A fast and sensitive method for measuring picomole levels of total free amino acids in very small amounts of biological tissues.
In the present study we describe a simple and fast method to measure the concentration of total free amino acids in very small amounts of biological tissues. The procedure described here is based on the reaction of free amino acids with o-phthaldialdehyde (OPA) in the presence of a reducing agent, beta-mercaptoethanol (MET), to give a complex which can be measured by fluorescence. It is a very rapid process and has the same reliability as the conventional ninhydrin method of Moore and Stein but is about 500 times more sensitive. The sensitivity of the new protocol is such to permit the determination with high reliability of very small amounts of free amino acids at picomole levels, either in a standard amino acid mixture or in biological tissues, without chromatographic separation of the amino acids. It is particularly useful when the amount of the sample is very low, e.g. on a single pituitary or pineal gland of small animals or on single cells, such as oocytes or eggs, as well as single ganglions or axons of marine invertebrates.
Fisher
G H
GH
Department of Chemistry and School of Natural and Health Sciences, Barry University, Miami Shores, Florida 33161, USA. gfisher@mail.barry.edu
Arias
I
I
Quesada
I
I
D'Aniello
S
S
Errico
F
F
Di Fiore
M M
MM
D'Aniello
A
A
D000596:Amino Acids / Q000032:analysis* / Q000737:chemistry; D000818:Animals; D001671:Biochemistry / Q000379:methods*; D017952:Ganglia, Invertebrate / Q000737:chemistry / Q000378:metabolism; D008623:Mercaptoethanol / Q000737:chemistry; D010063:Ovum / Q000737:chemistry / Q000378:metabolism; D010870:Pineal Gland / Q000737:chemistry / Q000378:metabolism; D010902:Pituitary Gland / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D005680:gamma-Aminobutyric Acid / Q000032:analysis / Q000737:chemistry; D009764:o-Phthalaldehyde / Q000737:chemistry
D000596:Amino Acids; D005680:gamma-Aminobutyric Acid; D008623:Mercaptoethanol; D009764:o-Phthalaldehyde