Peptide analysis by isoelectric focusing in polyacrylamide gels

Why does ribonuclease irreversibly inactivate at high temperatures?

“…Thermoinactivated ribonuclease was reactivated in accordance with the general approach of Klibanov and Mozhaev (1978). Reduction was carried out by using 0.07 M dithiothreitol in a 6 M aqueous solution of guanidine hydrochloride according to the method of Bibring and Baxandall (1978). Following desalting on Sephadex G-25, the reduced enzyme was allowed to reoxidize for 18 h in the presence of a mixture of 0.5 mM each of reduced and oxidized glutathione and 1 mM ethylenediaminetetraacetic acid (EDTA) in 1 M tris( hydroxymethy1)aminomethane hydrochloride (Tris.HC1) buffer at pH 8.0 (Chavez & Scheraga, 1980).…”

“…H P L C amino acid analysis (Fernstrom & Fernstrom, 1981) of acid hydrolysates of reduced and carboxymethylated (Bibring & Baxandall, 1978) samples of native and thermoinactivated ribonuclease revealed that no significant changes accompany irreversible thermo- inactivation at p H 4 and 90 O C . There is no appreciable decrease in S-(carboxymethyl)cysteine, indicating that destruction of cystine residues, which is responsible in part for ribonuclease thermoinactivation at neutral pH, does not occur under mildly acidic conditions.…”

“…Samples of native and thermoinactivated ribonuclease were reduced and carboxymethylated prior to acid hydrolysis for amino acid analysis, hydrazinolysis, digestion with carboxy-peptidase Y, dansyl chloride end group analysis, or free amino group titration with trinitrobenzenesulfonic acid. The carboxymethylation method employed was based on that described by Bibring and Baxandall (1978): To a solution of 1 mg of native or thermoinactivated ribonuclease in 4.8 mL of 0.5 M Tris.HC1 buffer containing 6 M guanidine hydrochloride and 0.3% EDTA (pH 8.6) was added 50 mg of dithiothreitol (to give a final concentration of 0.07 M) and the mixture was incubated overnight under an argon atmosphere. Addition of 0.6 mL of 1 M iodoacetic acid in 1 N NaOH, followed by a 20-min incubation under argon and desalting on Sephadex G-10, yielded S-carboxymethylated ribonuclease.…”

Mutation in a structural gene for a beta-tubulin specific to testis in Drosophila melanogaster.

“…First, an increasing body of data suggests that microheterogeneity of tubulin subunits is -a key control point for microtubule assembly; in particular, in several species the tubulins present in the 9+2 axoneme of cilia or flagella differ from those present in the cytoplasm or those which comprise the mitotic apparatus (1)(2)(3)(4)(5)(6)(7)(8). Therefore, it might be expected in Drosophila that there are tubulins required for the sperm tail axoneme that are unique to spermiogenesis.…”

The Complete Amino Acid Sequence of the Zn2+‐Containing d‐Alanyl‐d‐Alanine‐Cleaving Carboxypeptidase of Streptomyces albus G

“…After unfolding the protein in 6 M guanidiniuni chloride and cleavage of the S-S bridges with dithiothreitol, alkylation of the SH groups in the reduced protein was carried out by treatment with iodoacetic acid, giving rise to S-carboxymethyl cysteine (CmCys) residues (SCM-protein), [5] or 4-vinyl-pyridine, giving rise to S-[2-(4'-pyridy1)ethyllcysteine (PeCys) residues (SPE-protein) [6]. The efficacy of each of the treatments was checked by amino acid analysis to detect dithiothreitol added to the chlorobutane to a final concentration of 0.001 was from Calbiochem.…”