- content of the sample glutinating units)
G-ioo
I I[ II~
5.1 5.1 o
lO.6 lo. 5 2.8
2 10 10
i8. 4 o o
25 5O-lOO o.8 0.8
o o i5.t
DEAE-Sephadex 1
ii.o
1[ l[I 1V
8. 5 o o
15.I 13. 5 6. 3 3.8
6,2 5,8 i.i 0.8
o
* As galactose.
lated per milligram of lyophilized preparation. Their peroxidase activities with Hb, however, were much smaller than that of native Hp. The strongest inhibitory haemagglutination activities were found in glycopeptide I (Sephadex G-Ioo) and in glycopeptides I I I and IV (DEAE-Sephadex). The activities were higher than that of H p T before fractionation but lower than that of native Hp. Glycopeptides I I I and IV (DEAE-Sephadex) contained no NANA and rather low amounts of hexose and hexosamine. On the other hand, glycopeptides I and I I (DEAE-Sephadex), which were rich in sugars, neither acted as Hb activators nor were haemagglutination inhibitors. DISCUSSION
It is now known that all inhibitors of influenza virus haemagglutination are glycoproteins, the carbohydrate groups of which are terminated by sialic acid residues. The enzymic release of sialic acid results in a reduction in the inhibitory titre 16. This opinion was confirmed in the experiment on the action of neuraminidase on Hp. However, in addition to the presence of sialic acid residues, mucoproteins must possess other structural features in order to qualify as efficient inhibitors. It has been suggested that such an essential factor might be the molecular size of sialomucoproteins. Strong inhibitors have molecular weights of the order of Io5-1o6. MORAWIECKI AND LISOWSKA17 stated that after polymerization of orosomucoid its virus inhibitory activity increased from 5" IO-I to 8. IO 4/*g/4 haemagglutinating units. Recently it has been firmly established that the phenotype HpI_ 1 produces a protein or proteins which appear homogeneous ; however both other types are believed to be serial polymers of basic units differing only slightly in amino acid composition 18. In the present work all the three main genetic types of H p exhibited indentical inhibitory influenza virus haemagglutination activity. Neither molecular Biochim. Biophys. Acta, 121 (1966) 4 2 - 5 °
PROPERTIES OF TRYPTIC GLYCOPEPTIDES OF HAPTOGLOBIN
49
size nor binding of Hb to Hp affected the inhibitory activity. Binding of Hb does not occur through the carbohydrate moiety of Hp, but it is the cause of the same inhibitory activities of Hps and of their complexes with Hb. In view of the opinion on the role of sialic acid in the mechanism of the inhibitory action of sialomucoproteins, it is of interest that some glycopeptides obtained by tryptic digestion of Hp that did not contain any NANA in their carbohydrate moiety (glycopeptides III and IV (DEAE-Sephadex) still exerted almost the same inhibition as native Hp containing 5 To of NANA and much higher inhibition than for example glycopeptide I (DEAE-Sephadex) which contained about IO % of NANA). It seems likely that other sugar replaces NANA in the combination of influenza virus with Hp and with the particulate cellular receptors at the surface of the red-blood cell. Hp resulted in a marked increase in the number of terminal NH2-groups (ninhydrin reaction) as well as in a corresponding loss of peroxidase and haemagglutination inhibitory activities. GOTTSCHALK AND FAZEKAS DE ST. GROTH 16 reported that sialoglycopeptides resulting from tryptic digestion of ovine submaxillary glycoprotein were devoid of measurable inhibitory activity. According to the results of the present paper, although the direct product of tryptic digestion (HpT) showed low inhibitory activity as well as peroxidase activity in the complex with Hb, however, some glycopeptides after separation were found to be almost as strong inhibitors as the native Hp and exhibited measurable peroxidase activity when complexed with Hb. During tryptic digestion, in spite of the fragmentation of the haptoglobin molecule, the level of protein as measured by the tannin micromethod 9 remained constant during the course of the experiment. Tannic acid precipitates polypeptides of molecular weight above 2000*. It may be concluded that the amount of peptides and glycopeptides of molecular weight below 2000 arising from Hp by tryptic action did not exceed i0 % of the total protein, this amount being less than the standard error of the method. CHEFTEL et al. TM isolated glycopeptides resulting from hydrolysis of Hp by proteolytic enzymes. Their results cannot be compared with those presented in this paper because conditions of hydrolysis and different methods of separation of glycopeptides were different. The mechanism of activation of Hb by the action of Hp has not been elucidated. Hp is believed to ensure the regularity of the kinetics of catalysis by the H p - H b complex--which may be described as an enzyme--especially proportionality between velocity and concentration of the peroxidase and applicability of the Michaelis equation to the enzyme. The physico-chemical basis of the phenomenon has been a subject of interest (see refs. 20, 21). In the present work only one glycopeptide, a product of the action of trypsin on Hp, was found to retain the ability to activate Hb (glycopeptide I (Sephadex G-IOO) corresponding to IV (DEAE-Sephadex)). It would be interesting to have information about the size and composition of the smallest fragment of Hp molecule able to change Hb into "true" peroxidase. In view of the experiments on the reductive cleavage of disulphide bonds of Hp 2~, as well as those of the present paper, it seems likely that in the formation of the H p - H b complex having the properties of true peroxidase, the main role is played not by the secondary structure but rather by the primary one, namely by some essential amino acid corn* P e r s o n a l c o m m u n i c a t i o n from W. MEJBAUM-I'{ATZENELLENBOGEN.
