Immobilization of SH group-containing compounds in the presence of a sulfite-containing antioxidant

Immobilization of SH group-containing compounds on a solvent-insoluble support is carried out in the presence of an antioxidant to prevent oxidation of SH groups to S--S bonds. This improves immobilization efficiency and suppresses deterioration of inherent characteristics of the SH group-containing compound. Antioxidants include sodium pyrosulfite (sodium disulfite), sodium sulfite, sodium hydrogensulfite, sodium hydrosulfite and L-ascorbic acid. SH group-containing compounds include cysteine, peptides or proteins containing cysteine and thiol compounds such as ethanethiol, aminoethanethiol, benzylthiol and thiophenol. Preferably, the SH group-containing compound has a molecular weight not more than 3.times.10.sup.4. The support may be activated by a functional group such as glycidyl, imidocarbonato, tosyl, tresyl, carboxyl, amino, azido or hydroxyl. The support can be inorganic such as glass beads or organic such as a synthetic polymer or a polysaccharide.

TECHNICAL FIELD
 The present invention relates to a method for immobilizing an SH
 (sulfhydryl) group-containing compound on a solvent-insoluble support.
 BACKGROUND ART
 As a method for immobilizing a compound on a solvent-insoluble support for
 use as a ligand for specific adsorption, many investigations have been
 undertaken in the diverse field of immobilized enzymes, affinity
 chromatography, ion exchange chromatography, etc. and some methods are
 used on the industrial application.
 As a representative method for immobilization known in the field of
 immobilized enzymes, there can be mentioned: (1) the method which
 comprises forming the imidocarbonate on the support by the cyanogen
 bromide activating method and, then, causing it to react with the amino
 group of the compound to serve as a ligand; (2) the so-called acid azide
 derivative method which comprises, sequentially, esterification of the
 carboxyl group on a support, hydrazide formation, conversion to the azide
 and substitution thereof with the amino group of the compound to serve as
 a ligand; (3) the so-called diazo method which comprises forming a
 diazonium salt on a support and letting it react with the amino group of
 the compound to serve as a ligand; (4) the so-called condensation reagent
 method which comprises condensing the amino or carboxyl group on a support
 with the carboxyl or amino group of the compound to serve as a ligand in
 the presence of a condensing agent; (5) the so-called alkylation method
 which comprises modifying a support with bromoacetyl or
 4,6-dichloro-s-triazinyl and causing the resulting derivative to react
 with the amino group of the compound to serve as a ligand; and (6) the
 so-called matrix-crosslinking method which comprises crosslinking the
 amino group on a support and the amino group of the compound to serve as a
 ligand with glutaraldehyde followed by reduction (Atsuo Tanaka & Takuo
 Kawamoto: Modern Chemistry, 24 to 30, July 1992).
 However, those methods have the drawbacks, namely
 (a) when the compound to serve as a ligand contains an SH group, the
 efficiency of immobilization is relatively low; and
 (b) when the compound to serve as a ligand is an SH group-containing
 peptide or protein, in particular, the inherent activity of the compound
 (enzyme activity, binding activity, etc.) is sometimes sacrificed.
 The inventors of the present invention discovered that when a peptide or
 protein to serve as a ligand contains an SH group, the above-mentioned
 phenomena (a) and (b) occur during its immobilization on a
 solvent-insoluble support. Similar phenomena are also observed with
 compounds other than peptides and proteins provided that they contain SH
 groups. There is no information in the available literature to the effect
 that the above phenomena occur when a compound to serve as a ligand is
 immobilized on an inert support.
 SUMMARY OF THE INVENTION
 In the above state of the art, the present invention has its objects to
 provide a method for immobilization which is capable of improving the
 efficiency of immobilization in the reaction between an SH
 group-containing compound and a solvent-insoluble support as well as
 suppressing the deterioration of the inherent characteristic of the SH
 group-containing compound due to immobilization.
 For accomplishing the above object, the inventors of the present invention
 did an extensive investigation concerning various conditions of the
 reaction and endeavored to establish a method for immobilization which is
 capable of improving the efficiency of immobilization and suppressing the
 deterioration of the inherent characteristics of SH group-containing
 compounds. As a result, the inventors discovered the involvement of SH
 group, as a major factor, in the above-mentioned phenomena (a) and (b).
