C: Food Chemistry & Toxicology JFS C: Food Chemistry and Toxicology Determination of Total Protein Content in Gelatin Solutions with the Lowry or Biuret Assay P. ZHOU AND J.M. REGENSTEIN ABSTRACT: Gelatins can be obtained from different sources and prepared using different processes, and the end product gelatin may vary in amino acid composition and molecular weight distribution. In the present study, the variation in “protein color” development among gelatins in colorimetric total protein content measurements was investigated at 540 nm using the Biuret assay and at 650 nm using the Lowry assay, with bovine serum albumin as the reference protein. In both the Biuret and Lowry assays, the color response varied significantly among gelatins. The difference in imino acid content was the major factor responsible for this variation, which probably influenced the gelatin helix → coil phase transition and resulted in the difference in gelatin associate state. Based on their “protein color” development abilities in both Biuret and Lowry, gelatins were classified into 2 major groups with the hierarchical cluster analysis: 1 group included all cold water fish gelatins, while the other included gelatins from warm water fish, avian, and mammalian species. Keywords: Biuret, gelatin, hydroxyproline, imino acids, Lowry, protein content Introduction G elatin is an important functional biopolymer that is widely used in foods to improve elasticity, consistency, and stability. It is a class of proteinaceous substances that are derived from the parent protein collagen by procedures involving the destruction of cross- linkages between polypeptide chains of collagen, in some cases, with some amount of breakage of polypeptide bonds (Johns and Courts 1977). Gelatin can be obtained from mammalian, avian, and fish species. The principal raw materials used in gelatin production today are cattle bones, cattle hides, and pork skins. In addition, fish skin and bone and chicken bone could also serve as the sources of raw material for gelatin manufacture. Due to the various sources and manufacture processes of gelatin, the chemical properties of gelatin may vary in 2 ways: first, the differences in amino acid composition, which is similar to that of the parent collagen and, therefore, directly reflects the influence of species and type of tissue; and second, the differences in molecular weight distribution, which result from the variation in the nature or severity of the extraction conditions. During gelatin or collagen preparation, or its chemical and phys- iochemical property determinations, the total protein content often has been monitored by the Lowry assay (Sato and others 1986, 1987; Aidos and others 1999; Sadowska and others 2003), the Biuret as- say (Young and Lorimer 1960; Nomura and others 1996; Yoshimura and others 2000; Zhou and Regenstein 2004, 2005), or the Kjeldahl method (Montero and others 1999; Muyonga and others 2004a, 2004b). Although the Kjeldahl method could be applied in the pro- tein content determination, it is time and sample consuming. It is, therefore, not the 1st choice in a research laboratory for routine de- terminations (Sapan and others 1999). Furthermore, in this method, although it is well recognized that a factor is needed to calculate the total protein from the nitrogen content, most data are calculated using the standard 6.25 conversion factor counting all nitrogen in the sample, making it only an approximation of the actual protein MS 20060229 Submitted 4/24/2006, Accepted 8/9/2006. The authors are with Dept. of Food Science, College of Agriculture and Life Sciences, Cornell Univ., Stocking Hall, Ithaca, NY 14853 Direct inquiries to author Regenstein (E- mail: jmr9@cornell.edu). present. For gelatin, this value was not only very different from other proteins but also different among different gelatins (Leach and Eas- toe 1977). On the other hand, the Biuret and Lowry assays are 2 commonly used colorimetric total protein determination methods, and both are simple, rapid, and relatively precise (AOAC 1990). With colorimetric total protein determination methods, there are some factors that may cause a variation in the color response of dif- ferent proteins and these factors need to be taken into consideration. In the Lowry assay, factors such as amino acid composition might in- fluence the development of color (Chou and Goldstein 1960; Sapan and others 1999). In the Biuret assay, although the amino acid com- position is not a significant factor, the protein association state might result in a variation with respect to the color response (Sapan and others 1999). It has been well known that due to its specific amino acid composition and protein conformation, the color response of gelatin was very different from those of other proteins in both the Lowry and Biuret assays (Gornall and others 1949; Lowry and others 1951). The studies on gelatin showed that among gelatins, there were variations in amino acid composition and molecular weight distri- bution (Eastoe and Leach 1977), which could result in variations in the “protein color” response among different gelatins. Thus, the objective of this study is to identify the variation of color response among gelatins from various sources and preparations us- ing both the Lowry and Biuret assays, and to determine the main factors responsible for the variation. Materials and Methods Gelatins Cattle hide gelatin (MCS; SKW Biosystems, Inc., Waukesha, Wis., U.S.A.), pork skin gelatin (MPS; Kind & Knox Gelatine, Inc., Sioux City, Iowa, U.S.A.), pork bone gelatin (MPB; Kind & Knox Gelatine, Inc.), chicken bone gelatin (ACB, Food Industry Technol- ogy, Miami Beach, Fla., U.S.A.), and tilapia skin gelatin (FWT; Rous- selot, Dubuque, Iowa, U.S.A.) were obtained as commercial food grade gelatins. Catfish skin gelatin (FWC), cod skin gelatin (FCC), North Atlantic pollock skin gelatin (FCS), and cusk skin gelatin (FCT) were prepared by an alkaline extraction process. Various Alaska C474 JOURNAL OF FOOD SCIENCE—Vol. 71, Nr. 8, 2006 C  2006 Institute of Food Technologists doi: 10.1111/j.1750-3841.2006.00151.x Further reproduction without permission is prohibited