alpha-chymotrypsin and sodium-cyanoborohydride

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.

Topics: Amino Acid Sequence; Amino Acids; Animals; Ascorbic Acid; Binding Sites; Borohydrides; Cattle; Chromatography, High Pressure Liquid; Chymotrypsin; Crystallins; Electrophoresis, Polyacrylamide Gel; Glucose; Glycosylation; Kinetics; Macromolecular Substances; Mass Spectrometry; Molecular Sequence Data; Pepsin A; Peptide Fragments

When the retinal Schiff base in chymotryptically cleaved bacteriorhodopsin is reduced to a secondary retinylamine by prolonged exposure to 10% (wt/vol) sodium cyanoborohydride, at pH 10, in the absence of light, approximately 45% of the retinal is found linked to Lys-41 and 22% to Lys-40, and the remainder is scattered over various sites on the large chymotryptic fragment, including the physiological site at Lys-216. The retinal-binding site is destroyed or blocked by the reduction conditions, but the bacteriorhodopsin lattice remains intact. The results demonstrate that artifactual linkage to Lys-40/41 is possible under special conditions. Under these conditions, the epsilon-amino groups of Lys-40/41 show an enhanced ability to form retinylidene linkages with the retinal released by the physiological linkage site at Lys-216, due to some combination of close proximity to the normal linkage site, and increased reactivity with respect to other lysine epsilon-amino groups. The results are of interest for the characterization of the two newly discovered rhodopsin-like proteins, halorhodopsin and slow rhodopsin.

Topics: Amino Acids; Bacteriorhodopsins; Borohydrides; Chemical Fractionation; Chymotrypsin; Cyanogen Bromide; Darkness; Halobacterium salinarum; Hydrogen-Ion Concentration; Light; Polylysine; Protein Binding; Purple Membrane; Retinaldehyde; Schiff Bases; Tritium