The Research Of Metallothionein Detection Method

The Metallothionein discovered until now already had more than 50 years history, passed through the multi-country scientist's unceasing research in this more than 50 years, has established many kinds of examination method, like the Metal-binding method the Radioimmunoassay method, the Enzyme-linked immunosorbent assay method, Differential pulse polarography, the Western blot analysis method,the -SH coloration and the high performance liquid chromatography and so on.Although at present examines the method to be many, but simultaneously has some insufficiencies, like sensitivity low, reproduction quality difference, quantitative not precise and so on short comings.Therefore, so far, but also does not have the establishment standard, unified also the simple examination method.This experiment embarked from this angle, has established with the high performance liquid chromatography examination Metallothionein hydrolysate cysteine, thus carried on, the quota method qualitatively to the Metallothionein.This method has the examination sensitivity high, the operation simple, the quota precise and so on the merits, suits the conventional laboratory operation.The Metallothionein includes the massive cysteine, this atRicle obtains the cysteine through the hydrolisis Metallothionein. Uses Pre-column Derivatization, with O-phthalaldehyde (OPA) took the derivation reagent, then uses the high performance liquid chromatography to carry on the determination again, thus establishment standard, unification examination method.First chooses the cysteine best stable condition of existence.Respectively examined pH is 2.05, 3.05, 5.00, 7.00, 9.00 condition cysteine but whether can exist stably has not transformed as the cystine; Next determination cysteine picks out lowly limits.Respectively examined the density is 2mg/L, 4mg/L, 5mg/L, the 10mg/L cysteine standard solution.When S/N>3, determined picks out lowly for the cysteine limits; Determines the cysteine accuracy once more.Examined 10mg/L, 100mg/L, the 1000mg/L density cysteine 10 specimen handling stabilities separately; Determined once more the cysteine grows after the retention time.Grows after the different density cysteine has the linearity, also mixes the sign with 17 kind of amino acids to grow after the retention time to carry on the contrast; Finally determined in the sample joins 20ug/L, 200ug/L, the 2000ug/L different density Metallothionein standard solution returns-ratio.The experimental result indicated that, 1. cysteine may exist stably under the pH=2.05 condition, but has not transformed as the cystine.2. works as when the cysteine the density is 10mg/L, S/N>3 determined 10mg/L is the cysteine picks out lowly limits.3. under the different density the cysteine RSD<10%, conforms to the rating.4. grows after through the different density cysteine standard solution the atlas to mix the sign with 17 kind of amino acids to grow after the atlas to carry on the contrast to be possible to know, the cysteine standard solution grows after the retention time is 8.8min.In 5. samples joins 20ug/L, 200ug/L, the 2000ug/L Metallothionein standard solution determines its returns-ratio respectively is 44.3%, 66.8%, 83.6%.This experiment simultaneously also uses the amino acid analyzer determination Metallothionein the hydrolysate, also carries on the contrast with the high performance liquid chromatographyy.First determines the Metallothionein the best oxidized time.Separately examined the Metallothionein standard solution after to oxidize 1h, 3h, 6h, 12h, the 16h content; In next determination sample joins 20ug/L, 200ug/L, 2000ug/L Metallothionein standard solution its returns-ratio.The experimental result indicated that, after 1. Metallothionein standard solution oxidizes 16h the content to be highest, the result is best, determined 16h is the Metallothionein best oxidized time.2. when in the sample joins the density is 20ug/L, 200ug/L, the 2000ug/L Metallothionein standard solution determines its returns-ratio respectively is 58.9%, 70.5%, 89.4%.Namely returns-ratio which obtains with the high performance liquid chromatography and the amino acid analyzer determination basic consistent, thus has proven the high performance liquid chromatographyy determination Metallothionein feasibility.Also for the establishment unification, highly effective, the simple examination method has laid the foundation.