Chemical degradation of /sup 3/H-labeled substance P in tris buffer solution

Journal Article · Thu Sep 01 00:00:00 EDT 1988 · Anal. Biochem.; (United States)

DOI:https://doi.org/10.1016/0003-2697(88)90215-1· OSTI ID:6582664

Higa, T; Desiderio, D M

Substance P (SP) is an important neuropeptide that has been implicated in several physiological processes, and it is necessary to devise an analytical procedure to measure endogenous SP with a combination of high sensitivity and maximum molecular specificity. However, the unique chemical nature of SP (polarity, chemical stability, ease of oxidation, peptide bond lability) plays a significant role in its analysis, such as in receptor assays, immunoassays, chromatography, and mass spectrometry. In this study, we evaluated in polypropylene and glass assay tubes the effects on the recovery and stability of tritiated SP ((3H)SP) of several pertinent experimental parameters such as buffer, pH, multiple freeze-thaw cycles, and incubation temperature and time. Bovine serum albumin (BSA) effectively reduced the absorption of (3H)SP to polypropylene and glass tube surfaces. Following multiple (6X) freeze-thaw cycles of solutions in BSA-precoated tubes, the recovery of radioactive (3H)SP remained high (greater than 75%) after the last cycle, whereas recovery was minimal in uncoated or siliconized glass tubes. A high level of radioactivity recovery was maintained for 14 days of storage of (3H)SP in triethylamine formate (TEAF) solution in BSA-precoated tubes at 4 and -20 degrees C, but decreased at 37 degrees C to less than 80% in only 3 h. Following storage in Tris-HCl (pH 7.4) buffer, a combination of HPLC and mass spectrometric analyses revealed that a significant amount of peptide bond cleavage occurred to produce the two peptides ArgProLys (RPK) and ArgProLysProGlnGln (RPKPQQ), with only a small amount of remaining intact SP. That decomposition was not observed in triethylamine formate TEAF (pH 3.14) buffer solutions.

• Research Organization: Univ. of Tennessee, Memphis (USA)
• OSTI ID: 6582664
• Journal Information: Anal. Biochem.; (United States), Vol. 173:2
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
PEPTIDES
METABOLISM
ALBUMINS
BUFFERS
FREEZING
LIQUID COLUMN CHROMATOGRAPHY
MASS SPECTROSCOPY
SCINTILLATION COUNTING
SPECIFICITY
STABILITY
THAWING
TRACER TECHNIQUES
TRITIUM COMPOUNDS
CHROMATOGRAPHY
COUNTING TECHNIQUES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
ORGANIC COMPOUNDS
PROTEINS
SEPARATION PROCESSES
SPECTROSCOPY
550501* - Metabolism- Tracer Techniques