 Moreover, in view of the fact that those phenomena are liable to occur in
 reactions conducted in aqueous solution in the high pH (&gt;7) region, the
 inventors assumed that, under the conditions of immobilization reactions,
 SH groups are oxidized, for instance, to give the S--S bond.
 The S--S bond may at times be formed within the molecule of the compound to
 serve as a ligand or may also occur between the molecules. It can be
 thought that when the reaction site (functional group) necessary to be
 utilized for immobilization of the compound to serve as a ligand is SH
 group, the formation of the S--S bond inactivates the reaction site and
 that, when the reaction site is a functional group other than SH group,
 the reaction site is hidden in the interior of the molecule as the S--S
 bond is formed. It can be thought that the decrease in immobilization
 efficiency occurs probably in such situations. Moreover, it can also be
 assumed that the formation of the S--S bond causes the deterioration of
 the inherent characteristic of the SH group-containing compound.
 Based on the above experience and assumption, the inventors paid attention
 to the importance of avoiding formation of the S--S bond and, after many
 investigations, found that the above-mentioned object can be accomplished
 by conducting the reaction between a solvent-insoluble support and an SH
 group-containing compound in the presence of an antioxidant. The present
 invention has been developed on the basis of the above finding.
 The present invention, therefore, is directed to a method for immobilizing
 an SH group-containing compound which comprises reacting a
 solvent-insoluble support with the above-mentioned compound in the
 presence of an antioxidant.

DETAILED DESCRIPTION OF THE INVENTION
 The present invention is now described in detail.
 According to the present invention, the solvent-insoluble support means a
 support which is not soluble in a solvent to be employed. In the preferred
 embodiment of the present invention, the solvent-insoluble support is a
 solvent-insoluble support in case of using water as the solvent, that is
 to say a water-insoluble support.
 The water-insoluble support mentioned above is not particularly restricted
 but includes inorganic supports such as glass beads, silica gel, etc.;
 organic supports such as synthetic polymers, e.g. crosslinked polyvinyl
 alcohol, crosslinked polyacrylate, crosslinked polyacrylamide, crosslinked
 polystyrene, etc. and polysaccharides, e.g. crystalline cellulose,
 crosslinked cellulose, crosslinked agarose, crosslinked dextran, etc.; and
 organic-organic, organic-inorganic or other complex supports as obtained
 by using said support materials in combination.
 Furthermore, when an SH group-containing compound immobilized on a
 solvent-insoluble support is to be used in chromatography or as an
 adsorbent, the solvent-insoluble support is preferably a hydrophilic
 support. This is because a hydrophilic support is comparatively low in
 nonspecific adsorption and satisfactory to adsorption specificity caused
 by ligand (immobilized SH group-containing compound). The hydrophilic
 support in this specification means a support which shows a contact angle
 of not greater than 60.degree. with respect to water when the compound
 constituting the support is tested in a board form.
 The hydrophilic support mentioned above is not particularly restricted but
 includes, for example, supports which comprise various polysaccharides and
 derivatives thereof, such as cellulose, acetylcellulose, chitosan, chitin,
 agarose, dextran, etc.; and such other supports as polyvinyl alcohol,
 co(ethylene-vinyl acetate) polymer hydrolyzate, polyacrylamide,
 polyacrylic acid, polymethacrylic acid, poly(methyl methacrylate),
 polyacrylic acid-polyethylene graft, polyacrylamide-polyethylene graft,
 and glass, among others.
 Among those hydrophilic supports, hydroxyl group-containing supports are
 preferred in adsorption affinity and selectivity. Particularly when the
 support immobilized on an SH group-containing compound is used as an
 adsorbent, a porous cellulosic gel is one of the most suitable kinds of
 supports because it has the following beneficial characteristics; thus,
 (1) Because such gels are comparatively high in mechanical strength and
 tough, they are not disintegrated or give dusts in stirring and other
 mechanical procedures and when they are packed into columns, no compaction
 or plugging occurs even when a fluid is passed at a high flow rate, thus
 permitting operations at high flow rates. Moreover, its pore structures
 are not easily altered by autoclaving, for instance.
