A simple and sensitive spectrophotometric method for the quantitative determination of solid supported amino groups

A simple and sensitive method for the quantitative determination of free amino groups on solid support is described. This approach is a modification of Ngo's [(1986) J. Biochem. Biophys. Methods 12, 349-354] method reported earlier. The method is based on the reaction of the solid support with an excess of 5'-O-(4,4'-dimethoxytrityl)-thymidine-3'-O-(2,4-dinitrophenyl) succinate (DTDS) in the presence of a catalytic amount of 4-dimethylaminopyridine. After removing the excess reagent, solid support is treated with perchloric acid to release 4,4'-dimethoxytrityl cation into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption at 498 nm (epsilon 498 = 70,000), is then determined spectrophotometrically. A comparative study of DTDS, N-succinimidyl-3-(2-pyridyldithio)propionate and 4,4-dimethoxytrityl chloride is also included. The method was found to be very useful to determine those amino groups which are available for functionalization of solid supports, especially, monitoring the functionalization of solid supports for affinity chromatography and synthesis of biopolymers.     

Competitive labelling, a method for determining the reactivity of individual groups in proteins. The amino groups of porcine elastase

1. A method is described for determining the ionization constants and reactivities of individual amino groups in proteins. The principle is that in the presence of a trace amount of radioactive label, the various reactive groups in a protein molecule will compete for the label and the amount incorporated into any one group will be determined by its nucleophilicity, pK and micro-environment. The relative amounts of label incorporated into various groups will be proportional to their second-order rate constants and by comparing these rate constants with those expected on the basis of a linear free-energy relationship obtained with a series of standard compounds, the micro-environment can be defined for a particular amino group. 2. The method consists of treating a protein and an internal standard with a limiting amount of radioactive reagent and then with an excess of unlabelled reagent to yield a chemically homogeneous but heterogeneously labelled compound. After appropriate enzymic digestion peptides containing each labelled group are isolated and their rates of reaction, relative to the internal standard, are determined from their specific radioactivities. The entire procedure is repeated at several pH values. 3. When the method was applied to the amino groups of porcine elastase by using tritiated acetic anhydride as the labelling reagent, the N-terminus was found to have pK(a) 9.7 and a much lower than normal reactivity. Lysine-87 and lysine-224 were found to have pK(a) 10.3 and normal reactivities. At pH values greater than 10.5 there are discontinuities in all the titration curves, indicating that the entire molecule is undergoing a structural reorganization.     

A simple spectrophotometric determination of solid supported amino groups

A simple spectrophotometric method for the quantitative determination of solid phase supported amino groups is described. The method involves reacting the solid support with an excess of activated acylating agent, N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) and an efficient acylation catalyst, 4-dimethylaminopyridine, and after thoroughly removing the unreacted SPDP, the solid support is reacted with an excess of dithiothreitol to quantitatively release pyridine-2-thione from the solid support to the solution. After an appropriate dilution, the released pyridine-2-thione which has a strong absorbance at 343 nm, is quantified by reading its absorbance in a spectrophotometer at 343 nm.     

A simple method for stabilizing protein-sulfhydryl groups during SDS-gel electrophoresis

Sulfhydryl-containing proteins can oxidize during SDS-polyacrylamide gel electrophoresis to produce broad bands with irreproducible mobilities. Oxidation affects molecular weight estimates and decreases resolution. Alkylation of reduced proteins prior to electrophoresis eliminates the problems caused by reoxidation. A simple protocol involves reducing proteins with low concentrations of dithioerythritol and alkylating with a slight excess of iodoacetamide. These reactions are insensitive to atmospheric oxygen and to reagent concentrations provided that sulfhydryl groups are first completely reduced and then completely alkylated. The reagents need not be removed from the proteins prior to electrophoresis.     

The role of the exocyclic amino groups of conserved purines in hammerhead ribozyme cleavage.

A series of 2 stranded hammerhead ribozymes has been synthesized in which single conserved adenosine residues have been replaced by nebularine or single guanosine residues by inosine. Comparison of the rates of trans-cleavage for the modified structures suggests the presence of interstrand non-Watson-Crick hydrogen bonding interactions including a GA:AG double mismatch. The exocyclic amino group of one of the guanosine residues is essential for cleavage.     

Synthesis and application of a fluorescent imido ester for specific labelling of amino groups in proteins

2-(5'-Dimethylaminonaphthalene-1'-sulfonamido)methylimidic acid methyl ester has been synthesized for fluorescence labelling of amino groups in proteins. The incorporation of the dansyl group serving as an extrinsic fluorescent probe is determined spectrophotometrically. Glucose dehydrogenase (beta-D-glucose: NAD(P+) 1-oxidoreductase, EC 1.1.1.47) from Bacillus megaterium having a reactive lysine residue which belongs to the active site has been labelled. To give proof of the selectivity of the modification, the enzyme preparation having 1.3 dansyl groups per subunit has been digested with trypsin and the major labelled peptide has been isolated and sequenced.     

