Technical Information

Many reviews of chemical modification of proteins exist.^5,6,6a   Some brief examples, extracted from the literature are provided here to illustrate typical conjugation protocols for surfaces, polypeptides, IgG's, or oligonucleotides.  

Part I  Conjugation with sulfhydryl reactive probes.
Part II Conjugation with amino reactive probes.


PART I:                                Conjugation With Sulfhydryl Reactive Probes

Typical sulfhydryl modification with maleimide:

Use of maleimide derivatives for modification of sulfhydryl groups is described extensively in the literature⁵.  The maleimide group is specific for sulfhydryls at ca. pH 6.5-7.5.  Reaction with sulfhydryl groups generates a stable 3-thiosuccinimidyl ether linkage (1).  The maleimide ring itself can hydrolyze in aqueous buffer to a non-reactive cis-maleamic acid derivative (2) over long reaction times or extremes in pH (pH>8.0), while competing electrophilically for free sulfhydryl groups^7,8.  A study of the rate of hydrolysis of N-ethylmaleimide via UV absorbtion, in 20 mM phosphate, 20 mM acetate buffer, pH 7.5, 25 °C revealed a K_{(decomposition)}=1.69 X 10^-3 moles/min, with t_1/2=410 min or 6.8 h.  Optimum derivatization of sulfhydryl groups with maleimides is usually achieved within 2.0-2.5 h, pH 7.0-8.0.

Typical sulfhydryl modification with iodoacetamide:

The use of bromo or iodoacetyl compounds for modification of sulfhydryl groups on cysteine side chains is described in the literature^2,3.   Modification proceedure is similiar to that employed for maleimide, pH 6.5-8.0.   In the absence of free sulfhydryl groups, or those sterically occulded, iodoacetyl groups can react with the imidazole side chain of histidine⁹.  Reaction with haloacetyl derivatives should be shielded from light.

Example:  Bromoacetyl modification of BSA⁴:

To 30 mg of BSA dissolved in 5 mL of 0.1 M NaHCO₃, was added 0.2 mL of a 0.7 M solution of Bu₃P in 2-propanol.  Stir 30 min/25 °C.  Add 30 mg of bromoacetyl derivative, stir 1h.  Dialyze for 12 h/4 °C against 0.1 M NH₄HCO₃ with three changes of same over 2-days.  Isolate conjugate by lyophilization.

Typical sulfhydryl modification with vinylsulfone:

Vinylsulfone is a slightly softer electrophile than maleimide.   Sulfhydryl addition to vinylsulfones generates a stable b-thiosulfonyl linkage (4).   Sulfhydryl modification with vinylsulfones has the advantage that the vinylsulfone itself is stable in aqueous solution for days at pH 9.0¹, thus allowing extended reaction times for modification without hydrolysis.  Sulfhydryl addition to vinylsulfone, unlike maleimides, does not generate stereoisomers which in some instances may complicate analysis and/or bioactivity of the conjugate.

Example:  Vinylsulfone labeling of RNase¹:  

RNase (1.35 mg/mL)  in 0.2 M, pH 8.0 borate buffer.  Vinylsulfone in DMF (10 mg/mL) stock solution is added to RNase at 22 °C.  Reaction is allowed to proceed until optimum modification is achieved (1-2.5 h).  Quench aliquotes with 1.5 M b-mercaptoethanol prior to analysis.  Product is isolated by gel filtration.

Note I: [Reduction of RNase:  14 mg protein (M_r=13,700) dissolved in  300 mL of 25 % b-mercaptoethanol in 4.5 M guanidinium chloride.  Heat 100 °C/5 min.  The reduced protein (four cystine disulfide bonds reduced to eight cysteine thiol groups) is isolated by gel filtration chromatography using 0.05 M tris, 0.2 M guanidinium chloride pH 4.3.] 

Typical sulfhydryl modification with pyridyl disulfides:

2-Pyridyl disulfide derivatives react with free sulfhydryl groups of polypeptides or IgG to form disulfide conjugates (5) with release of 2-pyridylthione (6).   Thiolation can be quantitated by measurement of 2-pyridylthione (l_max=343 nm, e=8.08 X 10³ M^-1 cm^-1).  Conjugates can be cleaved back to their original sulfhydryl groups by addition of excess free thiols such as DTT, DTE, or BME.   Cleavage can also be achieved by use of non-thiol reducing agents such as Bu₃P or TCEP·HCl.

Example:  Modification of BSA with labeled RNase¹⁰:

Reduced BSA (80 mg containing 490 nmol of mercaptoalbumin by Ellman's Assay) was dissolved in 1.95 mL 0.1 M phosphate, 0.1 M NaCl buffer pH 7.5.  2-Pyridyldithio derivative of RNase (500 nmol) was added in same buffer.  Reaction is usually complete within 2 h, and can be followed by UV measurement of released 2-pyridylthione.   Gel filtration through Sepharose 6B using 0.3 M NaCl provides pure adduct.   For labeling 2-pyridyldithio-small molecule derivatives, dissolve in organic co-solvent if necessary.

Bibliography

1. Morpurgo, M, Veronese, F.M., Kachensky, D., Harris, J.M (1996) Bioconjugate Chem. 7, 363-368.
2. Inman, J.K., Highet, P.F., Kolodny, N., Robey, F.A. (1991) Bioconjugate Chem. 2, 458-463.
3. Bernatowicz, M.S., Matseuda, G.R. (1986) Anal. Biochem. 155, 95.
4. Kolodny, N., Robey, F.A., (1990) Anal. Biochem. 187, 136-140.
5. Means, G.E., Feney, R.E. (1990) Bioconjugate Chem. 1, 2-12.
6. Brinkley (1992) Bioconjugate Chem. 3, 2.
6a. Hermanson, G.T. (1996) Bioconjugate Techniques,  Academic Press.
7. Ishi, Y., Lehrer, S.S. (1986) Biophys. J. 50, 75-80.
8. Smyth, D.G., Blumenfeld, O.O., Konigsberg, W. (1964) Biochem. J. 91, 589.
9. Gurnd, F.R.N., (1967) Methods Enzymol. 11, 532-541.  See also ref in ref #5.
10.  Carlsson, J., Drevin, H., Axen, R. (1978) Biochem. J. 173, 723-737.
11. Kohno, T., et. al. (1994) unpublished esults with PEG-20K-maleimide.

Additional helpful references:  Analysis for extent of sulfhydryl modification.

1. Riddles, P.W., Blakeley, R.L., Zerner, B. (1983) Methods Enzymol. 91, 49-60.
2. Riddles, P.W., Blakeley, R.L., Zerner, B. (1979) Anal. Biochem. 67, 493-502.
3. Ellman, G.L. (1959) Arch. Biochem. Biophys. 82, 70-77.
4. Ellman, G.L. (1958) Arch. Biochem. Biophys. 74, 443.
5.

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PART II:                              Conjugation With Amine Reactive Probes

Typical amine modification with NHS or sulfo-NHS ester:

Typical amine modification with isocyanates:


Typical amine modification with isothiocyanates:


Additional helpful references:  Analysis for extent of amine modification.


1. Udenfriend, S., et.al. (1972) Science 178, 871. (Use of fluorescamine.)
2. (Use of TNBS.)