This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proline-rich proteins (PRPs) are a family of salivary proteins present in abundant amounts in human parotid and submandibular/sublingual secretions and account for 10-40% of the proteins in these secretions (Kousvelari et al., 1980; Baum et al., 1982; Mandel & Bennick 1983). On the basis of their chemical properties, they are divided into three groups, acidic, basic and glycosylated PRPs. The acidic PRPs are a group of eight structurally closely related molecules with pIs between 4 and 5 (Oppenheim et al., 1971; Bennick and Connell, 1971; Hay et al., 1988). PIFs, PRP1, PRP2 contain 150 residues and have the same amino acid sequence except at positions 4 and 50, which are either aspartic acid or asparagine. PIFf, PRP3 and PRP4 are composed of 106 residues and are identical with the N-terminal 106 residues of PIFs, PRP1 and PRP2, respectively. Genetic studies revealed that the sequence of PRP ?Pa? is identical to PIFs except for the residue at position 27 where leucine substitutes for isoleucine and the residue at position 103 where cysteine substitutes for arginine (Azen et al., 1987). Another acidic PRP characterized by genetic analysis was the ?Db? protein that would contain an extra 21 amino acid repeat of residues 61-81 of PIFs resulting in a 171-amino acid residue polypeptide chain (Azen et al., 1987). The acidic PRPs exhibit unusual biochemical characteristics. They are rich in the amino acids glycine, proline, glutamine and glutamic acid. Amino acid sequencing and chemical analysis has shown that both the serines at residues 8 and 22 are phosphorylated in PIFs, PIFf, PRP1, PRP2, PRP3 and PRP4. Due to the high degree of sequence homology and close pI values of PRPs, separation of these proteins requires chromatography with extremely high resolution and usually involves at least two steps, using a combination of gel filtration and anion-exchange chromatographic techniques. This purification scheme is not only cumbersome, but often results in protein contaminants and unwanted protein loss. Furthermore, these procedures make it impractical to quantitate different PRPs in a number of individuals. The present investigation has resulted in the development of a one step procedure to separate the 8 acidic PRP isoforms and characterized the primary structure and post-translational modifications of the Pa and Db proteins. A manuscript reporting these results will be submitted shortly.