 (2) Composed of cellulose, those gels are hydrophilic and contain many
 hydroxyl groups available for ligand binding and, moreover, they are
 relatively low in nonspecific adsorption.
 (3) Because those gels are comparatively high in strength even when the
 void volume is increased, large adsorption capacities fully comparable to
 those of soft gels can be obtained.
 The cellulosic gel in this specification means any cellulose derivative the
 backbone chain of which is composed of cellulose, such as cellulose or
 acetylcellulose. However, the present invention is not restricted to the
 use of such gels.
 The SH group-containing compound according to the present invention means a
 compound to be immobilized on a solvent-insoluble support and having one
 or more --SH (sulfhydryl) group within its molecule. The SH group in such
 a compound may assume an ionized form, e.g. --S.sup.- Na.sup.+, --S.sup.-
 K.sup.+, depending on the kind of solution in which it occurs, or may be
 added in the form of a salt, e.g. --SNa or --SK, at the time of reaction.
 However, since these forms are essentially the same, the term SH group is
 used herein in the generic sense, including the above cases.
 The SH group-containing compound mentioned above is not particularly
 restricted but includes, for example, cysteine, peptides or proteins
 containing cysteine as a constituent amino acid and thiol compounds (high,
 medium or low molecular compounds having SH group in the backbone chain or
 in side chains, inclusive of ethanethiol, aminoethanethiol, benzylthiol,
 thiophenol and compounds having such moieties in side chains or termini).
 The SH group-containing compound mentioned above is preferably at least
 one member selected from the group consisting of amino acid, peptide and
 protein.
 The molecular weight of said SH group-containing compound was a subject of
 our intensive study. The study revealed that as the molecular weight is
 increased, the effect of the invention is detracted. Thus, as far as SH
 group-containing compounds having molecular weights up to 3.times.10.sup.4
 are concerned, a significant difference was found in the effect of the
 invention according to whether an antioxidant is added or not. On the
 other hand, for proteins which are high polymers having molecular weights
 of the order of 1.5.times.10.sup.5, the difference in the effect of the
 invention was relatively small. Therefore, the molecular weight of said SH
 group-containing compound is preferably not greater than 3.times.10.sup.4.
 The antioxidant according to the present invention means a substance which
 has the property to prevent or suppress oxidation of a compound in a
 reaction system and includes, for example, a substance which either
 directly inhibits oxidation of a compound or becomes oxidized to thereby
 inhibit oxidation of a compound in a reaction system and a substance
 having the property to purge dissolved oxygen from the reaction system. Of
 course, a similar effect may be achieved by decreasing dissolved oxygen
 substantially through degassing or cooling of the reaction solvent but in
 consideration of workability and universality of application, addition of
 an antioxidant is a better choice leading to a better outcome more
 effectively and safely.
 The antioxidant mentioned above includes a large variety of substances,
 among them, also includes SH group-containing compounds. Mercaptoethanol
 is a representative example. However, when the immobilization reaction
 proceeds as the solvent-insoluble support is subject to nucleophilic
 reaction as it is the case with a support having glycidyl (epoxy) groups,
 the antioxidant mercaptoethanol itself may react and be immobilized. When
 such cases are taken into consideration, the use of an antioxidant having
 nucleophilicity is subject to limitation in terms of the scope of
 application. Of course, although even such an antioxidant is sufficiently
 useful for certain reactions, a widely useful antioxidant which can be
 used without such fear is preferred.
 In the present invention, said antioxidant is preferably at least one
 member selected from the group consisting of sodium pyrosulfite (sodium
 disuflite), sodium sulfite, sodium hydrogensulfite, sodium hydrosulfite
 and L-ascorbic acid. Those antioxidants are advantageous in that they can
 be utilized in the immobilization reaction of a large majority of SH
 group-containing compounds with a solvent-insoluble support.
 In the present invention, the molar concentration ratio of the antioxidant
 to the SH group-containing compound is preferably 1000:1 to 1:1000.
 Outside of this range, the concentration of the antioxidant is preferably
 1 .mu.mol/L to 1 mol/L. It is more preferable that both conditions be
 satisfied. In such cases, the antioxidant expresses its antioxidant
 activity sufficiently to maximize the immobilization rate of the SH
 group-containing compound and the activity retention thereof after
 immobilization. It should be noted that the molar concentration mentioned
 above is the molar concentration in a reaction system.
