Ribonuclease sequence

In the synthesis of the enzyme ribonuclease by the Merrifield method, the 124 amino acids were arranged in the ribonuclease sequence through 369 reactions and some 12,000 individual operations of the automated peptide-synthesis machine without isolation of any intermediates. 

Monolithic capillary microreactor Trypsin Covalent linking Hybrid silica monolith-based immobilized trypsin microreactor for on-line protein digestion of 4-protein mixture BSA, myoglobin, P-globulin, and ribonuclease Sequence coverage were 8,26,10, and 54%, respectively Proteins extracted from E. coli digested and lOlproteins identified. [131]... 

Memfield successfully automated all the steps m solid phase peptide synthesis and computer controlled equipment is now commercially available to perform this synthesis Using an early version of his peptide synthesizer m collaboration with coworker Bemd Gutte Memfield reported the synthesis of the enzyme ribonuclease m 1969 It took them only SIX weeks to perform the 369 reactions and 11 391 steps necessary to assemble the sequence of 124 ammo acids of ribonuclease... 

If polyribonucleotides are treated simultaneously with methoxylamine and bisulphite, cytidine residues are converted into 5,6-dihydro-7V4-methoxycytidine-6-sulphonate,154 and uridine into 5,6-dihydrouridine-6-sulphonate.155 Treatment with dilute ammonia regenerates the uridine residues, leaving the dihydrocytidine derivatives unaffected. When only the cytidine residues have been derivatized, pancreatic ribonuclease becomes uridyl ribonuclease, since it is unable to cleave the chain on the 3 -side of the modified cytidine.154 This allows the isolation of blocks of modified cytidine residues. T2 ribonuclease may also be used. Alternatively, a ribonuclease from Physarum polycephalum has been found to hydrolyse CpX links very slowly, allowing the isolation of cytidine blocks.156 If both uridine and cytidine residues are modified, T2 ribonuclease acts as puryl ribonuclease, allowing the isolation of cumulative blocks of pyrimidines.155 This ability to alter the specificity of nuclease cleavage is a useful tool in sequence analysis. 

An example of this effect is provided by ribonuclease A (RNase A). At pH 8 and 37°, the rate of deamidation of Asn67 was more than 30-fold lower in the native than in the unfolded protein [111]. Deamidation of the native RNase A was also ca. 30-fold slower than of an octapeptide whose sequence is similar to that of the deamidation site, although the reaction mechanisms were similar [108][123],... 

Studies of proteolytic fragments of staphylococcal nuclease (Tan-iuchi and Anfinsen, 1969) and RNase A (Taniuchi, 1970) seemed to support this view. Taniuchi (1970), in summary remarks, said Thus, the minimum information of the specific folding of a protein requiring almost the entire amino acid sequence is observed with both staph-yloccocal nuclease and bovine pancreatic ribonuclease. ... 

Christian Anfinsen United States ribonuclease, amino acid sequencing and biological activity ... 

Ribonuclease A (RNase A) was selected as the target enzyme for solid-phase synthesis because its sequence was known (Scheme S), 22 25 and an X-ray structure had been deduced. 24 Importantly, it had been shown that this 124-residue protein could be reduced and unfolded and then reoxidized to re-form the four disulfide bonds with recovery of full enzymatic activity. 25 ... 

An example of a noncovalent system has already been discussed in the case of ribonuclease A (see Section 5.1.6). 29 32 Thus, much information was gained from the relatively easy synthesis of many peptide analogues with sequences derived from either end of the 124-residue protein, and their combination with the protein segments RNase(21-124), RNase(l-118), or RNase(21-118) produced by selective enzymatic digestions. 

---