THE U.S. FIRST LADIES GOWNS: A BIOCHEMICAL STUDY OF SILK PRESERVATION
MARY A. BECKER, POLLY WILLMAN, & NOREEN C. TUROSS
New silk fibroin goes into solution only with very aggressive treatments, such as those using LiBr or LiI, which degrade the molecule, rendering it partially or totally soluble (Lucas et al. 1958). Urea was chosen as the solvent for this study because of its known denaturing properties (Stryer 1988) that allow the protein molecules to assume a random-coil chain conformation with no evidence of main chain peptide cleavage.
All samples were extracted in 1 ml of 7 M urea, 0.05 Tris(tris(hydroxymethyl) aminomethane) solution (pH 8) for one week with constant rocking and occasional vortex mixing to facilitate dispersion of the solution throughout the fabric. After one week, the urea-Tris solution was removed, and the remaining fabric was rinsed a minimum of five times with deionized water. The fabric samples were prefrozen, then lyophilized to dryness. Percent weight loss was calculated using the comparison of preextraction to postextraction dry weights.
5.2 AMINO ACID ANALYSIS
Microgram-sized fabric samples were weighed and put into hydrolysis tubes, dissolved in 200–400 μl of 6N HCl, flushed with nitrogen, and tightly capped. The samples were then hydrolyzed at 110°C for 22 hours. After hydrolysis, samples were dried in a vacuum evaporator at 50°C and stored dry at −20°C until just prior to chromatographic separation. All samples from the First Ladies gowns were filtered through 0.45 μm Millipore filters to remove particulates prior to injection.
Asparagine and glutamine residues are deamidated to aspartic and glutamic acid upon acid hydrolysis (Robinson and Rudd 1974) and therefore reported as the sum of both the acid and amide (Asx and Glx).
Amino acid compositions of 45 silk fabric samples from the First Ladies gowns were determined using reverse-phase high-pressure liquid chromatography (RP-HPLC) on a Millipore Waters Pico-Tag Amino Acid Analysis system. Amino acids were precolumn derivatized with phenylisothiocyanate (PITC) and separated by RP-HPLC (Bidlingmeyer et al. 1984; Cohen et al. 1986). Amino acids were eluted using a gradient buffer system and detected by UV absorbance at 254 nm.
Amino acid compositions of the artificially aged silk fabrics were determined by ion-exchange high-pressure liquid chromatography (IE-HPLC) (Benson and Hare 1975; Stathoplos 1989). Primary amino acids were separated by ion-exchange and detected by postcolumn derivatization with o-phthaldialdehyde /2-mercaptoethanol (OPA) to form fluorescent-substituted isoindole products. Two runs were required to adequately separate the basic amino acids from the interfering buffer peaks (Hare 1977). For analysis, hydrolyzed samples were diluted to a concentration of 1 μg/μl. The compositions are based on daily calibrations to standard solutions of each amino acid reported.
5.3 DESALTING THE PROTEIN-UREA EXTRACTIONS
Urea was removed from the silk that came into solution using a rapid desalting procedure (Cooper 1977). Rapid desalt columns were prepared from a slurry of polyacrylamide gel (Bio-Gel P-6DG) and 50 mM ammonium acetate (pH 7.2) loaded into 10 ml disposable pipettes. A column was calibrated for a 2 mL sample with bromphenol blue (670 MW) and blue dextran (2,000,000 MW) dissolved in urea. Samples were loaded onto the individual columns, eluted with 50 mM ammonium acetate, and collected into vials. Sample collection was terminated just prior to the elution volume of bromphenol blue, before the urea eluted. Samples were lyophilized, resuspended in distilled water, and then relyophilized to obtain a final dry weight.