Biochim. Biophys. Acta, 121 (1966) 4 2 - 5 °
5°
W. DOBRYSZYCKA, E. LISOWSKA
position and sequence in the peptide chain fragment of Hp necessary for binding and activating Hb. A further analytical approach to these interesting questions would appear worthwhile. REFERENCES I 2 3 4 5 6 7 8 9 lO It 12 13 14 15 16 17 18 19 20 21 22
M. POLONOVSKI AND M. F. JAYLE, Compt. Rend. Soc. Biol., 129 (1938) 457M. F. JAYLE, Bull. Soc. Chim. Biol., 33 (1951) 876]N/[.E. RAFELSON, Jr., L. CLOAREC, J. MORETTI AND M. F. JAYLE, Nature, 191 (1961) 279. S, FAZEKAS DE ST. GROTH AND A. GOTTSCHALK, Biochim. Biophys. Aeta, 78 (1963) 248. C. B. LAURELL, Clin. Chim. Acta, 4 (1959) 79. H. SMITH, P. EDMAN AND J. A. OWEN, Nature, 193 (I962) 286. J. KILLANDER, Biochim. Biophys. Acta, 93 (1964) I. E. B. MCQUARRIE AND H. N. BENIAMS, Proc. Soc. Exptl. Biol. Med., 86 (1954) 627. W. 1V~EJBAUM-KATZENELLENBOGEN,Acta Biochim. Polon., 2 (1955) 279. L. CLOAREC,Contribution ~ l'Etude Physico-Chimique des Haptoglobines Humaines, h/Iprinaeric R. F o u l o n et Cie., Paris, 1964, p. 6. S. MOORE AND W. H. STEIN, J. Biol. Chem., 211 (1954) 907. Z. DISCHE, in D. GLICK, Methods of Biochemical Analysis, Vol. 2, lnterscience, New York, 1955, p. 313 . C. J. M. RONDLE AND \V. T. J. MORGAN, Biochem. J., 61 (I955) 586. L. SVENNERHOLM, Biochim. Biophys. Acta, 24 (1957) 004i. TAMM AND F. L. HORSFALL, J. Expll. Med., 95 (1952) 71. A. GOTTSCHALK AND S. FAZEKAS DE ST. GROTH, Biochim. Biophys. Acta, 43 (196o) 513 • A. MORAWlECKI AND E. LISOWSKA, Biochem. Biophys. Res. Commun., 18 (1965) 606. L. CLOAREC, Contribution ~'~l'Etude Physico-Chimique des Haptoglobines Humaines, hnprinlerie R. F o u l o n et Cie, Paris, 1964, p. 39. R. I. CHEFTEL, L. CLOAREC, J. MORETTI AND M. F. JAYLE, Bull. Soc. Chim. Biol., 47 (1965) 385 . J. MORETTI AND J. YON, Biochim. Biophys. Acta, 46 (1961) 545. \¥. DOBRYSZYCKA, Arch. Immunol. Terapii Experimentalis, 14 (1966) 561. E. LISOXVSKAAND \¥. DOBRYSZYCKA, Abstracts Third Meeting .FEES, Meeting edition, P,,lish Scient. Public. \Varsaw, 1966, No. FI52, p. 256.
Biochim. Biophys. Acta, I2 t (1966) 42-5 o