 In reacting the solvent-insoluble support with the SH group-containing
 compound according to the method for immobilization of the present
 invention, the efficiency of immobilization can be improved by activating
 the solvent-insoluble support somehow in advance.
 In the preferred embodiment of the present invention, the solvent-insoluble
 support is a support activated with at least one kind of functional group
 selected from the group consisting of glycidyl (epoxy), imidocarbonate,
 tosyl, tresyl, carboxyl, amino, azido and hydroxyl. When the reaction of
 the support with an SH group-containing compound is curried out in the
 presence of an antioxidant, not only the immobilization rate of the SH
 group-containing compound but also the activity retention rate thereof
 after immobilization is high, therefore the use of such an activated
 solvent-insoluble support is effective.
 BEST MODE FOR CARRYING OUT THE INVENTION
 The following examples are merely intended to illustrate the present
 invention in further detail and should by no means be construed as
 defining the scope of the invention.
 EXAMPLE 1
 Immobilization of Aminoethanethiol on Sephacryl S1000 (Sephacryl-AET-a)
 (1) Epoxy-activation of Sephacryl S1000
 To 83 ml of Sephacryl S1000 (Amersham Pharmacia Biotech) was added a
 sufficient amount of water to make 186 ml, and 113 ml of 2 M sodium
 hydroxide-water was further added. The temperature was then adjusted to
 40.degree. C. To this mixture was added 38 ml of epichlorohydrin (Wako
 Pure Chemical Ind.), and the reaction was carried out at 40.degree. C. for
 2 hours. After completion of the reaction, the reaction mixture was
 thoroughly washed with water on a glass filter to provide epoxy-activated
 Sephacryl S1000.
 (2) Immobilization of aminoethanethiol and quantitation of immobilized
 aminoethanethiol
 In 15 ml of 0.05 M borate buffer (pH 10.0) were dissolved 1.20 mg of
 2-aminoethanethiol (Wako Pure Chemical Ind.) and 150 mg of sodium sulfite
 (Wako Pure Chemical Ind.), and the solution was readjusted to pH 10 with
 0.01 N sodium hydroxide-water and made up to 25 ml. To 5 ml of the above
 epoxy-activated Sephacryl S1000 was added the above aminoethanethiol
 solution (whole amount), and after 18 hours of shaking at 37.degree. C.,
 the mixture (aminoethanethiol:sodium sulfite=1:76, sodium sulfite=47.6
 mmol/L) was washed with a sufficient amount of physiological saline on a
 glass filter to provide Sephacryl-AET-a (0.20 mg/ml-gel). The amount of
 immobilized aminoethanethiol was determined by the TNBS
 (trinitrobenzenesulfonic acid) colorimetric assay of amino groups using
 the supernatants before and after the reaction.
 EXAMPLE 2
 Immobilization of Cysteine on Tresyl-Toyopearl (Toyopearl-Cys-a) and
 Quantitation of Immobilized Cysteine
 In 4 ml of a coupling buffer (pH 8.2, 0.5 M sodium chloride-0.1 M carbonate
 buffer) were dissolved 0.50 mg of L-cysteine hydrochloride monohydrate
 (Wako Pure Chemical Ind.) and 1.0 mg of sodium pyrosulfite (Wako Pure
 Chemical Ind.). Then, 800 mg of AF-tresyl-Toyopearl 650 was added in dry
 state and the reaction was conducted at room temperature overnight.
 The reaction mixture was washed with 0.5 M sodium chloride-water, then a
 block buffer (pH 8.0, 0.5 M sodium chloride-0.1 M Tris-HCl buffer) was
 added, and the reaction was carried out at room temperature for 2 hours
 (cysteine:sodium pyrosulfite=1:1.89, sodium pyrosulfite=1.3 mmol/L). This
 reaction mixture was further washed with 0.5 M sodium chloride-water to
 provide Toyopearl-Cys-a (0.11 mg/ml-gel). The amount of immobilized
 cysteine was determined by said amino group assay as in Example 1.