A spectrophotometric method for the estimation of amino groups on polymer supports

A simple and sensitive method for the estimation of polymer-supported amino groups is reported. The polymer support is treated either with N-succinimidyl-4-O-(4,4'-dimethoxytrityl)-butyrate or 2,4-dinitrophenyl-4-O-(4,4'-dimethoxytrityl)-butyrate and a catalytic amount of 4-dimethylaminopyridine. After removal of the excess reagent through washing, a weighed quantity of the polymer support is treated with perchloric acid to release the 4,4'-dimethoxytrityl cation from the solid support into the solution. The released 4,4'-dimethoxytrityl cation, which has a strong absorption (epsilon 498 = 70,000/M) at 498 nm, is determined spectrophotometrically. A comparative study of these reagents with N-succinimidyl-3-(2-pyridyldithio)-propionate, 4,4'-dimethoxytrityl chloride, and sodium 2,4,6-trinitrobenzenesulfonate methods is also included.     

A method for histochemical demonstration of protein-bound amino groups by light microscopy

We have established an efficient method for the histochemical demonstration of protein-bound amino groups by light microscopy, using a ninhydrin or alloxan-thiocarbohydrazide-silver proteinate (NHY or ALX-TCH-SP) sequence followed by a physical development (PD) procedure. As a result of the present experimental studies on Carnoy's solution-fixed paraffin sections of a series of rat tissues, including three types of major salivary glands, liver, pancreas, stomach, duodenum, jejunum, colon, kidney, prostate, and spleen, the sensitivity and specificity of the new method were found to be sufficient. In the tissues tested, protein-bound amino groups were visualized by distinct brownish or blackish reaction products. Comparisons of the particular method with the NHY or ALX-Schiff methods employed hitherto have substantiated the fact that the former method leads to apparently higher visibility of reaction products than the latter.     

A simple biosynthetic method for stereospecific resonance assignment of prochiral methyl groups in proteins

A new method for stereospecific assignment of prochiral methyl groups in proteins is presented in which protein samples are produced using U-[¹³C]glucose and subsaturating amounts of 2-[¹³C]methyl-acetolactate. The resulting non-uniform labeling pattern allows proR and proS methyl groups to be easily distinguished by their different phases in a constant-time two-dimensional ¹H-¹³C correlation spectra. Protein samples are conveniently prepared using the same media composition as the main uniformly-labeled sample and contain higher levels of isotope-enrichment than fractional labeling approaches. This new strategy thus represents an economically-attractive, robust alternative for obtaining isotopically-encoded stereospecific NMR assignments of prochiral methyl groups.     

A dot-blot screening procedure for mutated ras oncogenes using synthetic oligodeoxynucleotides

To analyze human tumors for the presence of mutated ras oncogenes, a procedure was developed based on selective hybridization of mutation-specific oligodeoxynucleotide probes to genomic DNA [Bos et al., Nucl. Acids Res. 12 (1984) 9155–9163]. We have improved this procedure both in sensitivity and speed by including an in vitro amplification step of ras-specific sequences. This amplification step has first been described by Saiki et al. [ Science 230 (1985) 1350–1353] and results in a more than 10⁴-fold increase in the sequence which might contain the mutation. Furthermore, we have improved the selectivity of our hybridizations. As a result, mutated ras oncogenes can now be detected with a dot-blot screening procedure requiring less than 1 μg of tumor DNA.     

A simple procedure for constructing 5'-amino-terminated oligodeoxynucleotides in aqueous solution.

A rapid method for the synthesis of oligodeoxynucleotides (ODNs) terminated by 5'-amino-5'-deoxythymidine is described. A 3'-phosphorylated ODN (the donor) is incubated in aqueous solution with 5'-amino- 5'-deoxythymidine in the presence of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC), extending the donor by one residue via a phosphoramidate bond. Template- directed ligation of the extended donor and an acceptor ODN, followed by acid hydrolysis, yields the acceptor ODN extended by a single 5'-amino-5'-deoxythymidine residue at its 5'terminus.     

A simple method for the separation of bile acid groups by ion-exchange chromatography

A simple ion-exchange chromatographic method for the separation of bile acid mixtures is described. The method employs Dowex 1 as anion exchanger and mixtures of aqueous ethanol and hydrochloric acid as eluants. The use of mixtures in which both the ethanol and acid concentrations are varied has permitted a clear separation of the major bile acid groups (nonconjugated, glycine conjugated, and taurine conjugated bile acids).