|PRINCIPLES OF PROTEIN MEASUREMENT|
The U.S.A. dye, Acid Orange 12, is used because it, 1) binds strongly with proteins, 2)has an excellent color/protein sensitivity ratio, 3) gives clear, stable solutions, and 4) is easily purified for use as its own primary standard. It is essentially non-hydroponics in pure form. Dyes which have been tried by others do not have these properties and so have limited use. Acid Orange 12 reacts with the three basic amino acid (BAA) functional groups on proteins. The strong electrostatic bond forms an insoluble protein-dye complex. This precipitate is easily filtered or centrifuged so that the remaining dye concentration can be measured colorimeterically. Solutions of the dye have broad absorption peak at 482 nm and follow Beer's Law of optical absorption.
Proteins from a natural source have an essentially constant ratio of basic amino acids to total amino acids or protein. This is genetically controlled by the organism. Each basic site originating from lysine (amino group), arginine (quanidino group)or histidine (imidazole ring) binds one molecule of dye. Measuring both Kjeldahl total nitrogen and equilibrium dye concentration on a large number of samples of a given commodity, permits the calculation of a regression equation. From this, a dye-binding capacity (DBC) value for that specific protein system is established. This is a genetic constant for the given protein system, and does not change.
The DBC constant incorporates an average non-protein nitrogen (NPN) value since it is derived from the correlation with numerous Kjeldahl total nitrogen measurements. Individual samples may have appreciably different NPN levels. A total nitrogen measurement for such a sample would be less indicative of the true protein value. Once the DBC is established for a given protein system, the dye-binding method of estimating protein is independent and absolute. It is not responsive to variations in NPN.
When the ratio of BAA to protein is not genetically controlled, such as in random mixtures of commodities, or when the percentage of NPN is changed, as in the ultra-filtration concentration of whey proteins, protein measured by dye-binding may not correlate as well with Kjeldahl total nitrogen measurements. Denaturation of lysine by overheating or Maillard type reactions will also lower the correlation between protein measured by dye-binding and Kjeldahl total nitrogen. The UDY dye-binding method is being used on a large number of protein systems. These include, but are not limited to, all grains, oilseeds, forages, legumes, meat and dairy products.
The protein-dye reaction requires some controls over certain factors that influence the equilibrium dye concentration. Temperature of the sample-dye mixture has a small but significant effect on the degree of protein-dye association. Different ions and buffer systems have potentially large effects on the DBC value. A mass action effect is significant at low dye concentrations. Mass action and ionic effects are negligible or absent entirely in the normal concentration range used for the buffer system now used in UDY Reagent Dye Solution. Starch from some commodities binds a small, but significant, amount of dye. This does not cause loss of accuracy because the starch content and its dye-binding are essentially constant. Correlations coefficients over 99% are normally obtained between equilibrium dye concentration and total nitrogen measurements.
Lysine maybe blocked from reacting with the dye. Reactive or nutritionally available lysine can be calculated from the difference in results when the lysine epsilon amino groups are blocked and not blocked. Lysine measurements provide a better nutritional index than total nitrogen for some protein systems because lysine is often the limiting amino acid in protein utilization. The procedure used is included in the Procedure-Table section.
LysIndex, a measure of dye bound per gram of sample, is another convenient means of estimating nutritional quality. Since lysine is one of three BAA which react with the dye, LysIndex is related to the amount of nutritionally available lysine. Although it is not directly proportional to the lysine content, its measurement is more practical. LysIndex is as easily measured as protein by dye-binding while the lysine measurement, per se,is more involved. The table of settings for the Digital Colorimeter for LysIndex measurement is in the Procedure-Tables section.
LysIndex is especially more nutritionally significant than total protein for swine, poultry, and other non-ruminants. It is, therefore, the index of choice and should be used for all feeding applications -- especially non-ruminants. Recent research has indicated that when proteins are degraded by excessive heat in drying or other processing or storage conditions which damage the lysine, nutritional utilization is affected for both ruminants and non-ruminants. Previous notions that protein quality is unimportant for ruminants are incorrect.
Casein in milk, undenatured whey protein in non-fat dry milk, and true protein in whey can be measured rapidly and accurately by dye-binding procedures.
Protein testing by dye-binding does not depend on the protein first being dissolved before it will interact with the dye. When the particle size of solids is reduced sufficiently, dye diffusion enables proteins to react completely. The UDY Extract-R-Reactor accelerates the diffusion and reaction. This enables complete reaction to be achieved in less than one minute for most materials. When the samples are fluid or semi-fluid, the proteins generally react completely in less than 5 seconds with thorough hand shaking.