 EXAMPLE 3
 Immobilization of an IgG-binding Peptide (MP47C on a Porous Support (Kac)
 Using Sodium Hydrosulfite (Kac-MP47C-a) and the Evaluation of IgG-binding
 Activity
 (1) Production of MP47C peptide
 To acquire MP47C peptide shown in the sequence listing under SEQ ID NO:1, a
 DNA coding for MP47C peptide was designed as shown in the sequence listing
 under SEQ ID NO:2 and synthesized so that it could be ligated to the pUCNT
 vector [Japanese Kokai Publication Hei-4-212692] utilizing Nde I and Hind
 III restriction enzyme sites for the 5'-end and 3'-end, respectively.
 The DNA having the above sequence was ligated to the pUNT vector after
 cleavage with the restriction enzymes Nde I and Hind III (Takara Shuzo)
 using DNA Ligation Kit Ver. 2 (Takara Shuzo) according to the protocol to
 construct a pUCNT MP47C vector (FIG. 1).
 Using the routine technique, this pUCNT MP47C vector DNA was introduced
 into Escherichia coli HB101 (Funakoshi Hanbai) and a transformant was
 selected by using resistance to the antibiotic ampicillin as a marker.
 From this transformant, the plasmid DNA was extracted and the gene sequence
 was analyzed in the routine manner to confirm that this pUCNT MP47C vector
 had the designed DNA sequence. Then, this transformant was shake-cultured
 in 6 L of L-broth (5 g/L NaCl, 10 g/L bactotrypsin, 5 g/L yeast extract)
 at 37.degree. C. for 20 hours and the cells were harvested by
 centrifugation (Hitachi RPR9-2 rotor, 4.degree. C., 6000 rpm., 20 min).
 The pellet obtained was suspended in 300 ml of TE buffer (20 mM Tris-HCl,
 1 mM EDTA; pH 7.5) and sonicated (BRANSON 250, 6 min..times.3, on ice) and
 the supernatant was recovered by centrifugation (Hitachi RPR16 rotor,
 4.degree. C., 15000 rpm, 20 min). The supernatant thus recovered was
 heat-treated at 70.degree. C. for 10 minutes, then centrifuged again
 (Hitachi RPR16 rotor, 4.degree. C., 15000 rpm, 20 min) to recover 300 ml
 of a supernatant. The supernatant was purified by high performance liquid
 chromatography (column: Waters .mu. BONDASPHERE 5.mu. C18 300A,
 19.0.times.150 mm). Thus, the column was first activated by passing 40 ml
 of acetonitrile at a flow rate of 5 ml/min and, then, 300 ml of a sample
 was passed at the same flow rate. The column was then washed with 200 ml
 of 0.1% TFA+64% acetonitrile and the objective MP47C peptide was eluted
 with 200 ml of 0.1% TFA+40% acetonitrile and recovered. The eluate was
 concentrated to 100 ml using an evaporator and lyophilized to provide 1.3
 g of high-purity product.
 (2) Epoxy-activation of a cellulose gel
 To 90 ml of the applicant's prototype gel Kac, which is a cellulosic porous
 hard gel having an exclusion limit molecular weight of not less than
 5.times.10.sup.6 for globular proteins was added a sufficient amount of
 water to make 180 ml. Then, 60 ml of 2 M sodium hydroxide-water was added
 and the temperature was adjusted to 40.degree. C. To this solution was
 added 21 ml of epichlorohydrin and the reaction was conducted at
 40.degree. C. with stirring for 1 hour. After completion of the reaction,
 the reaction product was thoroughly washed with water to provide an
 epoxy-activated cellulose gel.
