Pentamines as substrate for human spermine oxidase

Abstract

Substrate activities of various linear polyamines to human spermine oxidase (hSMO) were investigated. The activities were evaluated by monitoring the amount of H2O2 released from sample polyamines by hSMO. H2O2 was measured by a HPLC method that analyzed fluorescent dimers derived from the oxidation of homovanillic acid in the presence of horseradish peroxidase. Six triamines were tested and were found not to be hSMO substrates. Of sixteen tetramines tested, spermine (Spm) was the most active substrate, followed by homospermine and N-butylated Spm. Pentamines showed a characteristic pattern of substrate activity. Of thirteen pentamines tested, 3343 showed higher substrate activity than Spm, and 4343 showed similar activity to Spm. The activities of the other pentamines were as follows: 3443, 4443, 4344, 3344, 4334, 4444, and 3334 (in decreasing order). Product amines released from these pentamines by hSMO were then analyzed by HPLC. Triamine was the only observed product, and the amount of triamine was nearly equivalent to that of released H2O2. A marked difference in the pH dependency curves between tetramines and pentamines suggested that hSMO favored reactions with a non-protonated secondary nitrogen at the cleavage site. The Km and Vmax values for Spm and 3343 at pH 7.0 and 9.0 were consistent with the higher substrate activity of 3343 compared to Spm, as well as with the concept of a non-protonated secondary nitrogen at the cleavage site being preferred, and 3343 was well degraded at a physiological pH by hSMO.

Fig 1. Measurement of H₂O₂ release from Spm by hSMO

The experiment was performed in triplicate with the data presented as mean ± standard deviation. The experiment was performed in the absence and presence of 0.25 mM Spm or preincubated with MDL72527.

Fig. 2. Substrate activities of polyamines

Polyamines (0.25 mM) were incubated with hSMO for 10 min under the conditions described in Materials and Methods. The experiment was performed in triplicate, the data were normalized to that of Spm, and presented as mean ± standard deviation.

Fig 3. Measurement of triamines released from indicated polyamines by hSMO

Indicated polyamines (0.25 mM) were incubated with hSMO for 30 min. The experiment was performed in triplicate, the data were normalized to 34 released from Spm, and presented as mean ± standard deviation.

Fig. 4. Tetramines and pentamines exhibit distinct patterns of pH-dependency

H₂O₂ release was determined using the indicated polyamines (0.25 mM) as a substrate at pH 7.0 - 9.0. The experiment was performed in triplicate, the data were normalized to that of Spm at pH 9.0, and presented as mean ± standard deviation.