Krishnan Rajagopalan

Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers

Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which provide base stacking interactions to the purine ring of ATP (Rajagopalan et al. (1996) Proc. Natl. Acad. Sci. USA 93, 6019-6024). Biotin was coupled to either the phosphate or the ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity probe and shown to insert into the affinity site efficiently. Several monoclonal and polyclonal antibodies, as well as enzymatic and recombinant antibody fragments and light chain dimers were photoaffinity biotinylated and used in ELISA, FACS and Western blots. The selectivity of this site-specific biotinylation method also allows for biotinylation of antibodies in culture supernatants and immune sera without prior purification. Because the biotinylation takes place under physiological conditions and within a short time period, photobiotinylation would be the preferred method for antibodies which are easily damaged by classical non-site specific random biotinylation chemistry.

Photoaffinity labeling of rat steroid 5α-reductase (isozyme-1) by a benzophenone derivative of a 4-methyl-4-azasteroid

[1,2-3H]N-4(Benzylbenzoyl)-3-oxo-4-aza-4-methyl-5 alpha-androstane-17 beta-carboxamide ([3H]-4MABP) has been synthesized as a photoaffinity probe of the steroid-binding domain of rat steroid 5 alpha-reductase isozyme-1 (5 alpha R-1). Reversible binding of the probe to 5 alpha R-1 in microsomal preparations yielded a reversible dissociation constant (Kd) of -3 nM, whereas inhibition experiments indicated that the probe had a 50% inhibition concentration of 4.4 nM and was a competitive inhibitor of the enzyme (Ki approximately 3 nM) with respect to testosterone. SDS-PAGE analysis of microsomal, detergent-solubilized, and (6.5%) polyethylene glycol-precipitated fractions of 5 alpha R-I photolyzed with [3H]4MABP in the presence of NADPH showed that the radioactivity was incorporated into a single protein band with a mass of 26 kDa (apparent molecular weight of 5 alpha R-1). UV photolysis was accompanied by an irreversible loss in enzyme activity, consistent with its covalent modification. Increasing the time of UV irradiation and concentration of [3H]4MABP indicated that the half-life and apparent Kd for its photo insertion were approximately 3 min and 7.5 nM, respectively. Photolysis in the presence of a 20-fold excess of N,N-diethyl-4-aza-4-methyl-3-oxo-5 alpha-androstane-17 beta-carboxamide or the 3-carboxysteroid SKF-105111 resulted in partial protection of 5 alpha R-1 from the probe, whereas minimal incorporation of radioactivity was observed in the absence of NADPH or in the presence of NADP+. The results indicate that [3H]4MABP is an effective probe of the steroid (D-ring) binding domain of 5 alpha R-1.

Analysis of the steroid binding domain of rat steroid 5α-reductase (isozyme-1) the steroid D-ring binding domain of 5α-reductase

Abstract We have previously shown that the photoactive 4-azasteroid, [1,2 3 H]N-4(benzylbenzoyl)-3-oxo-4-aza-4-methyl-5α-androstan-17β-carboxamide is an effective probe of rat steroid 5α-reductase (isozyme-1) (5αR-1). In the current investigation, PEG-fractionated (6.5%) detergent-solubilized preparations containing 5αR-1 activity were ultraviolet (UV)-photolyzed with [ 3 H]-4MABP and subsequently purified by 8.75% preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fractions corresponding to the radioactive peak following the dye front were analyzed by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and showed the presence of a single, labeled, 26 KDa protein band, the apparent molecular weight of 5αR-1. TCA precipitation of the labeled fractions, followed by long-term digestion of the TCA pellet with chymotrypsin and high-performance liquid chromatography analysis, indicated that the majority of the radioactivity eluted with a peak retention time of 55–56 min. Rechromatography of this fraction using a modified gradient (elution 54–55 min), followed by sequence analysis, yielded a single N-terminal tetrapeptide with the sequence, -L-E-G-F-, corresponding to residues 15–18 of the 5αR-1 sequence. Site-directed mutagenesis studies indicated that mutant F18L showed an ∼12-fold increase in the K m for testosterone, whereas the K m for reduced nicotinomide adenine dinucleotide phosphate remained virtually unaltered.