 (3) Immobilization of MP47C peptide and quantitation of the immobilized
 peptide
 In 0.5 ml of 0.05 M borate buffer (pH 10.0) were dissolved 10 mg of MP47C
 peptide and 50 .mu.g of sodium hydrosulfite (Wako Pure Chemical Ind.), and
 the solution was readjusted to pH 10 and made up to 1.0 ml with 0.01 N
 sodium hydroxide-water. The MP47C solution (whole amount) was added to 1
 ml of the above epoxy-activated cellulose gel, and after 16 hours of
 shaking at 37.degree. C. (MP47C peptide:sodium hydrosulfite=1:372, sodium
 hydrosulfite=0.58 mmol/L), the reaction mixture was washed with a
 sufficient amount of PBS (10 mM phosphate buffer containing 150 mM sodium
 chloride) to provide Kac-MP47C-a (8 mg/ml-gel). The amount of immobilized
 MP47C peptide was calculated from the ratio of areas before and after the
 reaction on HPLC. .mu.-bondasphere C18 (Nippon Waters, 3.9 mm ID.times.150
 mm H) was used as the column and (A): 0.1% TFA/H.sub.2 O and (B):80%
 acetonitrile/0.1% TFA/H.sub.2 O were used as the mobile phase. Thus,
 elution was started at (A):(B)=95:5 and the proportion of (B) was
 increased on a gradient of 3%/min. The flow rate was 1 ml/min.
 (4) Evaluation of the IgG-binding activity of Kac-MP47C-a
 Kac-MP47C-a or the ligand-free support (Kac) (1 ml) was taken in a vial,
 and after addition of 6 ml of healthy human serum, the vial was shaken at
 37t for 2 hours. Then, this suspension was centrifuged at 5000 rpm for 1
 minute and the IgG concentration of the supernatant was determined by
 turbidimetric immunoassay with Shionogi Biolaboratories. The IgG
 adsorption rate was calculated by the following formula.
EQU Adsorption rate (%)={(Ir-It)/Ir}.times.100
 Ir: IgG concentration of Kac support supernatant
 It: IgG concentration of Kac-MP47C-a adsorbent supernatant
 As a result, the IgG adsorption rate of Kac-MP47C-a was found to be 61%.
 EXAMPLE 4
 Immobilization of a Portion (Fab) of Anti-IgG Antibody on Tresyl-activated
 Sepharose 4B (Sepharose 4B-Fab-a)
 (1) Immobilization of a portion (Fab) of anti-IgG antibody on
 tresyl-activated Sepharose 4B and quantitation of immobilized Fab
 Using a small amount of 1 mM HCl-water, 4 g of tresyl-activated Sepahrose
 4B (Amersham Pharmacia Biotech) was caused to swell for 15 minutes. The
 support was then washed 30 with 1 mM HCl-water and further with a coupling
 buffer (pH 8.0, 0.5 M NaCl-0.1 M NaHCO.sub.3). In 2 ml of the coupling
 buffer, 2 mg of the Fab prepared by papain digestion (PIERCE, ImmunoPure
 Fab Preparation Kit) from anti-human IgG (Fab) antibody (Binding Site) and
 4.2 .mu.g of sodium hydrogensulfite were dissolved. To this solution was
 added 0.4 ml of the above washed gel, and the reaction was conducted at
 25.degree. C. for 4 hours. The reaction product was washed with coupling
 buffer, then a block buffer (pH 9, 1 M ethanolamine-0.5 M sodium
 chloride-0.1 M NaHCO.sub.3) was added, and the reaction was conducted at
 room temperature for 2 hours (Fab: sodium hydrogensulfite=1:1, sodium
 hydrogensulfite=20 .mu.mol/L). The reaction product was washed with two
 different buffers for aftertreatment (pH 4.0, 0.5 M sodium chloride-0.1 M
 acetic acid-sodium acetate buffer; pH 8.0, 0.5 M sodium chloride-0.1
 M-Tris-HCl buffer) alternately 3 times each to provide Sepharose 4B-Fab-a
 (4.0 mg/ml-gel). The immobilization amount was calculated from the
 concentrations of the anti-IgG Fab in the supernatants before and after
 the reaction as determined with Micro BCAProtein Assay Reagent Kit
 (PIERCE).
 (2) Evaluation of the IgG-binding activity of Sepharose 4B-Fab-a
 Sepharose 4B-Fab-a or the ligand-free support (Sepharose 4B), 0.2 ml, was
 taken in a vial and after addition of 0.6 ml of healthy human serum, the
 vial was shaken at 37.degree. C. for 2 hours. Then, this suspension was
 centrifuged at 5000 rpm for 1 minute and the IgG concentration of the
 supernatant was determined by turbidimetric immunoassay with Shionogi
 Biolaboratories. The IgG absorption rate was calculated by the following
 formula.
