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A kinetic mechanism for the digestion of collagen by bacterial collagenase has been proposed which is consistent with experimental data. Arguments are presented justifying the application of a homogeneous approach to the digestion of insoluble collagen. The mechanism takes account of increased kinetic blockages with aging and enzyme complexity. It is shown to be applicable to collagen from various tissues, including relatively impure preparations. However, the mechanism is not applicable to collagen from the immature period of the lifespan. Kinetic blockages are shown to increase three- to four-fold from age 20 to 70 years, within the main helical region of the collagen molecule. There is no increase of kinetic blockages in the N-terminal region of the molecule over this age range.

Nonenzymatically glycosylated proteins gradually form fluorescent cross-linked protein adducts--a process termed "browning." The rate of this reaction increases with the glucose concentration. Assaying for the presence of browning products in long-lived proteins should therefore provide information on long-term metabolic control. We measured collagen-linked fluorescence typical for nonenzymatic browning in skin-biopsy specimens from 41 subjects with longstanding Type I diabetes and from 25 controls. Fluorescence correlated with age and (weakly) with the duration of diabetes. Mean age-adjusted fluorescence values were twice as high in diabetic subjects as in control subjects (P less than 0.0001) and increased with the severity of retinopathy, nephropathy, and arterial and joint stiffness. The correlation was significant for retinopathy (r = 0.42; P less than 0.01), arterial stiffness (r = 0.41; P less than 0.01), joint stiffness (r = 0.34; P less than 0.05), and the sum of all complications (r = 0.47; P less than 0.01). Fluorescence also correlated with systolic (r = 0.42; P less than 0.01) and diastolic (r = 0.36; P less than 0.05) blood pressures. If one can assume that the fluorescence results from a browning product of glucose, our data suggest that there is an overall correlation between the severity of diabetic complications and cumulative glycemia over many years.

The digestion of human collagen by bacterial collagenase was studied in order to relate collagen structure to the digestion kinetics. The digestion was shown to be first order in collagen and in collagenase on given material from run to run. There was no product inhibition and no enzyme deactivation. However, the rate changed with age, and also within a run as a function of conversion. It was shown that the normalized time (time X enzyme concentration), to reach a given amount of digestion can be plotted as a linear function of the sample age, from 20 to 70 years. The digestion broke down into two regions. The first was a rapid first order region which amounted to about 10% of the collagen. The second region could be graphed as a second order plot to obtain a straight line. This implied that the rate of digestion was functionally second order in collagen; first order in concentration and first order in conversion of collagen.