Liberation of Hydroxyproline from Gelatin by Acid Hydrolysis
A review of t'he literature on gelatin hydrolysis reveals a lack of consistency in the techniques employed. Acids of varying concentrations, enzymes, and nonexchange resins arc among the many methods employed (l-7). Although such techniques as enzymic degradation of proteins are used for obvious reasons, too often methods are reported that require prolonged, arbitrarily chosen hydrolysis times that can result in destruction of labile amino acids.
The purpose of this study was to find a simple, convenient technique for the complete liberat'ion of hydroxyproline from gelatin by acid hydrolysis.
Methods and Results. Hydrolysis was accomplished by sealing the gelatin samples in 4 ml of HCI of the desired normality in Pyrex test tubes. These were placed into wells in a preheated aluminum block, and the time and temperature of hydrolysis varied as desired.
Each hydrolyzate was adjusted to approximately pH 6.5 with 6 AT NnOH and diluted to 1000 ml; duplicate samples were analyzed quantitatively for hydroxyproline after the method of Woessner (8). Pilot studies were conducted using gelatin in 4.0 ml of 6.5 N HCI at a temperature of 130Β°C for 1 hr. Nitrogen determinations of the desiccated gelatin used gave values of 18.54 t 0.2%. Under optimal conditions, 30 mg of the gelatin yielded from 3.93 to 4.11 pg/ml hydroxyproline in a 1 : 1000 dilution following hydrolysis, with a mean value of 4.02 pg. Using these values of time, temperature, and normality, we found that the maximum weight of gelatin that would liberate the theoretical maximum of hydroxyproline was between 240 and 300 mg, with 100% being obtained at 240 mg, dropping to 92% for 300 mg.