EQU Adsorption rate (%)={(Ir-It)/Ir}.times.100
 Ir: IgG concentration of Sepharose 4B support supernatant
 It: IgG concentration of Sepharose 4B-Fab-a adsorbent supernatant
 As a result, the IgG adsorption rate of Sepharose 4B-Fab-a was found to be
 82%.
 Comparative Example 1
 Immobilization of Aminoethanethiol on Sephacryl S1000 and Quantitation of
 Immobilized Aminoethanethiol (Sephacryl S1000-AET-b)
 Except that sodium sulfite was not used, the procedure of Example 1 was
 otherwise repeated to immobilize aminoethanethiol on Sephacryl S1000 and
 provide Sephacryl S1000-AET-b (0.13 mg/ml-gel).
 Comparative Example 2
 Immobilization of Cysteine on Tresyl-Toyopearl and Quantitation of
 Immobilized Cysteine (Toyopearl-Cys-b)
 Except that sodium pyrosulfite was not used, the procedure of Example 2 was
 otherwise repeated to immobilize cysteine on tresyl-Toyopearl and provide
 Toyopearl-Cys-b (0.07 mg/ml-gel).
 Comparative Example 3-1
 Immobilization of an IgG-binding Peptide (MP47C) on a Porous Support (Kac)
 (Kac-MP47C-b)
 (1) Immobilization of MP47C and quantitation of immobilized MP47C
 Except that sodium hydrosulfite was not used, the procedure of Example 3
 was otherwise repeated to immobilize MP47C on Kac and provide Kac-MP47C-b
 (5 mg/ml).
 (2) Evaluation of the IgG-binding activity of Kac-MP47C-b
 Except that Kac-MP47C-b was used in lieu of Kac-MP47C-a, the procedure
 described in Example 3 under "Evaluation of the IgG-binding activity of
 Kac-MP47C-a" was faithfully followed to evaluate the IgG-binding activity.
 As a result, the IgG-adsorption rate of Kac-MP47-b was found to be 38%.
 Comparative Example 3-2
 Immobilization of an IgG-binding Peptide (MP47C) on a Porous Support (Kac)
 (Kac-MP47C-c) and Evaluation of the IgG-binding Activity of Kac-MP47C-c
 (1) Immobilization of MP47C and quantitation of immobilized MP47C
 Except that the amount of MP47C was changed to 20 mg, the procedure of
 Comparative Example 3-1 was otherwise repeated to immobilize MP47C on Kac
 and provide Kac-MP47C-c (8 mg/ml).
 (2) Evaluation of the IgG-binding activity of Kac-MP47C-c
 Except that Kac-M47C-c was used in lieu of Kac-MP47C-a, the procedure
 described in Example 3 under "Evaluation of the IgG-binding activity of
 Kac-MP47C-a" was faithfully followed to evaluate the IgG-binding activity.
 As a result, the IgG adsorption rate of Kac-MP47C-c was found to be 47%.
 Comparative Example 4-1
 Immobilization of a Portion (Fab) of anti-IgG Antibody on Tresyl-activated
 Sepharose 4B (Sepharose 4B-Fab-b)
 (1) Immobilization of Fab on tresyl-activated Sepharose 4B and quantitation
 of immobilized Fab
 Except that sodium hydrogensulfite was not used, the procedure of Example 4
 was otherwise repeated to immobilize a portion (Fab) of anti-IgG antibody
 on Sepharose 4B and provide Sepharose 4B-Fab-b (2.5 mg/ml-gel).
 (2) Evaluation of the IgG-binding activity of Sepharose 4B-Fab-b
 Except that Sepharose 4B-Fab-b was used in lieu of Sepharose 4B-Fab-a, the
 procedure described in Example 4 under "Evaluation of the IgG-binding
 activity of Sepharose 4B-Fab-a" was faithfully followed to evaluate the
 IgG-binding activity. As a result, the IgG adsorption rate of Sepharose
 4B-Fab-b was found to be 65%.
 Comparative Example 4-2
 Immobilization of a Portion (Fab) of anti-IgG Antibody on Tresyl-activated
 Sepharose 4B (Sepharose 4B-Fab-c)
 (1) Immobilization of Fab on tresyl-activated Sepharose 4B and quantitation
 of immobilized Fab
 Except that the amount of Fab was changed to 3 mg, the procedure of
 Comparative Example 4-1 was otherwise repeated to immobilize a portion
 (Fab) of anti-IgG antibody on Sepharose 4B and provide Sepharose 4B-Fab-c
 (4.0 mg/ml-gel).
