Hydroxyproline products

Prolylhydroxylase, the collagen synthesis enzyme that has low activity in silicon-deficient tissue, requires iron that cycles between Fe " " and Fe " ". The apoenzyme will bind aluminum (less strongly than iron), and the enzyme is then, of course, inactive. An experiment was conducted [6] in which the apoenzyme was presented with iron first and then aluminum, aU other essential cofactors being present Activity, as measured by hydroxyproline production, was reduced by 20% of the control level. When the apoenzyme was presented with aluminum first and then iron, activity was reduced by 55% of the control levels. Silicic acid alone had no effect on the activity, which remained at the control level (Table 58.1). 

Very few post-translational modifications have been found on tropoelastin. However, hydroxylation of 25% of the proline residues is observed [10]. The enzymatic modification of proline to hydroxyproline (Hyp) is performed by prolyl hydroxylase [11]. The purpose of this hydroxylation remains unclear and it is even proposed that Hyps in tropoelastin are a by-product of collagen hydroxylation as this occurs in the same cellular compartment [8]. 

Plaquet et al. (PI) found in the urine of rachitic children peptides consisting of proline, hydroxyproline, and glycine, which they believed to be the products of collagen degradation. Two similar peptides containing considerable amounts of proline and hydroxyproline were isolated from the urine of a patient with rheumatoid arthritis by Mechanic et al. (Ml). One of these peptides consisted of three proline, two hydroxyproline, and nine glutamic acid residues, the second one consisted of four proline, four hydroxyproline, and one glutamic acid residues. The N-terminal amino acid in the first peptide was demonstrated to be hydroxyproline. 

Primary amino acids will react with o-phthalaldehyde in the presence of the strongly reducing 2-mercaptoethanol (pH 9-11) to yield a fluorescent product (emission maximum, 455 nm excitation maximum, 340 nm). Peptides are less reactive than a-amino acids and secondary amines do not react at all. As a result, proline and hydroxyproline must first be treated with a suitable oxidizing agent such as chloramine T (sodium A-chloro-p-toluene-sulphonamide) or sodium hypochlorite, to convert them into compounds which will react. Similarly cystine and cysteine should also be first oxidized to cysteic acid. 

All primary amines react with fluorescamine under alkaline conditions (pH 9-11) to form a fluorescent product (Figure 10.12) (excitation maximum, 390 nm emission maximum, 475 nm). The fluorescence is unstable in aqueous solution and the reagent must be prepared in acetone. The secondary amines, proline and hydroxyproline, do not react unless they are first converted to primary amines, which can be done using A-chlorosuccinimide. Although the reagent is of interest because of its fast reaction rate with amino acids at room temperature, it does not offer any greater sensitivity than the ninhydrin reaction. 

Hydroxyproline assay. Hydroxyproline was determined according to Jamall et al. (1981). The assay employs p-dimethylaminobenzaldehyde (Ehrlich s reagent), which forms colored products with pyrroles originating from hydroxyproline oxidation. The values thus determined for hydroxyproline mass were multiplied by 8.0 to obtain the corresponding collagen mass. 

Analogous results were obtained in the more complicated case of 15, i.e., the acetal derived from (2S,47 )-4-hydroxyproline and trimethylacetaldehyde85. Alkylation was carried out by means of the dianion 16 and the product 17 was formed in moderate yields but with a d.r. >98 2. 

Orthophthalaldehyde (OPA) in combination with a thiol is the reagent of choice for derivatization, despite its inability to react with proline, hydroxyproline, and the sulfur-containing amino acids. Another drawback of the reagent is the instability of the reaction products, making an automated derivatization system coupled to an automated injector, and constant retention times an absolute necessity. Taking into account these considerations, the HPLC analysis will be of use to every biochemical genetics laboratory for biological fluids other than urine. The system has also a... 

The red pigment isolated in crystalline form from the reaction products of 1-methyl-3,4-benzoquinone and 4-hydroxyproline ethyl ester was identified as 4-(4 -hydroxy-2 -carbethoxy-l -pyrrolidyl)-5-methyl-l,2-benzoquinone (115).283 This highly colored compound should probably be formulated as a zwitterion (116). Similar pigments can be obtained by the interaction of o-benzoquinone with proline, hydroxyproline, pyrrolidine, and glycine. Suzuki has shown... 

There are many types of vertebrate collagen. Typically they contain about 35% Gly, 11% Ala, and 21% Pro and 4-Hyp (4-hydroxyproline, an uncommon amino acid see Fig. 3-8a). The food product gelatin is derived... 

Figure B2.2.2 Reaction of ninhydrin with proline or hydroxyproline, producing the yellow product.

Yamaguchi et al. also showed that Rb-L-prolinate catalyzes enantioselective addition of nitroalkanes to several acyclic and cyclic enones [25, 26]. For acyclic enone acceptors the best result, i.e. 74% yield and 68% ee of the S product, was achieved in the addition of 2-nitropropane to -3-penten-2-one (40a, Scheme 4.13) [25]. Screening of several proline derivatives and cyclic amino acids of other ring size resulted in the identification of the O-TBDMS-derivative of 4-hydroxyproline as the best catalyst for addition of nitrocyclohexane to cycloheptenone. In this particular reaction 74% yield and 86% ee were achieved [26]. 

---