Faculty Bibliography

Dog serum treated with the oxidant chloramine-T is rapidly and selectively depleted of its ability to inhibit porcine pancreatic elastase or dog neutrophil elastase. Trypsin inhibitory capacity of serum is not affected. Purified dog alpha-1-proteinase inhibitor (alpha-1-PI) is similarly oxidized with an apparent rate constant of 1.1 x 10(3) M-1 sec-1. Reversal of the oxidative inactivation using dithiothreitol was demonstrated. Cigarette smoke also directly affects the inhibitory capacity of both serum and pure alpha-1-PI. These studies form a basis for developing a model of functionally deficient alpha-1-PI by taking advantage of oxidative inactivation of normal proteinase inhibitor levels

The principal canine plasma protease inhibitor, alpha-1-antiproteinase, has been purified 90-fold with a 25% yield to apparent homogeneity. The purification scheme includes anion-exchange chromatography, to separate away the bulk of the serum albumin; affinity chromatography by insolubilized concanavalin A, to remove most of the other serum proteins as well as traces of albumin; and, finally, sizing on Sephacryl-S-200. Unique to this purification scheme is the batch use of insolubilized hemoglobin--Sepharose beads to remove the ubiquitous contaminant haptoglobin. The purified material has an apparent molecular weight of 58 000, 11.2% carbohydrate, and an E280nm1% = 5.82, and can be separated by isoelectric focusing into at least two distinct forms with pI values of 4.40 and 4.52. In addition, canine alpha-1-antiproteinase is immunologically distinct from human alpha-1-antiproteinase

Affinity chromatography on columns containing globin mRNA, R17 phage mRNA, or double-stranded RNA linked to cellose is used to demonstrate unequivocally that the eukaryotic initiation factor (eIF-2) that forms a ternary complex with Met-tRNAf and GTP also binds tightly to these RNA species. Affinity chromatography of reticulocyte ribosomal wash yields over 100-fold purification of Met-tRNAf-binding factor. This factor is eluted as one of the most tightly bound proteins, and is active in protein synthesis even after passage over a column of double-stranded RNA-cellulose. eIF-2 binds mRNA and double-stranded RNA in distinctly different modes, protecting essentially all sequences in double stranded RNA, but very few in mRNA, against digestion with ribonuclease. Apparently, eIF-2 recognized the A conformation of double-stranded RNA, but not its sequence. By contrast, globin, Mengo virus, R17 and vesicular stomatitis virus mRNA are shown to possess a high-affinity binding site for eIF-2 that is absent in negative-strand RNA of vesicular stomatitis virus, an RNA that cannot serve as messenger. The results support the concept that eIF-2, the initiation factor that binds Met-tRNAf, recognizes an internal sequence in mRNA essential for protein synthesis

The weaker basicity at the 2-amino and 4-amino sites of pyridines, pyrimidines, and purines, when compared to the endocyclic nitrogen atoms, precludes estimates of the pK values for the ionization of the conjugate acids of exocyclic amino groups by direct titration. A kinetic method is proposed for obtaining estimates of these microconstants for the aromatic exocyclic amino groups of heterocyclic compounds [K(c) = (ArNH(2))a(H(+))/(ArNH(3)(+))], based upon the rates of reaction with formaldehyde to form the N-hydroxymethylamine. The Bronsted equation, pK(c) = log k(0)(u)- 1.61)/0.87, where k(0)(u) is the pH-independent rate constant for N-hydroxymethylation with respect to unhydrated formaldehyde, is based on aromatic exocyclic amines (pK(c) = -6.0-2.0) and provides, with values of K(0)(u), ranges of values of pK(c) at 25 degrees C for series of adenine, guanine, and cytidine derivatives of -2.8 to -2.2, -1.8 to -1.6, and -2.2 to -1.7, respectively. From the same Bronsted equation, the estimates of the microscopic proton dissociation constants of amides for N-protonation of benzamide and urea are estimated at -8.4 and -3.7, respectively.

The products of the reaction of exocyclic aromatic amines with formaldehyde are hydroxymethylamines and not imines; rate and equilibrium constants for carbinolamine formation have been determined for a series of substituted anilines and 1 -methyl-4-aminopyridiniuim ion. The hydroxymethylamine formation constants (K (1)) from hydrated plus unhydrated formaldehyde and aromatic amines of varying basicity (about pK(a)(1) 6 to -6) are in the range 3-27 M(-)(1) and show a relatively small dependence upon basicity (beta(nuc) values 0.04 and 0.16 for substituted anilines and N-methylanilines, respectively). The formation constants for cationic hydroxymethylamines from cationic amines and formaldehyde (K(2)) are about two orders of magnitude less favorable than those for the neutral species and, reciprocally, the pK(a) (2)values of N-hydroxymethylanilinium ions are about two pH units lower than their parent anilinium ions. Hydroxymethylamine formation occurs by general and specific acid and general and specific base catalyzed pathways, in addition to a pH-independent pathway. For the anilines, the Bronsted plots for general acid catalysts are nonlinear with the break occurring at a lower pK value than expected from the pK(0) value estimated for the alcoholic group of >N(+)HCH(2)0H of ~9. A stepwise preassociation or spectator mechanism is suggested to account for these data and involves a preassociation of catalyzing acid, amine, and aldehyde in an encounter complex ( K (E) ) ,the formation of (k2) and proton transfer from the catalyst to an unstable zwitterionic tetrahedral intermediate (k(3)), T+/-, with in the encounter complex, and dissociation or rotation (k(4)) before product formation. With strongly acidic catalysts k(2)is the rate-determining step and with more weakly acidic catalysts k(3)is the rate-determining step. There appears to be a significant degree of stabilization of the transition state for C-N bond formation by general acids and hydronium ions. For the most weakly basic amine, 1-methyl-4-aminopyridiniuiomn , the hydronium ion and general acid catalyzed pathways appear to be more concerted based upon a linear Bronsted plot (alpha = 0.28) as a consequence of the decreased stability of T+/-. The rate constants for the uncatalyzed conversion of T+/- to a neutral tetrahedral intermediate are 1-5 X IO(7) s(-)(1) and are consistent with a solvent mediated intramolecular proton-transfer ("proton switch") mechanism of carbinolamine formation. For a more limited series of general base catalysts (pK = 9-11.5), the rate constants for the proton-transfer processes calculated from the observed rate constants and estimated equilibrium constants (K(n),) for the formation of T+/- from amine and hydrated and unhydrated formaldehyde are in the range of 10(7.4)-10(9)M(-)(1)s(-)(1) for monofunctional general bases. These values approach those expected for diffusion-limited processes but the limited data with linear Bronsted plots (beta values of 0.17 +/- 0.05, 0.06 +/- 0.02, and 0.13 +/- 0.01 for the 4-cyano-, 4-nitro-, and 3-fluoro-4-nitroanilines, respectively) for general base catalysts do not unequivocally rule out a concerted mechanism of catalysis with proton removal by the base from the nucleophile as the N-C bond is formed.