 (2) Evaluation of the IgG-binding activity of Sepharose 4B-Fab-c
 Except that Sepharose 4B-Fab-c was used in lieu of Sepharose 4B-Fab-a, the
 procedure described in Example 4 under "Evaluation of the IgG-binding
 activity of Sepharose 4B-Fab-a" was faithfully followed to evaluate the
 IgG-binding activity. As a result, the IgG adsorption rate of Sepharose
 4B-Fab-c was found to be 72%.
 The immobilization amounts and activities of SH group-containing compounds
 in the above-mentioned Examples and Comparative Examples are summarized in
 Table 1.
 TABLE 1
 Immobiliza-
 tion amount Activity
 Abbreviation (mg/mL-gel) (%)
 Example 1 Sephacryl-AET-a 0.20 --
 Compar. Ex. 1 Sephacryl-AET-b 0.13 --
 Example 2 Toyopearl-Cys-a 0.11 --
 Compar. Ex. 2 Toyopearl-Cys-b 0.07 --
 Example 3 Kac-MP47C-a 8 61
 Compar. Ex. 3-1 Kac-MP47C-b 5 38
 Compar. Ex. 3-2 Kac-MP47C-c 8 47
 Example 4 Sepharose4B-Fab-a 4 82
 Compar. Ex. 4-1 Sepharose4B-Fab-b 2.5 65
 Compar. Ex. 4-2 Sepharose4B-Fab-c 4 72
 It will be apparent from Table 1 that in the examples using the method for
 immobilization of the present invention, SH group-containing compounds
 could be immobilized on solvent-insoluble supports in good yields and the
 inherent activity of the SH group-containing compound could be well
 sustained.
 Constituted as described above, the present invention provides a novel
 method for immobilization by which SH group-containing compounds can be
 immobilized on solvent-insoluble supports in good yields and with good
 retention of the inherent activities of such SH group-containing
 compounds.
 SEQUENCE LISTING
 &lt;100&gt; GENERAL INFORMATION:
 &lt;160&gt; NUMBER OF SEQ ID NOS: 2
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 1
 &lt;211&gt; LENGTH: 58
 &lt;212&gt; TYPE: PRT
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: MP47C peptide
 &lt;400&gt; SEQUENCE: 1
 Met Thr Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly Glu
 1 5 10 15
 Thr Thr Thr Lys Ala Val Asp Ala Glu Thr Ala Glu Lys Ala Phe Lys
 20 25 30
 Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Val Trp Thr Tyr Asp Pro
 35 40 45
 Ala Thr Lys Thr Phe Thr Val Thr Glu Cys
 50 55
 &lt;200&gt; SEQUENCE CHARACTERISTICS:
 &lt;210&gt; SEQ ID NO 2
 &lt;211&gt; LENGTH: 184
 &lt;212&gt; TYPE: DNA
 &lt;213&gt; ORGANISM: Artificial Sequence
 &lt;220&gt; FEATURE:
 &lt;223&gt; OTHER INFORMATION: DNA coding for MP47C peptide
 &lt;400&gt; SEQUENCE: 2
 cat atg acc acc tat aaa ctg gtt atc aac ggt aaa acc ctg aaa ggt 48
 Met Thr Thr Tyr Lys Leu Val Ile Asn Gly Lys Thr Leu Lys Gly
 1 5 10 15
 gaa acc acc acc aag gct gtt gac gct gaa acc gct gaa aaa gca ttt 96
 Glu Thr Thr Thr Lys Ala Val Asp Ala Glu Thr Ala Glu Lys Ala Phe
 20 25 30
 aaa cag tat gct aac gac aac ggt gtc gac ggt gtt tgg acc tat gac 144
 Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Val Trp Thr Tyr Asp
 35 40 45
 ccc gct acc aaa acc ttt acc gtt acc gaa tgc taagctt 184
 Pro Ala Thr Lys Thr Phe Thr Val Thr Glu Cys
 50 55