3.1. Preparations of Samples for NMR Studies

To determine the 3-D structure of PSTD using NMR spectroscopy, we synthesized a 35 amino acid peptide (L245-S279 from the gene sequence encoding the human CMP-N-acetylneuraminate-α-2,8-polysialyltransferase, ST8Sia IV, containing the 32-amino acid PSTD (from residue K246 to R277) sequence. The sequence of the 35-amino acid-PSTD peptide (L245-S279) is as follows: 245LKNKLKVRTAYPSLRLIHAVRGYWLTNKVPIKR277PS279.

The 35 amino acid-PSTD from ST8Sia IV was chemically synthesized by DG Peptides (Hangzhou City, Zhejiang Province, China). Its molecular weight was determined to be 4117.95 and its purity established to be 99.36%.

CMP-Sialic acid (CMP-Sia: C₂₀H₂₉N₄O₁₆P₂Na), sialic acid (Sia: C₁₁H₁₉NO₉), DP 3: C₃₃H₅₀N₃O₂₅Na₃), and polySia were purchased from Santa Cruz Biotechnology. The molecular weight of CMP-Sia, Sia and DP3, are 636.43, 309.11, and 957.73, respectively.

The DP of polySia, when accurately determined in the absence of acidic conditions, which occurs in the DMB method for determining the chain length of polySia, is polydisperse, with DP’s ranging from ~ 5 to > 400 Sia residues [56]. The molecular weight of the sodium salt of the polySia/”Colominic acid” (Cat. No. CAS 70431-34-4: sc-239576, purified from E. coli K1) used in this study, while polydisperse, is ~31 KDa, with the average DP of ~ 95. In the present study, this sample is designated as “polySia” [56].

For both the 1-D and 2-D NMR experiments, the concentration of the 35 amino acid-PSTD peptide was 2.0 mM. The concentration of CMP-Sia, Sia and DP3 was 1 mM. For all of the NMR studies, the concentration of polySia was (0.1 mM), which was dissolved in 25%TFE (v/v), 10% D₂O (v/v), and 65% (v/v) 20 mM phosphate buffer (pH 6.7). Following this, 2-Dimethyl-2-silapentane-5-sulfonic acid (DSS) was added to all samples to serve as a reference standard.