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以胰蛋白酶对酪蛋白的Ｋｍ值测定为例，讨论了底物浓度选择在酶Ｋｍ值测定中的重要性。底物酪蛋白的浓度分为三组：高浓度（１～５ｍｇ／ｍｌ），低浓度（０．１～１ｍｇ／ｍｌ）和极低浓度（０．０２～０．０８ｍｇ／ｍｌ），高底物浓度（＞８Ｋｍ）的测定结果不能有效地区分多酶（作用于同一底物的多种酶）的存在，也不符合作图法要求的准确度，所得到的Ｋｍ值可能是不准确的或错误的。因此，测定Ｋｍ值时所用的底物浓度必须在０．２～５Ｋｍ范围内，必要时应包括更广泛的浓度范围（包括高底物浓度）。
Taking the Km value of trypsin to casein as an example, the importance of substrate concentration selection in the determination of enzyme Km was discussed. The concentration of substrate casein was divided into three groups: high concentration (1-5mg / ml), low concentration (0.1-1mg / ml) and very low concentration (0.02-0.08mg / ml) The results of the concentration determination (> 8Km) do not effectively distinguish the presence of multiple enzymes (multiple enzymes acting on the same substrate) nor the accuracy required by the mapping method. The Km values obtained may be inaccurate or Incorrect. Therefore, the concentration of substrate used to determine the Km value must be in the range of 0.2 to 5 Km and a broader range of concentrations (including high substrate concentrations) should be included if necessary.