Digestion mixtures

It must be emphasised that these experiments are only roughly quantitative, for owing to the alight opalescence of the digesting mixture, accurate end-px>ints using phenolphthalein can be obtained only by using an instrument known as a comparator. 

Structure of GFP and its chromophore. To study the chro-mophore of GFP, a sample of GFP was denatured by heating it at 90°C. It was digested with papain, and then a peptide containing the fluorophore was isolated and purified from the digested mixture. The structural study of the peptide has indicated that the chromophore of GFP is an imidazolone derivative shown below (Shimomura, 1979). This chromophore structure was confirmed later by Cody etal. (1993) in a hexapeptide isolated from GFP. It is intriguing that the structure of the GFP chromophore is a part of the structure of coelenterazine. 

The unseparated digest mixture was studied directly by mass spectrometry using matrix-assisted laser desorption ionization (MALDI) and this showed six of the polypeptides detected by LC-MS and three of the expected polypeptides that had not been detected by LC-MS. In contrast, MALDI did not show three polypeptides observed by LC-MS. 

Microsequencing many peptides in a digested mixture may be coupled with bioinformatics to reveal information on all the component species. 

Eberlein and Kattner [194] described an automated method for the determination of orthophosphate and total dissolved phosphorus in the marine environment. Separate aliquots of filtered seawater samples were used for the determination orthophosphate and total dissolved phosphorus in the concentration range 0.01-5 xg/l phosphorus. The digestion mixture for total dissolved phosphorus consisted of sodium hydroxide (1.5 g), potassium peroxidisulfate (5 g) and boric acid (3 g) dissolved in doubly distilled water (100 ml). Seawater samples (50 ml) were mixed with the digestion reagent, heated under pressure at 115-120 °C for 2 h, cooled, and stored before determination in the autoanalyser system. For total phosphorus, extra ascorbic acid was added to the aerosol water of the autoanalyser manifold before the reagents used for the molybdenum blue reaction were added. For measurement of orthophosphate, a phosphate working reagent composed of sulfuric acid, ammonium molyb-... 

Organic carbon can be determined in calcareous soils after the carbonates have been removed by treatment with sulphuric acid-iron(II) sulphate solution and the samples oven-dried at 105°C. However, as in all other wet-combustion methods, chloride ions interfere [7]. Interference from small amounts of chloride ions (up to 4mg of Ch as KC1 or NaCl) was reduced by adding 2.5% of mercury II oxide or silver I sulphate to the acid digestion mixture. 

Presence of the His392-T3rr415 (y, yes n, no) covalent bond was determined by MALDI-MS analysis of trypic digest mixtures. Where no indication is given, no determination was made. 

Another method of bomb dissolution involves placing the sample and digestion mixture in a sealed PTFE bomb and then encasing this in a stainless-steel jacket. This may then be placed in a conventional oven for a period of several hours. This technique, although cheaper, takes substantially longer. 

For platinum compounds, the nitric/perchloric acid digestion mixture used in NIOSH procedure S-191 was replaced with aqua regia to solubilize platinum metal. Platinum dioxide was solubilized by first heating the compound to >380°C to convert the Pt02 to the aqua regia-soluble forms of platinum metal and platinum monoxide. 

An elegant study of the kinetics has been performed on single-stranded biosynthetic polymers (58), prepared by the method of Bollum et al. (59). Three polymers were used (1) d([3H]pA)B(pA)r2e, (2) d([3H]pA)5(pA)uo ([2- C]pA) - and (3) d([3H]PT)t(PA)in. The digestion was carried out in the presence of Mg2+ and allowed to proceed in a pH stat until 10% of internucleotide bonds were hydrolyzed. The reaction was stopped by heating on a steam bath. The size of the products in the digestion mixture was determined by means of chromatography on a column of Bio-Gel P-60 previously calibrated with oligomers. 

Perform immediately the purification of the digest mixture on a reversed-phase column or by fast desalting by micro SPE using a Ci8 Zip-Tip (Millipore). 

Dry the target under gentle vacuum and wash (also used for desalting if cmde hemolymph or digestion mixture) the sample preparation twice with 1 piL of 1 % TFA. Remove the washing solution after a few seconds using forced air, and dry the sample under vacuum. 

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