Ribonuclease A and

Isab, A.A. and Sadler, P.J. (1977) Reactions of gold(III) ions with ribonuclease A and methionine derivatives in aqueous solution. Biochimica et Biophysica Acta, 492, 322-330. 

The ease that certain protein mixtures can be separated using reverse micelle extraction was clearly demonstrated by Goklen and Hatton [46], Goklen [31], and Jarudilokkul et al. [25], who investigated a series of binary and ternary protein mixtures. In two cases, they were able to quantitatively extract cytochrome c and lysozyme from a ternary mixture of these proteins with ribonuclease A. Woll and Hatton [24] investigated the separation of a mixture of ribonuclease A and concanavalin A, and showed that the system behaved ideally and that there was no interaction between the proteins. 

ESI-MS has been used for the quantification of a number of substrates and products of enzymatic reactions [56,57]. Hsieh et al. report the use of ion spray mass spectrometry (a technical variation of electrospray ionization) coupled to HPLC for the kinetic analysis of enzymatic reactions in real time [58]. The hydrolysis of dinucleotides with bovine pancreatic ribonuclease A and the hydrolysis of lactose with 3-galactosidase were monitored and the resulting data were used for the estimation of and v x of these reactions. Another field of application of electrospray mass spectrometry is the screening of combinatorial libraries for potent inhibitors [31,59]. 

The solid-phase method was tested with the synthesis of many small peptides,[19] including bradykinin, angiotensin, desaminooxytocin, and the 20-residue S-peptide of ribonuclease A, and was found to be useful and efficient. 

A relative of the kinases is adenylate cyclase, whose role in forming the allosteric effector 3, 5 -cyclic AMP (cAMP) was considered in Chapter 11. This enzyme catalyzes a displacement on Pa of ATP by the 3 -hydroxyl group of its ribose ring (see Eq. 11-8, step a). The structure of the active site is known.905 Studies with ATPaS suggest an in-line mechanism resembling that of ribonuclease (step a, Eq. 12-25). However, it is Mg2+ dependent, does not utilize the two-histidine mechanism of ribonuclease A, and involves an aspartate carboxylate as catalytic base.906 All isoforms of adenylate cyclase are activated by the a subunits of some G proteins (Chapter 11). The structures907 of Gsa and of its complex with adenylate kinase905 have been determined. The Gsa activator appears to serve as an allosteric effector. 

In a follow-up study, the same authors examined the applicability of the same device for relevant protein samples and investigated the main contributions to band broadening [82]. As a consequence of the small depth of the beds, zone spreading caused by Joule heating was shown to be negligible (see Sect. 3.1.1). Cross fields of up to 100 V/cm were applied for the separation of human serum albumin, ribonuclease A and bradykinin. The feasibility of fraction collection was demonstrated with four collected fractions of a whole rat plasma sample. Off-line analysis of these four isolated fractions by CE indicated the separation of serum albumins and globulins. 

Retrieve pdb file of ribonuclease A and use it as the reference coordinates to perform homology modeling of sheep ribonuclease A with the following amino acid sequence ... 

Lewis and Rees1681 determined values of D equal to 2.44, 2.44, and 2.43 for the proteins lysozyme, ribonuclease A, and superoxide dismutase, respectively. Protein regions... 

FIGURE 5 Displacement chromatography of proteins on IMAC using imidazole as the displacer. Column SO X 5 mm i.d. Cu2+ charged metal chelate Sepharose (10 /urn) mobile phase I M NaCI, 25 mM phosphate buffer, pH 7.0 feed l.8mL of 0.89 mM ribonuclease A and 0.69 mM myoglobin in the carrier displacer 10 mM imidazole in the carrier flow rate 0.1 mL/min fraction size 100 /cL fractions. (Vunnum et a/.58)... 

The purification of many enzymes has been achieved by use of gel-filtration chromatography, ion-exchange chromatography, or other chromatographic procedures. Eluates from such columns may be analyzed for carbohydrate and protein content, as well as for enzymic activity. Such analyses have revealed that, for glycoenzymes, the enzymic activity, the carbohydrate content, and the protein content are distributed identically in the eluates from the columns. Results of this type of experiment for ribonuclease B are shown in Fig. 1. In these experiments,8 ribonuclease B and a hydrolyzate of the enzyme were subjected to gel filtration on Sephadex, and the eluates were analyzed for protein and for carbohydrate by appropriate colorimetric methods. A control experiment containing ribonuclease A and D-man-nose was treated similarly. The data in Fig. 1 show that the carbohydrate component in the unhydrolyzed sample of ribonuclease B... 

Fig. 1.—Distribution of Carbohydrate and Protein Components in (A) Ribonuclease B, (B) a Hydrolyzate of Ribonuclease B, and (C) a Mixture of Ribonuclease A and D-Mannose, in Effluents from Gel-filtration Columns.8...

Also, HPLC methods with electrochemical or fluorescent detection are used (H19, M3). In proteins, dityrosine can be estimated by immunochemical methods employing dityrosine-specific antibodies (K5). Measurements of o,o -dityrosine and o-tyrosine levels in rat urine express dityrosine contents in skeletal muscle proteins, and have been proposed as the noninvasive oxidative stress test in vivo. One should be aware, however, that A-formylkynurenine, also formed in protein oxidation, has similar fluorescence properties as dityrosine (excitation 325 nm, emission at 400-450 nm) (G29). Also, oxidation of mellitin when excited at 325 nm produces an increase in fluorescence at 400—450 nm, despite the fact that mellitin does not contain tyrosine. Oxidation of noncontaining Trp residues ribonuclease A and bovine pancreatic trypsin inhibitor with "OH produces loss of tyrosine residues with no increase in fluorescence at 410 nm (S51). There are also methods measuring the increased hydrophobicity of oxidized proteins. Assays are carried out measuring protein binding of a fluorescent probe, 8-anilino-l-naphthalene-sulfonic acid (ANS). Increase in probe binding reflects increased surface hydrophobicity (C7). 

K. A. Palmer and H. A. Scheraga, /. Comput. Chem., 13, 329 (1992). Standard-Geometry Chains Fitted to X-ray Derived Structures Validation of the Rigid-Geometry Approximation, II. Systematic Searches for Short Loops in Proteins Applications to Bovine Pancreatic Ribonuclease A and Human Lysozyme. 

Three levels of protein-nucleic acid recognition have been observed. Nature provides three examples of protein-nucleic acid interactions which we shall consider. The nucleic acid component can be (1) a single nucleotide, e.g., a coenzyme or a substrate, (2) a single-stranded DNA or RNA as in ribonucleases A and T, or (3) a double-stranded DNA or RNA as in the highly specific complexes with repressors in the tRNA-synthetase complex, or in the unspecific nuclease DNase I. 

Specific recognition of single-strand nucleic acid has been investigated in detail for the ribonucleases A and Tj (RNase A and RNase Tj). While RNase A distinguishes pyrimidine nucleotides C and U from the purine nucleotides A and G, RNase Tt specifically recognizes G. 

The small proteins (55-129 amino acids, e. g. the ribonucleases A and T, as well as lysozyme) synthesized in the years 1968-1973 did not satisfy chemical purity demands. E. Bayer, Angew. Chem. 1991, 103, 117. 

G. Reid, J. L. Stephenson, Jr., and S. A. McLnckey, Tandem mass spectrometry of ribonuclease A and B N-Unked glycosylation site analysis of whole protein ions, Anal. Chem. 74 (2002), 577-583. 

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