A chemical affinity system exhibiting antibody-like properties is described. The system exploits bioconjugates with appended phenylboronic acid (PBA) moieties and a support-bound phenylboronic acid complexing reagent derived from salicylhydroxamic acid (SHA) for protein immobilization on a chromatographic support. The structure of the PBA.SHA complex was characterized by 11B NMR and mass spectrometry and compared with complexes derived from model compounds. Protein modification reagents were synthesized from 3-aminophenylboronic acid and utilized to prepare bioconjugates from alkaline phosphatase (AP) and horseradish peroxidase (HRP). AP obtained from one source afforded PBA bioconjugates exhibiting significant loss of enzymatic activity, whereas AP obtained from a second source afforded PBA bioconjugates exhibiting only a modest loss of enzymatic activity. Conversely, HRP afforded PBA bioconjugates exhibiting no loss of enzymatic activity. SHA-modified Sepharose was prepared by reaction of methyl 4-[(6-aminohexanoylamino)methyl]salicylate with CNBr-activated Sepharose 4B, followed by treatment with aqueous alkaline hydroxylamine. PBA-AP and PBA-HRP conjugates were efficiently immobilized on SHA-Sepharose at pH 8.3. PBA-AP conjugates were retained after washing with acidic buffers at pH 6.7, 4.2, and 2.5, whereas PBA-HRP conjugates were retained after washing with buffer at pH 6.7, but were eluted to some extent at and below pH 4.2. The results are interpreted in terms of multivalent interactions involving boronic acid complex formation between the enzyme bioconjugates and immobilized complexing reagent.

We have used a nonspecific protein cleaving reagent to map the interactions between subunits of the multisubunit enzyme RNA polymerase (Escherichia coli). We developed suitable conditions for using an untethered Fe-EDTA reagent, which does not bind significantly to proteins. Comparison of the cleaved fragments of the subunits from the core enzyme (a!2313') and the holoenzyme (core + .70) shows that absence of the Or70 subunit is associated with the appearance of several cleavage sites on the subunits fJ (within 10 residues of sequence positions 745, 764, 795, and 812) and 3' (within 10 residues of sequence positions 581, 613, and 728). A cleavage site near 13 residue 604 is present in the holoenzyme but absent in the core, demonstrating that a conformational change occurs when cr70 binds. No differences are observed for the a subunit.Gene transcription in living cells is a complex process, with large numbers of protein factors involved in selecting the correct initiation site on DNA and initiating and carrying out RNA synthesis to the appropriate end. The proteins responsible for transcription in both prokaryotic and eukaryotic cells are actively being identified and their functions explored (1-8).Bacterial RNA polymerase is a well-studied example. It is composed of five protein subunits (Ct2f1'o), of which one (o-) is a member of a group of related proteins that convey different DNA-binding specificities. The most common a subunit in Escherichia coli has a mass of 70 kDa and is designated a70.It has long been known that oa70 binds reversibly to the at23' core enzyme (9-11), but the actual binding site has been difficult to identify. This problem has been addressed by chemical cross-linking (12-14) and by molecular genetics (15), but identification of the particular amino acid residues on the core subunits involved in oJ7() binding remains a challenge.

Phenylboronic acid bioconjugates prepared from alkaline phosphatase by reaction with either 2,5-dioxopyrrolidinyl 3-[N-[3-(1,3,2-dioxaboran-2-yl)phenyl]carbamoyl]propanoate (PBA-XX-NHS) or 2,5-dioxopyrrolidinyl 6-[[3,5-di-(1,3,2-dioxaboran-2-yl)phenyl]carbonylamino]hexanoate (PDBA-X-NHS) were compared with respect to the efficiency with which they were immobilized on salicylhydroxamic acid-modified Sepharose (SHA-X-Sepharose) by boronic acid complex formation. When immobilized on moderate capacity SHA-X-Sepharose (5.4 micromol of SHA/mL of gel), PDBA-alkaline phosphatase conjugates were shown to be stable with respect to both the alkaline (pH 11.0) and acidic (pH 2.5) buffers utilized to recover anti-alkaline phosphatase during affinity chromatography. Boronic acid complex formation was compared to covalent immobilization of alkaline phosphatase on Affi-Gel 10 and Affi-Gel 15. PDBA-AP.SHA-X-Sepharose was shown to afford superior performance to both Affi-Gel 10 and Affi-Gel 15 with respect to immobilization of alkaline phosphatase, retention of anti-alkaline phosphatase and recovery of anti-alkaline phosphatase under alkaline conditions. High capacity SHA-X-Sepharose (> or = 7 micromol of SHA/mL of gel) was shown to afford superior performance to moderate capacity SHA-X-Sepharose (4.5 micromol of SHA/mL of gel) with respect to stability at pH 11.0 and pH 2.5 when a PDBA-alphaHuman IgG conjugate with a low incorporation ratio of only 1.5:1 was immobilized on SHA-X-Sepharose and subsequently utilized for affinity chromatography of Human IgG. The results are interpreted in terms of either a bivalent or trivalent interaction involving boronic acid complex formation.

The reduction of Os(NH3)5(OTf)3 in D20 or H20 over zinc amalgam gives rise to a species that retains the ability to form (NH3)50sn adducts with the -acidic ligands acetone, 7V,iV-dimethyluracil (DMU), and acetonitrile for periods of hours in the absence of reducing agent. The i?2-DMU complex was shown not to be in equilibrium with free DMU, in contrast to a previous report. Kinetic trapping studies are described that demonstrate the following:(1) an "active" source of (NH3)50sn other than the aquo complex is formed and decomposes by a first-order process with a half-life of about 3 hat room temperature in D20; (2) the rate of the decomposition process increases markedly at lower pH; and (3) the decomposition rate in H20 is about twice that in D20. No direct spectroscopic or electrochemical evidence of species having osmium-hydrogen bonds was observed at neutral pH. Pentaammineosmium(II) is an extraordinarily versatile metal center for the binding and manipulation of -acidic ligands.1 We are interested in the -complexation of (NH3)5Os2+ with molecules of biochemical significance, but its chemistry in aqueous solution

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